http://2009.igem.org/wiki/index.php?title=Special:Contributions/JolandaWitteveen&feed=atom&limit=50&target=JolandaWitteveen&year=&month=2009.igem.org - User contributions [en]2024-03-29T05:51:22ZFrom 2009.igem.orgMediaWiki 1.16.5http://2009.igem.org/Team:Groningen/Ethics/SurveyTeam:Groningen/Ethics/Survey2009-10-21T23:48:15Z<p>JolandaWitteveen: /* Results */</p>
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<div class="intro introduction"><br />
==Survey==<br />
Ethics is an important issue to consider in the new field of synthetic biology. In order to gain more insight into the Dutch public opinion we decided to create a survey. <br />
The results of our survey suggest that in general people in the Netherlands are most concerned about the safety issues, however, they do trust researchers and developers to consider them.<br />
Surprisingly there appears to be no difference in these matters between people familiar with synthetic biology and those unfamiliar with it.<br />
</div><br />
<br />
===Considerations===<br />
The survey contains questions about the ethical issues surrounding synthetic biology and also about the ethical issues surrounding our project.<br />
We wanted to reach a mixed public in order to get an idea of the Dutch public opinion on the ethical issues of synthetic biology and our project. We did a convenience sampling so we handed out our survey among family, friends, secondary school pupils and fellow students. We have chosen for this sample construct because it is not expensive and it is easy to apply. We added a link to our survey on our wiki-website. In an attempt to exclude non-Dutch respondents we included the question “do you live or work in the Netherlands?” to our survey. Since we thought that it might make a difference if the participants already knew what synthetic biology was, we included the question “do you know what synthetic biology is?”. Being religious or not was also thought to possibly influence the opinion of the participant about for instance the playing God issue, so a question about religion was included as well. Just as age and gender could make a difference and therefore questions about that were included. <br />
We wanted to approach the Delphi method for brainstorming, also previous described by [[Team:Groningen/Literature#Zwart, SD, et al.2006|(Zwart, SD, et al.2006)]] in the Netherlands and recently used in the EU synth-ethics meeting, to gain more insights in ethics. Central in this method is the fact that participants can give their opinion anonymously and without consequences. The idea behind it is that if someone can speak freely this will open up the discussion. Although we did not organize a discussion, we did try to mimic this by including an essay question in the survey in which we asked the participants to give other ethical and safety issues surrounding our project. Since the survey is anonymously and we also aimed to ask all kinds of users to fill it out this survey approaches the open discussion method.<br />
The questions are based on the four ethical issues of synthetic biology described by [[Team:Groningen/Literature#Bhutkar, A2005|(Bhutkar, A2005)]], safety, security, playing God and intellectual property. All the respondents were shown a cover letter with information about the aim of the survey. The participants could decide to stop at any moment during the survey. After analyzing the data we send a debriefing to the respondents with the conclusion about our survey. For the confidentiality of the respondents we used the program ‘Examine’. In this program all the participants have a random number as ID. Also we have minimizes the number of people who could see or handle the data. The questions consists mostly of theorems, which were translated to a Likert scale afterwards.<br />
<br />
===Results===<br />
The survey was opened from 11-09-2009 until 07-10-2009 (27 days) and was completed by 262 respondents, 147 male and 115 female. <br />
The educational level of the respondents was mixed. As can be seen in figure 1 the different educational groups are all represented. There is a small bias to the educative level university. So the survey is, however, not a completely representative sample of the Dutch population, but it is still a good suggestion for the Dutch public opinion. <br />
We took this into account when we did the statistical analysis and choose, therefore, to do non-probability tests. We have chosen the ‘Mann-Whitney’ test for two independent samples, by questions with more than two independent groups, we use the ‘Kruskal-Wallis H.’ test. The power of the Mann-Whitney test was determined and appeared to be high enough.<br />
Both procedures are testing equality of population medians among groups. Sometimes if the data looked normal distributed (we have checked this with boxplots), we used an ANOVA two-tailed with the post-hoc ‘Bonferroni’ All test are done with a confidence level of 95% (α=0.05) [[Team:Groningen/Literature#Salkind1991|(Salkind 1991)]]. <br />
<br />
[[Image:Groningen_Figure1Survey.png|400px]]<br />
<br />
Figure 1: Educational level of the respondents of the ethical survey of the iGEM team Groningen 2009<br />
<br />
===Inferential statistics=== <br />
<br />
‘’Difference between different levels of education’’<br />
Grouping the university (biology), university (non-biology) and university (engineering) shows that the data can be biased towards the university level educated respondents. <br />
There, however, does not seem to be a difference in response between the different educational levels towards the ethical issues of our project, one exception being the response towards the risk of bio terrorism. The university level educated respondents see less risk of bio terrorism of our project than do the lower level educated respondents. (p=0,001)<br />
There does also seem to be a significant difference in response to the question “how do you feel about bacteria being manipulated for research” between respondents with educational level secondary school and university (biology) (p=0.014, two-way ANOVA with post-hoc bonferoni). University students are more positive towards manipulating bacteria for research then secondary school pupils. This can also be seen when a correlation test is done. The spearman’s rho correlation test was chosen because we use categorical variables and the data is normal distributed. A (weak) correlation was found (R=0,215 and p=0,001) between level of education and openness towards manipulating bacteria. Respondents with a higher level of education are more positive towards manipulating bacteria.<br />
<br />
<br />
''Difference between religious and not religious''<br />
It was predicted that being religious or not could influence the opinion of the respondents towards the playing God issue of our project. No difference, however, could be detected (p=0.96, two tailed Mann-Withney test). Both religious and non-religious respondents did not thought that using GMO’s for water or sludge cleaning is unethical in the sense that researchers are playing God (figure 2).<br />
<br />
<br />
[[Image:Groningen_Figure2Survey.png|400px]]<br />
<br />
Figure 2: Frequency table of the responses towards the question do you think using GMO’s for water or sludge cleaning unethical in the sense that researchers are playing God against being religious or not.<br />
<br />
''Difference between knowing and not knowing what synthetic biology is''<br />
<br />
Half of the respondents (51,1%) did not know what synthetic biology is, 48,9% of the respondents did. It was hypothesized that there would be a difference between respondents that know what synthetic biology is and those who do not, in how they feel about GMO’s being used in application. This, however, did not appear to be the case form our survey (p=0.236, two-tailed Mann-whitney test). <br />
<br />
''Difference between male and female''<br />
<br />
If male and female respondents were compared there appeared to be a difference how they feel about GMO’s being manipulated for research. Here a one-tailed test has been performed because it was presumed that women are, in general, more caring then men. A significant difference between the genders with a p-value of 0,0005 was found showing that women are more thoughtful about the usage of GMO’s for research then man.<br />
<br />
''Difference between older and younger respondents''<br />
<br />
It was also tested if age make a difference in how the participants feel about the ethical issues of our project. One significant difference was found between participants older than 50 and the group aged between 21 and 30. The older group (>50) appeared to be more afraid of misusage of GMO in cleaning water or sludge for bioterrorism than the younger group (p=0,022) <br />
<br />
<br />
===Descriptive statistics===<br />
<br />
No differences were found between other groups so the following results can be considered the same for all groups.<br />
The respondents were asked what they thought of bacteria being manipulated for research. The majority, 89.7%, answered “good” and only 5,8% answered “not good” because it can be dangerous or researchers are playing God. So making GMO’s does not seem to be considered unethical, using these GMO’s in practice was also not considered unethical, 93.5% was positive about it. Safety for the environment and human health was, however, a concern for 24,4% of the respondents. 69,1% of the respondents found, security and usefulness for society, a condition for the usage of GMO’s. The respondents were more critical about the usage of GMO’s for a specific application, in water or sludge cleaning. A majority of the respondents, 50,8%, thought that the usage of GMO’s can be dangerous for the environment or human health so this should be considered before usage. 29% of the respondents trusted the researchers in considering the safety and make the application more safe. Security of GMO’s for applications in the water and sludge cleaning is an issue that concerns 23,7% and they feel that this deserves some thought. Another 36,6% of the respondents also thought that security is an issue, however, there is always the risk of mis usage. The remaining 33,2% of the respondents did not think security is an issue in this application. Another important ethical issue according to [[Team:Groningen/Literature#Bhutkar, A2005|(Bhutkar, A2005)]], the playing God issue, does not seem to be a concern for the usage of GMO’s in water and sludge cleaning according to the respondents of our survey (92,4%). <br />
The fourth ethical issue, the intellectual property, was also considered for using GMO’s in water and sludge cleaning. Although 26,3% of the respondents thought it should be possible to patent the application of the iGEM team Groningen 2009 because it is important for development the majority (57.3%) of the respondents only agreed with patenting the whole system and not the DNA itself. 11.5% of the respondents were against patenting this system in which GMO’s are used (fig 3).<br />
<br />
[[Image:Groningen_Figure3Survey.png|400px]]<br />
<br />
Figure 3: The four ethical issues, as described by bhutkar, considered for the project of the iGEM team Groningen 2009.<br />
<br />
[[Image:Groningen_Figure4Survey.png|400px]]<br />
<br />
Figure 4: This figure shows what the most important ethical issues is in our project according to our respondents. The four issues, safety (A), security (B), intellectual property (C) and playing God (D) are derived from <br />
<br />
The survey included a question, which ethical issue was considered the most important for our project, two answers could be given. In the above figure it can be seen that most respondents, 40%, thought safety is the most important issue. Also in combination with other issues safety is considered to be very important. Also security is thought to be a risk in water and sludge cleaning, by 30% of the participants. As a second choice safety and security (A and B) are the most chosen answers. This shows that intellectual property and playing God play minor roles in the ethics of our project compared to the risks in safety and security. These are clearly the issues that should be taken into account in a possible application of our project.<br />
<br />
===Conclusion===<br />
The results of our survey suggest that in general people in the Netherlands are most concerned about the safety issues, however, they do trust researchers and developers to consider this in case GMO’s are used in application. Also the majority thinks that there is a security risk by using GMO’s for our project in a possible application. Less educated and older people would indicate a higher security risk than a higher educational level and younger people.<br />
<br />
In general manipulation of bacteria for research is considered good. Women, however, are more thoughtful about it than man. People with a higher level education are more open towards using bacteria for research than do lower level educated people. Unexpectedly we found no difference between people who did know what synthetic biology was and those who did not. So it appears that one is not more anxious about something because of a lack of awareness.<br />
Altogether comparing the four ethical issues of Bhutkar, safety, security, playing God and intellectual property, for synthetic biology in general and our project in particular shows that synthetic biological research is considered ok as long as done carefully and safety and security issues are considered.<br />
<br />
{{Team:Groningen/Footer}}</div>JolandaWitteveenhttp://2009.igem.org/Team:Groningen/Ethics/SurveyTeam:Groningen/Ethics/Survey2009-10-21T23:47:50Z<p>JolandaWitteveen: /* Results */</p>
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<div class="intro introduction"><br />
==Survey==<br />
Ethics is an important issue to consider in the new field of synthetic biology. In order to gain more insight into the Dutch public opinion we decided to create a survey. <br />
The results of our survey suggest that in general people in the Netherlands are most concerned about the safety issues, however, they do trust researchers and developers to consider them.<br />
Surprisingly there appears to be no difference in these matters between people familiar with synthetic biology and those unfamiliar with it.<br />
</div><br />
<br />
===Considerations===<br />
The survey contains questions about the ethical issues surrounding synthetic biology and also about the ethical issues surrounding our project.<br />
We wanted to reach a mixed public in order to get an idea of the Dutch public opinion on the ethical issues of synthetic biology and our project. We did a convenience sampling so we handed out our survey among family, friends, secondary school pupils and fellow students. We have chosen for this sample construct because it is not expensive and it is easy to apply. We added a link to our survey on our wiki-website. In an attempt to exclude non-Dutch respondents we included the question “do you live or work in the Netherlands?” to our survey. Since we thought that it might make a difference if the participants already knew what synthetic biology was, we included the question “do you know what synthetic biology is?”. Being religious or not was also thought to possibly influence the opinion of the participant about for instance the playing God issue, so a question about religion was included as well. Just as age and gender could make a difference and therefore questions about that were included. <br />
We wanted to approach the Delphi method for brainstorming, also previous described by [[Team:Groningen/Literature#Zwart, SD, et al.2006|(Zwart, SD, et al.2006)]] in the Netherlands and recently used in the EU synth-ethics meeting, to gain more insights in ethics. Central in this method is the fact that participants can give their opinion anonymously and without consequences. The idea behind it is that if someone can speak freely this will open up the discussion. Although we did not organize a discussion, we did try to mimic this by including an essay question in the survey in which we asked the participants to give other ethical and safety issues surrounding our project. Since the survey is anonymously and we also aimed to ask all kinds of users to fill it out this survey approaches the open discussion method.<br />
The questions are based on the four ethical issues of synthetic biology described by [[Team:Groningen/Literature#Bhutkar, A2005|(Bhutkar, A2005)]], safety, security, playing God and intellectual property. All the respondents were shown a cover letter with information about the aim of the survey. The participants could decide to stop at any moment during the survey. After analyzing the data we send a debriefing to the respondents with the conclusion about our survey. For the confidentiality of the respondents we used the program ‘Examine’. In this program all the participants have a random number as ID. Also we have minimizes the number of people who could see or handle the data. The questions consists mostly of theorems, which were translated to a Likert scale afterwards.<br />
<br />
===Results===<br />
The survey was opened from 11-09-2009 until 07-10-2009 (27 days) and was completed by 262 respondents, 147 male and 115 female. <br />
The educational level of the respondents was mixed. As can be seen in figure 1 the different educational groups are all represented. There is a small bias to the educative level university. So the survey is, however, not a completely representative sample of the Dutch population, but it is still a good suggestion for the Dutch public opinion. <br />
We took this into account when we did the statistical analysis and choose, therefore, to do non-probability tests. We have chosen the ‘Mann-Whitney’ test for two independent samples, by questions with more than two independent groups, we use the ‘Kruskal-Wallis H.’ test. The power of the Mann-Whitney test was determined and appeared to be high enough.<br />
Both procedures are testing equality of population medians among groups. Sometimes if the data looked normal distributed (we have checked this with boxplots), we used an ANOVA two-tailed with the post-hoc ‘Bonferroni’ All test are done with a confidence level of 95% (α=0.05) [[Team:Groningen/Literature#Salkind1991|Salkind 1991]]. <br />
<br />
[[Image:Groningen_Figure1Survey.png|400px]]<br />
<br />
Figure 1: Educational level of the respondents of the ethical survey of the iGEM team Groningen 2009<br />
<br />
===Inferential statistics=== <br />
<br />
‘’Difference between different levels of education’’<br />
Grouping the university (biology), university (non-biology) and university (engineering) shows that the data can be biased towards the university level educated respondents. <br />
There, however, does not seem to be a difference in response between the different educational levels towards the ethical issues of our project, one exception being the response towards the risk of bio terrorism. The university level educated respondents see less risk of bio terrorism of our project than do the lower level educated respondents. (p=0,001)<br />
There does also seem to be a significant difference in response to the question “how do you feel about bacteria being manipulated for research” between respondents with educational level secondary school and university (biology) (p=0.014, two-way ANOVA with post-hoc bonferoni). University students are more positive towards manipulating bacteria for research then secondary school pupils. This can also be seen when a correlation test is done. The spearman’s rho correlation test was chosen because we use categorical variables and the data is normal distributed. A (weak) correlation was found (R=0,215 and p=0,001) between level of education and openness towards manipulating bacteria. Respondents with a higher level of education are more positive towards manipulating bacteria.<br />
<br />
<br />
''Difference between religious and not religious''<br />
It was predicted that being religious or not could influence the opinion of the respondents towards the playing God issue of our project. No difference, however, could be detected (p=0.96, two tailed Mann-Withney test). Both religious and non-religious respondents did not thought that using GMO’s for water or sludge cleaning is unethical in the sense that researchers are playing God (figure 2).<br />
<br />
<br />
[[Image:Groningen_Figure2Survey.png|400px]]<br />
<br />
Figure 2: Frequency table of the responses towards the question do you think using GMO’s for water or sludge cleaning unethical in the sense that researchers are playing God against being religious or not.<br />
<br />
''Difference between knowing and not knowing what synthetic biology is''<br />
<br />
Half of the respondents (51,1%) did not know what synthetic biology is, 48,9% of the respondents did. It was hypothesized that there would be a difference between respondents that know what synthetic biology is and those who do not, in how they feel about GMO’s being used in application. This, however, did not appear to be the case form our survey (p=0.236, two-tailed Mann-whitney test). <br />
<br />
''Difference between male and female''<br />
<br />
If male and female respondents were compared there appeared to be a difference how they feel about GMO’s being manipulated for research. Here a one-tailed test has been performed because it was presumed that women are, in general, more caring then men. A significant difference between the genders with a p-value of 0,0005 was found showing that women are more thoughtful about the usage of GMO’s for research then man.<br />
<br />
''Difference between older and younger respondents''<br />
<br />
It was also tested if age make a difference in how the participants feel about the ethical issues of our project. One significant difference was found between participants older than 50 and the group aged between 21 and 30. The older group (>50) appeared to be more afraid of misusage of GMO in cleaning water or sludge for bioterrorism than the younger group (p=0,022) <br />
<br />
<br />
===Descriptive statistics===<br />
<br />
No differences were found between other groups so the following results can be considered the same for all groups.<br />
The respondents were asked what they thought of bacteria being manipulated for research. The majority, 89.7%, answered “good” and only 5,8% answered “not good” because it can be dangerous or researchers are playing God. So making GMO’s does not seem to be considered unethical, using these GMO’s in practice was also not considered unethical, 93.5% was positive about it. Safety for the environment and human health was, however, a concern for 24,4% of the respondents. 69,1% of the respondents found, security and usefulness for society, a condition for the usage of GMO’s. The respondents were more critical about the usage of GMO’s for a specific application, in water or sludge cleaning. A majority of the respondents, 50,8%, thought that the usage of GMO’s can be dangerous for the environment or human health so this should be considered before usage. 29% of the respondents trusted the researchers in considering the safety and make the application more safe. Security of GMO’s for applications in the water and sludge cleaning is an issue that concerns 23,7% and they feel that this deserves some thought. Another 36,6% of the respondents also thought that security is an issue, however, there is always the risk of mis usage. The remaining 33,2% of the respondents did not think security is an issue in this application. Another important ethical issue according to [[Team:Groningen/Literature#Bhutkar, A2005|(Bhutkar, A2005)]], the playing God issue, does not seem to be a concern for the usage of GMO’s in water and sludge cleaning according to the respondents of our survey (92,4%). <br />
The fourth ethical issue, the intellectual property, was also considered for using GMO’s in water and sludge cleaning. Although 26,3% of the respondents thought it should be possible to patent the application of the iGEM team Groningen 2009 because it is important for development the majority (57.3%) of the respondents only agreed with patenting the whole system and not the DNA itself. 11.5% of the respondents were against patenting this system in which GMO’s are used (fig 3).<br />
<br />
[[Image:Groningen_Figure3Survey.png|400px]]<br />
<br />
Figure 3: The four ethical issues, as described by bhutkar, considered for the project of the iGEM team Groningen 2009.<br />
<br />
[[Image:Groningen_Figure4Survey.png|400px]]<br />
<br />
Figure 4: This figure shows what the most important ethical issues is in our project according to our respondents. The four issues, safety (A), security (B), intellectual property (C) and playing God (D) are derived from <br />
<br />
The survey included a question, which ethical issue was considered the most important for our project, two answers could be given. In the above figure it can be seen that most respondents, 40%, thought safety is the most important issue. Also in combination with other issues safety is considered to be very important. Also security is thought to be a risk in water and sludge cleaning, by 30% of the participants. As a second choice safety and security (A and B) are the most chosen answers. This shows that intellectual property and playing God play minor roles in the ethics of our project compared to the risks in safety and security. These are clearly the issues that should be taken into account in a possible application of our project.<br />
<br />
===Conclusion===<br />
The results of our survey suggest that in general people in the Netherlands are most concerned about the safety issues, however, they do trust researchers and developers to consider this in case GMO’s are used in application. Also the majority thinks that there is a security risk by using GMO’s for our project in a possible application. Less educated and older people would indicate a higher security risk than a higher educational level and younger people.<br />
<br />
In general manipulation of bacteria for research is considered good. Women, however, are more thoughtful about it than man. People with a higher level education are more open towards using bacteria for research than do lower level educated people. Unexpectedly we found no difference between people who did know what synthetic biology was and those who did not. So it appears that one is not more anxious about something because of a lack of awareness.<br />
Altogether comparing the four ethical issues of Bhutkar, safety, security, playing God and intellectual property, for synthetic biology in general and our project in particular shows that synthetic biological research is considered ok as long as done carefully and safety and security issues are considered.<br />
<br />
{{Team:Groningen/Footer}}</div>JolandaWitteveenhttp://2009.igem.org/Team:Groningen/LiteratureTeam:Groningen/Literature2009-10-21T23:47:44Z<p>JolandaWitteveen: </p>
<hr />
<div>{{Team:Groningen/Header}}<br />
<br />
<div style="float:left" >{{linkedImage|GroningenPrevious.png|Team:Groningen/Glossary}}</div><br />
<div title="Arsie Says UP TO ACCUMULATION" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Protocols}}</div><br />
<br />
[[Category:Team:Groningen]]<br />
<br />
==Literature==<br />
Buoyancy related literature:<br />
*Buoyant density: [[Team:Groningen/Literature#Poole1977|Poole 1977]], [[Team:Groningen/Literature#Bylund1991|Bylund 1991]], '''[[Team:Groningen/Literature#Baldwin1995|Baldwin 1995]]'''<br />
*Gas vesicles: [[Team:Groningen/Literature#Bowen1965|Bowen 1965]], [[Team:Groningen/Literature#Walsby1979|Walsby 1979]], '''[[Team:Groningen/Literature#Walsby1994|Walsby 1994]]''', '''[[Team:Groningen/Literature#Li1998|Li 1998]]''', [[Team:Groningen/Literature#Sivertsen2008|Sivertsen 2008]], [[Team:Groningen/Literature#Holland2009|Holland 2009]]<br />
<br />
Other methods of arsenic purification.<br />
*General information about the subject:[[Team:Groningen/Literature#Frankenberger2001|Frankenberger 2001]],[[Team:Groningen/Literature#Stephan Hug|Stephan Hug]],[[Team:Groningen/Literature#Wlckramaslnghe2004|Wlckramaslnghe 2004]],[[Team:Groningen/Literature#Dong2008|Dong 2008]],'''[[Team:Groningen/Literature#EPA2000|EPA 2000]]''',<br />
*Ion exchange and Membranes:[[Team:Groningen/Literature#Oehmen2006|Oehmen 2006]],<br />
*Nanomaterials:[[Team:Groningen/Literature#Hristovski2007|Hristovski 2007]],[[Team:Groningen/Literature#Martinson2009|Martinson 2009]],[[Team:Groningen/Literature#Chang2009|Chang 2009]],<br />
*Precipitative Processes:[[Team:Groningen/Literature#Raje2005|Raje 2005]],<br />
<br />
Our metal related literature by subject:<br />
{|border="1" <br />
!<br />
!<br />
!Cu<br />
!Zn<br />
!As<br />
!Cd<br />
!Sb<br />
!Hg<br />
|-<br />
!rowspan="2" |Importers<br />
!GlpF<br />
|<br />
|<br />
|'''[[Team:Groningen/Literature#Meng2004|Meng 2004]]''', [[Team:Groningen/Literature#Rosen2009|Rosen 2009]]<br />
|<br />
|'''[[Team:Groningen/Literature#Meng2004|Meng 2004]]'''<br />
|<br />
|-<br />
!{{part|BBa_K190018|HmtA}}<br />
|[[Team:Groningen/Literature#Lewinson2009|Lewinson 2009]]<br />
|[[Team:Groningen/Literature#Lewinson2009|Lewinson 2009]]<br />
|<br />
|<br />
|<br />
|-<br />
!rowspan="1" |Exporters<br />
!ArsB<br />
|<br />
|<br />
|[[Team:Groningen/Literature#Tisa1989|Tisa 1989]], [[Team:Groningen/Literature#Carlin1995|Carlin 1995]],<br />
[[Team:Groningen/Literature#Dey1995|Dey 1995]] [[Team:Groningen/Literature#Rosen1996|Rosen 1996]], '''[[Team:Groningen/Literature#Meng2004|Meng 2004]]''', [[Team:Groningen/Literature#Summers2009|Summers 2009]]<br />
|<br />
|[[Team:Groningen/Literature#Tisa1989|Tisa 1989]], [[Team:Groningen/Literature#Carlin1995|Carlin 1995]], [[Team:Groningen/Literature#Rosen1996|Rosen 1996]], '''[[Team:Groningen/Literature#Meng2004|Meng 2004]]''', [[Team:Groningen/Literature#Summers2009|Summers 2009]]<br />
|<br />
|-<br />
!rowspan="6" |Accumulators<br />
!ArsR<br />
|<br />
|<br />
||[[Team:Groningen/Literature#Carlin1995|Carlin 1995]], [[Team:Groningen/Literature#Rosen1996|Rosen 1996]], '''[[Team:Groningen/Literature#Chen1997|Chen 1997]]''', '''[[Team:Groningen/Literature#Kostal2004|Kostal 2004]]''', [[Team:Groningen/Literature#Rensing2005|Rensing 2005]], [[Team:Groningen/Literature#Summers2009|Summers 2009]]<br />
|<br />
||[[Team:Groningen/Literature#Carlin1995|Carlin 1995]], [[Team:Groningen/Literature#Rosen1996|Rosen 1996]], [[Team:Groningen/Literature#Summers2009|Summers 2009]]<br />
<br />
|<br />
|-<br />
!ArsD<br />
|<br />
|<br />
|[[Team:Groningen/Literature#Chen1997|Chen 1997]], [[Team:Groningen/Literature#Lin2007-1|Lin 2007-1/2]], [[Team:Groningen/Literature#Summers2009|Summers 2009]]<br />
|<br />
|[[Team:Groningen/Literature#Chen1997|Chen 1997]], [[Team:Groningen/Literature#Lin2007-1|Lin 2007-1/2]], [[Team:Groningen/Literature#Summers2009|Summers 2009]]<br />
|<br />
|-<br />
!SmtA<br />
|[[Team:Groningen/Literature#Shi1992|Shi 1992]]<br />
|[[Team:Groningen/Literature#Shi1992|Shi 1992]], [[Team:Groningen/Literature#Turner1995|Turner 1995]], [[Team:Groningen/Literature#Robinson2001|Robinson 2001]], [[Team:Groningen/Literature#Blindauer2002|Blindauer 2002]]<br />
|<br />
|[[Team:Groningen/Literature#Shi1992|Shi 1992]]<br />
|<br />
|[[Team:Groningen/Literature#Shi1992|Shi 1992]]<br />
|-<br />
!MymT<br />
|[[Team:Groningen/Literature#Gold2008|Gold 2008]]<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|-<br />
!MT<br />
|<br />
|<br />
|[[Team:Groningen/Literature#Morris1999|Morris 1999]],[[Team:Groningen/Literature#Ngu2006|Ngu 2006]],[[Team:Groningen/Literature#Singh2008|Singh 2008]], [[Team:Groningen/Literature#Merrifield2004|Merrifield 2004]], [[Team:Groningen/Literature#Ngu2009|Ngu 2009]]<br />
|[[Team:Groningen/Literature#Deng2007|Deng 2007]]<br />
|<br />
|[[Team:Groningen/Literature#Chen1998|Chen1998]]<br />
|-<br />
!Unclassified<br />
|[[Team:Groningen/Literature#Brady1994|Brady 1994]]<br />
|[[Team:Groningen/Literature#Chang1998|Chang 1998]], [[Team:Groningen/Literature#Blindauer2001|Blindauer 2001]], [[Team:Groningen/Literature#Kao2008|Kao 2008]]<br />
|<br />
|[[Team:Groningen/Literature#Brady1994|Brady 1994]], [[Team:Groningen/Literature#Chang1998|Chang 1998]], <br />
|<br />
|[[Team:Groningen/Literature#Deng2008|Deng 2008]]<br />
|-<br />
!rowspan="1" |Promoters<br />
!<br />
|[[Team:Groningen/Literature#Mills1994|Mills 1994 ]], [[Team:Groningen/Literature#Khunajakr1999 |Khunajakr 1999]], [[Team:Groningen/Literature#Liu2004|Liu 2004]], [[Team:Groningen/Literature#Moore2005|Moore 2005]], [[Team:Groningen/Literature#Ettema2006 |Ettema2006 ]], [[Team:Groningen/Literature#Liu2006|Liu 2006]], [[Team:Groningen/Literature#Liu2008|Liu 2008]], [[Team:Groningen/Literature#Catini2008|Catini 2008]], [[Team:Groningen/Literature#Nawapan2009|Nawapan 2009]]<br />
|[[Team:Groningen/Literature#Thelwell1998|Thelwell 1998]], [[Team:Groningen/Literature#Liu2004|Liu 2004]], [[Team:Groningen/Literature#Moore2005|Moore2005]], [[Team:Groningen/Literature#Hirose2006 |Hirose 2006]], [[Team:Groningen/Literature#Kloosterman2008|Kloosterman 2008]]<br />
|[[Team:Groningen/Literature#Summers2009 |Summers 2009]]<br />
|[[Team:Groningen/Literature#Liu2004|Liu 2004]], [[Team:Groningen/Literature#Moore2005|Moore 2005]]<br />
|<br />
|<br />
|}<br />
<br />
;Alon 2007 {{anchor|Alon2007}}<br />
:{{star}} Uri Alon (2007). ''Introduction to systems biology: design principles of biological circuits'' Chapman & Hall/CRC. ISBN 978-1-58488-642-6<br />
<br />
;Baldwin 1995 {{anchor|Baldwin1995}}<br />
:W.W. Baldwin, Richard Myer, Nicole Powell, ''et al'' (August 1995). "[http://www.springerlink.com/content/nu1lkduf3w89fmd8 Buoyant density of Escherichia coli is determined solely by the osmolarity of the culture medium]". ''Archives of Microbiology'' '''164(2)''': 155-157<br />
<br />
;Beltramini 1981 {{anchor| Beltramini1981}}<br />
:M. Beltramini and K. Lerch (1981). "[http://www.ncbi.nlm.nih.gov/pubmed/6453726 Luminescence Properties of ''Neurospora'' Copper Metallothionein]". ''FEBS Letters'' '''127(2)''': 201-203<br />
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;Beyer 2004 {{anchor|Beyer2004}}<br />
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<br />
<br />
<br />
Miscellaneous:<br />
* [http://ginkgobioworks.com/cgi/primer.cgi Primer design Bioworks]<br />
* [http://openwetware.org/wiki/The_BioBricks_Foundation:Standards/Technical/Measurement Promotor measurement]<br />
* [http://openwetware.org/wiki/Main_Page OpenWetWare]<br />
* [http://www.hgsc.bcm.tmc.edu/projects/microbial/microbial-detail.xsp?project_id=105 E. coli DH10B genome] (with BLAST)<br />
{{Team:Groningen/Footer}}</div>JolandaWitteveenhttp://2009.igem.org/Team:Groningen/Ethics/SurveyTeam:Groningen/Ethics/Survey2009-10-21T22:19:17Z<p>JolandaWitteveen: /* Results */</p>
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<div>{{Team:Groningen/Header}}<br />
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<div style="float:left" >{{linkedImage|GroningenPrevious.png|Team:Groningen/Vision}}</div><br />
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==Survey==<br />
<br />
===Considerations===<br />
Ethics is an important issue to consider in the new field of synthetic biology. In order to gain more insights in the Dutch public opinion we decided to create a survey. <br />
The survey contains questions about the ethical issues surrounding synthetic biology and also about the ethical issues surrounding our project.<br />
We wanted to reach a mixed public in order to get an idea of the Dutch public opinion on the ethical issues of synthetic biology and our project. We did a convenience sampling so we handed out our survey among family, friends, secondary school pupils and fellow students. We have chosen for this sample construct because it is not expensive and it is easy to apply. We added a link to our survey on our wiki-website. In an attempt to exclude non-Dutch respondents we included the question “do you live or work in the Netherlands?” to our survey. Since we thought that it might make a difference if the participants already knew what synthetic biology was, we included the question “do you know what synthetic biology is?”. Being religious or not was also thought to possibly influence the opinion of the participant about for instance the playing God issue, so a question about religion was included as well. Just as age and gender could make a difference and therefore questions about that were included. <br />
We wanted to approach the Delphi method for brainstorming, also previous described by [[Team:Groningen/Literature#Zwart, SD, et al.2006|(Zwart, SD, et al.2006)]] in the Netherlands and recently used in the EU synth-ethics meeting, to gain more insights in ethics. Central in this method is the fact that participants can give their opinion anonymously and without consequences. The idea behind it is that if someone can speak freely this will open up the discussion. Although we did not organize a discussion, we did try to mimic this by including an essay question in the survey in which we asked the participants to give other ethical and safety issues surrounding our project. Since the survey is anonymously and we also aimed to ask all kinds of users to fill it out this survey approaches the open discussion method.<br />
The questions are based on the four ethical issues of synthetic biology described by [[Team:Groningen/Literature#Bhutkar, A2005|(Bhutkar, A2005)]], safety, security, playing God and intellectual property. All the respondents were shown a cover letter with information about the aim of the survey. The participants could decide to stop at any moment during the survey. After analyzing the data we send a debriefing to the respondents with the conclusion about our survey. For the confidentiality of the respondents we used the program ‘Examine’. In this program all the participants have a random number as ID. Also we have minimizes the number of people who could see or handle the data. The questions consists mostly of theorems, which were translated to a Likert scale afterwards.<br />
<br />
===Results===<br />
The survey was opened from 11-09-2009 until 07-10-2009 (27 days) and was completed by 262 respondents, 147 male and 115 female. <br />
The educational level of the respondents was mixed. As can be seen in figure 1 the different educational groups are all represented. There is a small bias to the educative level university. So the survey is, however, not a completely representative sample of the Dutch population, but it is still a good suggestion for the Dutch public opinion. <br />
We took this into account when we did the statistical analysis and choose, therefore, to do non-probability tests. We have chosen the ‘Mann-Whitney’ test for two independent samples, by questions with more than two independent groups, we use the ‘Kruskal-Wallis H.’ test. The power of the Mann-Whitney test was determined and appeared to be high enough.<br />
Both procedures are testing equality of population medians among groups. Sometimes if the data looked normal distributed (we have checked this with boxplots), we used an ANOVA two-tailed with the post-hoc ‘Bonferroni’ All test are done with a confidence level of 95% (α=0.05). <br />
<br />
[[Image:Groningen_Figure1Survey.png|400px]]<br />
<br />
Figure 1: Educational level of the respondents of the ethical survey of the iGEM team Groningen 2009<br />
<br />
===Inferential statistics=== <br />
<br />
‘’Difference between different levels of education’’<br />
Grouping the university (biology), university (non-biology) and university (engineering) shows that the data can be biased towards the university level educated respondents. <br />
There, however, does not seem to be a difference in response between the different educational levels towards the ethical issues of our project, one exception being the response towards the risk of bio terrorism. The university level educated respondents see less risk of bio terrorism of our project than do the lower level educated respondents. (p=0,001)<br />
There does also seem to be a significant difference in response to the question “how do you feel about bacteria being manipulated for research” between respondents with educational level secondary school and university (biology) (p=0.014, two-way ANOVA with post-hoc bonferoni). University students are more positive towards manipulating bacteria for research then secondary school pupils. This can also be seen when a correlation test is done. The spearman’s rho correlation test was chosen because we use categorical variables and the data is normal distributed. A (weak) correlation was found (R=0,215 and p=0,001) between level of education and openness towards manipulating bacteria. Respondents with a higher level of education are more positive towards manipulating bacteria.<br />
<br />
<br />
''Difference between religious and not religious''<br />
It was predicted that being religious or not could influence the opinion of the respondents towards the playing God issue of our project. No difference, however, could be detected (p=0.96, two tailed Mann-Withney test). Both religious and non-religious respondents did not thought that using GMO’s for water or sludge cleaning is unethical in the sense that researchers are playing God (figure 2).<br />
<br />
<br />
[[Image:Groningen_Figure2Survey.png|400px]]<br />
<br />
Figure 2: Frequency table of the responses towards the question do you think using GMO’s for water or sludge cleaning unethical in the sense that researchers are playing God against being religious or not.<br />
<br />
''Difference between knowing and not knowing what synthetic biology is''<br />
<br />
Half of the respondents (51,1%) did not know what synthetic biology is, 48,9% of the respondents did. It was hypothesized that there would be a difference between respondents that know what synthetic biology is and those who do not, in how they feel about GMO’s being used in application. This, however, did not appear to be the case form our survey (p=0.236, two-tailed Mann-whitney test). <br />
<br />
''Difference between male and female''<br />
<br />
If male and female respondents were compared there appeared to be a difference how they feel about GMO’s being manipulated for research. Here a one-tailed test has been performed because it was presumed that women are, in general, more caring then men. A significant difference between the genders with a p-value of 0,0005 was found showing that women are more thoughtful about the usage of GMO’s for research then man.<br />
<br />
''Difference between older and younger respondents''<br />
<br />
It was also tested if age make a difference in how the participants feel about the ethical issues of our project. One significant difference was found between participants older than 50 and the group aged between 21 and 30. The older group (>50) appeared to be more afraid of misusage of GMO in cleaning water or sludge for bioterrorism than the younger group (p=0,022) <br />
<br />
<br />
===Descriptive statistics===<br />
<br />
No differences were found between other groups so the following results can be considered the same for all groups.<br />
The respondents were asked what they thought of bacteria being manipulated for research. The majority, 89.7%, answered “good” and only 5,8% answered “not good” because it can be dangerous or researchers are playing God. So making GMO’s does not seem to be considered unethical, using these GMO’s in practice was also not considered unethical, 93.5% was positive about it. Safety for the environment and human health was, however, a concern for 24,4% of the respondents. 69,1% of the respondents found, security and usefulness for society, a condition for the usage of GMO’s. The respondents were more critical about the usage of GMO’s for a specific application, in water or sludge cleaning. A majority of the respondents, 50,8%, thought that the usage of GMO’s can be dangerous for the environment or human health so this should be considered before usage. 29% of the respondents trusted the researchers in considering the safety and make the application more safe. Security of GMO’s for applications in the water and sludge cleaning is an issue that concerns 23,7% and they feel that this deserves some thought. Another 36,6% of the respondents also thought that security is an issue, however, there is always the risk of mis usage. The remaining 33,2% of the respondents did not think security is an issue in this application. Another important ethical issue according to [[Team:Groningen/Literature#Bhutkar, A2005|(Bhutkar, A2005)]], the playing God issue, does not seem to be a concern for the usage of GMO’s in water and sludge cleaning according to the respondents of our survey (92,4%). <br />
The fourth ethical issue, the intellectual property, was also considered for using GMO’s in water and sludge cleaning. Although 26,3% of the respondents thought it should be possible to patent the application of the iGEM team Groningen 2009 because it is important for development the majority (57.3%) of the respondents only agreed with patenting the whole system and not the DNA itself. 11.5% of the respondents were against patenting this system in which GMO’s are used (fig 3).<br />
<br />
[[Image:Groningen_Figure3Survey.png|400px]]<br />
<br />
Figure 3: The four ethical issues, as described by bhutkar, considered for the project of the iGEM team Groningen 2009.<br />
<br />
[[Image:Groningen_Figure4Survey.png|400px]]<br />
<br />
Figure 4: This figure shows what the most important ethical issues is in our project according to our respondents. The four issues, safety (A), security (B), intellectual property (C) and playing God (D) are derived from <br />
<br />
The survey included a question, which ethical issue was considered the most important for our project, two answers could be given. In the above figure it can be seen that most respondents, 40%, thought safety is the most important issue. Also in combination with other issues safety is considered to be very important. Also security is thought to be a risk in water and sludge cleaning, by 30% of the participants. As a second choice safety and security (A and B) are the most chosen answers. This shows that intellectual property and playing God play minor roles in the ethics of our project compared to the risks in safety and security. These are clearly the issues that should be taken into account in a possible application of our project.<br />
<br />
===Conclusion===<br />
The results of our survey suggest that in general people in the Netherlands are most concerned about the safety issues, however, they do trust researchers and developers to consider this in case GMO’s are used in application. Also the majority thinks that there is a security risk by using GMO’s for our project in a possible application. Less educated and older people would indicate a higher security risk than a higher educational level and younger people.<br />
<br />
In general manipulation of bacteria for research is considered good. Women, however, are more thoughtful about it than man. People with a higher level education are more open towards using bacteria for research than do lower level educated people. Unexpectedly we found no difference between people who did know what synthetic biology was and those who did not. So it appears that one is not more anxious about something because of a lack of awareness.<br />
Altogether comparing the four ethical issues of Bhutkar, safety, security, playing God and intellectual property, for synthetic biology in general and our project in particular shows that synthetic biological research is considered ok as long as done carefully and safety and security issues are considered.<br />
<br />
<br />
{{Team:Groningen/Footer}}</div>JolandaWitteveenhttp://2009.igem.org/Team:Groningen/Ethics/SurveyTeam:Groningen/Ethics/Survey2009-10-21T22:11:07Z<p>JolandaWitteveen: /* Descriptive statistics */</p>
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<div>{{Team:Groningen/Header}}<br />
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<div style="float:left" >{{linkedImage|GroningenPrevious.png|Team:Groningen/Vision}}</div><br />
<div title="Arsie Says UP TO ACCUMULATION" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Parts}}</div><br />
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==Survey==<br />
<br />
===Considerations===<br />
Ethics is an important issue to consider in the new field of synthetic biology. In order to gain more insights in the Dutch public opinion we decided to create a survey. <br />
The survey contains questions about the ethical issues surrounding synthetic biology and also about the ethical issues surrounding our project.<br />
We wanted to reach a mixed public in order to get an idea of the Dutch public opinion on the ethical issues of synthetic biology and our project. We did a convenience sampling so we handed out our survey among family, friends, secondary school pupils and fellow students. We have chosen for this sample construct because it is not expensive and it is easy to apply. We added a link to our survey on our wiki-website. In an attempt to exclude non-Dutch respondents we included the question “do you live or work in the Netherlands?” to our survey. Since we thought that it might make a difference if the participants already knew what synthetic biology was, we included the question “do you know what synthetic biology is?”. Being religious or not was also thought to possibly influence the opinion of the participant about for instance the playing God issue, so a question about religion was included as well. Just as age and gender could make a difference and therefore questions about that were included. <br />
We wanted to approach the Delphi method for brainstorming, also previous described by [[Team:Groningen/Literature#Zwart, SD, et al.2006|(Zwart, SD, et al.2006)]] in the Netherlands and recently used in the EU synth-ethics meeting, to gain more insights in ethics. Central in this method is the fact that participants can give their opinion anonymously and without consequences. The idea behind it is that if someone can speak freely this will open up the discussion. Although we did not organize a discussion, we did try to mimic this by including an essay question in the survey in which we asked the participants to give other ethical and safety issues surrounding our project. Since the survey is anonymously and we also aimed to ask all kinds of users to fill it out this survey approaches the open discussion method.<br />
The questions are based on the four ethical issues of synthetic biology described by [[Team:Groningen/Literature#Bhutkar, A2005|(Bhutkar, A2005)]], safety, security, playing God and intellectual property. All the respondents were shown a cover letter with information about the aim of the survey. The participants could decide to stop at any moment during the survey. After analyzing the data we send a debriefing to the respondents with the conclusion about our survey. For the confidentiality of the respondents we used the program ‘Examine’. In this program all the participants have a random number as ID. Also we have minimizes the number of people who could see or handle the data. The questions consists mostly of theorems, which were translated to a Likert scale afterwards.<br />
<br />
===Results===<br />
The survey was opened from 11-09-2009 until 07-10-2009 (27 days) and was completed by 262 respondents, 147 male and 115 female. <br />
The educational level of the respondents was mixed. As can be seen in figure 1 the different educational groups are all represented. There is a small bias to the educative level university. So the survey is, however, not a completely representative sample of the Dutch population, but it is still a good suggestion for the Dutch public opinion. <br />
<br />
<br />
We consider this trough in the statistical analyses of the data with a non-probability test. We took this into account when we did the statistical analysis and choose, therefore, to do non-probability tests. We have chosen the ‘Mann-Whitney’ test for two independent samples, by questions with more than two independent groups, we use the ‘Kruskal-Wallis H.’ test. <br />
Both procedures are testing equality of population medians among groups. Sometimes if the data looked normal distributed (we have checked this with boxplots), we used an ANOVA two-tailed with the post-hoc ‘Bonferroni’ All test are done with a confidence level of 95% (α=0.05). <br />
<br />
[[Image:Groningen_Figure1Survey.png|400px]]<br />
<br />
Figure 1: Educational level of the respondents of the ethical survey of the iGEM team Groningen 2009 <br />
<br />
<br />
===Inferential statistics=== <br />
<br />
‘’Difference between different levels of education’’<br />
Grouping the university (biology), university (non-biology) and university (engineering) shows that the data can be biased towards the university level educated respondents. <br />
There, however, does not seem to be a difference in response between the different educational levels towards the ethical issues of our project, one exception being the response towards the risk of bio terrorism. The university level educated respondents see less risk of bio terrorism of our project than do the lower level educated respondents. (p=0,001)<br />
There does also seem to be a significant difference in response to the question “how do you feel about bacteria being manipulated for research” between respondents with educational level secondary school and university (biology) (p=0.014, two-way ANOVA with post-hoc bonferoni). University students are more positive towards manipulating bacteria for research then secondary school pupils. This can also be seen when a correlation test is done. The spearman’s rho correlation test was chosen because we use categorical variables and the data is normal distributed. A (weak) correlation was found (R=0,215 and p=0,001) between level of education and openness towards manipulating bacteria. Respondents with a higher level of education are more positive towards manipulating bacteria.<br />
<br />
<br />
''Difference between religious and not religious''<br />
It was predicted that being religious or not could influence the opinion of the respondents towards the playing God issue of our project. No difference, however, could be detected (p=0.96, two tailed Mann-Withney test). Both religious and non-religious respondents did not thought that using GMO’s for water or sludge cleaning is unethical in the sense that researchers are playing God (figure 2).<br />
<br />
<br />
[[Image:Groningen_Figure2Survey.png|400px]]<br />
<br />
Figure 2: Frequency table of the responses towards the question do you think using GMO’s for water or sludge cleaning unethical in the sense that researchers are playing God against being religious or not.<br />
<br />
''Difference between knowing and not knowing what synthetic biology is''<br />
<br />
Half of the respondents (51,1%) did not know what synthetic biology is, 48,9% of the respondents did. It was hypothesized that there would be a difference between respondents that know what synthetic biology is and those who do not, in how they feel about GMO’s being used in application. This, however, did not appear to be the case form our survey (p=0.236, two-tailed Mann-whitney test). <br />
<br />
''Difference between male and female''<br />
<br />
If male and female respondents were compared there appeared to be a difference how they feel about GMO’s being manipulated for research. Here a one-tailed test has been performed because it was presumed that women are, in general, more caring then men. A significant difference between the genders with a p-value of 0,0005 was found showing that women are more thoughtful about the usage of GMO’s for research then man.<br />
<br />
''Difference between older and younger respondents''<br />
<br />
It was also tested if age make a difference in how the participants feel about the ethical issues of our project. One significant difference was found between participants older than 50 and the group aged between 21 and 30. The older group (>50) appeared to be more afraid of misusage of GMO in cleaning water or sludge for bioterrorism than the younger group (p=0,022) <br />
<br />
<br />
===Descriptive statistics===<br />
<br />
No differences were found between other groups so the following results can be considered the same for all groups.<br />
The respondents were asked what they thought of bacteria being manipulated for research. The majority, 89.7%, answered “good” and only 5,8% answered “not good” because it can be dangerous or researchers are playing God. So making GMO’s does not seem to be considered unethical, using these GMO’s in practice was also not considered unethical, 93.5% was positive about it. Safety for the environment and human health was, however, a concern for 24,4% of the respondents. 69,1% of the respondents found, security and usefulness for society, a condition for the usage of GMO’s. The respondents were more critical about the usage of GMO’s for a specific application, in water or sludge cleaning. A majority of the respondents, 50,8%, thought that the usage of GMO’s can be dangerous for the environment or human health so this should be considered before usage. 29% of the respondents trusted the researchers in considering the safety and make the application more safe. Security of GMO’s for applications in the water and sludge cleaning is an issue that concerns 23,7% and they feel that this deserves some thought. Another 36,6% of the respondents also thought that security is an issue, however, there is always the risk of mis usage. The remaining 33,2% of the respondents did not think security is an issue in this application. Another important ethical issue according to [[Team:Groningen/Literature#Bhutkar, A2005|(Bhutkar, A2005)]], the playing God issue, does not seem to be a concern for the usage of GMO’s in water and sludge cleaning according to the respondents of our survey (92,4%). <br />
The fourth ethical issue, the intellectual property, was also considered for using GMO’s in water and sludge cleaning. Although 26,3% of the respondents thought it should be possible to patent the application of the iGEM team Groningen 2009 because it is important for development the majority (57.3%) of the respondents only agreed with patenting the whole system and not the DNA itself. 11.5% of the respondents were against patenting this system in which GMO’s are used (fig 3).<br />
<br />
[[Image:Groningen_Figure3Survey.png|400px]]<br />
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Figure 3: The four ethical issues, as described by bhutkar, considered for the project of the iGEM team Groningen 2009.<br />
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[[Image:Groningen_Figure4Survey.png|400px]]<br />
<br />
Figure 4: This figure shows what the most important ethical issues is in our project according to our respondents. The four issues, safety (A), security (B), intellectual property (C) and playing God (D) are derived from <br />
<br />
The survey included a question, which ethical issue was considered the most important for our project, two answers could be given. In the above figure it can be seen that most respondents, 40%, thought safety is the most important issue. Also in combination with other issues safety is considered to be very important. Also security is thought to be a risk in water and sludge cleaning, by 30% of the participants. As a second choice safety and security (A and B) are the most chosen answers. This shows that intellectual property and playing God play minor roles in the ethics of our project compared to the risks in safety and security. These are clearly the issues that should be taken into account in a possible application of our project.<br />
<br />
===Conclusion===<br />
The results of our survey suggest that in general people in the Netherlands are most concerned about the safety issues, however, they do trust researchers and developers to consider this in case GMO’s are used in application. Also the majority thinks that there is a security risk by using GMO’s for our project in a possible application. Less educated and older people would indicate a higher security risk than a higher educational level and younger people.<br />
<br />
In general manipulation of bacteria for research is considered good. Women, however, are more thoughtful about it than man. People with a higher level education are more open towards using bacteria for research than do lower level educated people. Unexpectedly we found no difference between people who did know what synthetic biology was and those who did not. So it appears that one is not more anxious about something because of a lack of awareness.<br />
Altogether comparing the four ethical issues of Bhutkar, safety, security, playing God and intellectual property, for synthetic biology in general and our project in particular shows that synthetic biological research is considered ok as long as done carefully and safety and security issues are considered.<br />
<br />
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{{Team:Groningen/Footer}}</div>JolandaWitteveenhttp://2009.igem.org/Team:Groningen/EthicsTeam:Groningen/Ethics2009-10-21T22:09:08Z<p>JolandaWitteveen: /* Intellectual property */</p>
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<div>{{Team:Groningen/Header}}<br />
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<div style="float:left" >{{linkedImage|GroningenPrevious.png|Team:Groningen/Safety}}</div><br />
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[[Category:Team:Groningen]]<br />
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= Ethical issues in synthetic biology =<br />
<br />
==Introduction==<br />
Synthetic biology is a relatively new field in biology in which engineering and biology are combined. It can be defined as the engineering of biology: the synthesis of complex, biologically based (or inspired) systems, which display functions that do exist in nature [[Team:Groningen/Literature#Serrano, L2007|(Serrano, L2007)]]. There are a number of approaches to synthetic biology; bioengineering, redesigning life and creating alternative life each with their own ethical concerns [[Team:Groningen/Literature#Deplazes, A2009|(Deplazes, A2009)]], [[Team:Groningen/Literature#Chopra, PK, A2006|(Chopra, PK, A2006)]]. <br />
<br />
==Safety==<br />
An important ethical issue for all disciplines is the safety issue, what are the risks posed by synthetic biology? [[Team:Groningen/Literature#Bhutkar, A2005|Bhutkar 2005]] distinguishes three safety risks. First, the risk of negative environmental impact this includes possible side effects, of genetically modified organism (GMO) that have to perform a task outside the lab, which can affect the environment negatively. Another risk is the risk to contaminate the natural genome pool, which includes genetic exchange between a GMO and a natural organism. The third risk mentioned by Bhutkar is the run-off risk, which includes possible unforeseen effects if a GMO has no controlled lifespan outside the lab. So the safety issue concerns the implication that GMOs could have on human health and the environment [[Team:Groningen/Literature#Kelle, A2009|(Kelle, A2009)]], [[Team:Groningen/Literature#Bhutkar, A2005|(Bhutkar, A2005)]]. Also local [[Team:Groningen/Safety|legislation]] and project specific [[Team:Groningen/Safety|safety]] risks should be considered.<br />
<br />
==Security==<br />
Also security issues are important ethical issues for bioengineering and when defining the difference between bio-safety and bio-security the easiest discrimination is that between mistakes and that of bad intentions. Synthetic biology is a [[Team:Groningen/Glossary#Dual-use|dual-use]] technology; it can be used for good goals or misused and cause harm. Misusage of the techniques and GMO's could result, among other things, in bioweapons and with the threshold for practicing biology getting lower and lower it is creating an unprecedented security problem [[Team:Groningen/Literature#Schmidt2008|(Schmidt, 2008)]]. More and more people will have the possibility to engineer biology and without proper regulatory oversight these socalled [[Team:Groningen/Glossary#Biohacker|biohackers]] can create potential hazards. A list of selected reading about security and possible interventions can be found [http://www.synbioproject.org/topics/synbio101/bibliography/governance/ here], [http://syntheticbiology.org/SB2.0/Biosecurity_resolutions.html here] and [http://www.jcvi.org/cms/research/projects/syngen-options/publications/ here]. Screening of ordered DNA and oligonucleotides for pathogenic agents and to license certain equipment and reagents could make it more controllable. This could be achieved by governmental regulation, however self regulation by the field of synthetic biology will be even better [[Team:Groningen/Literature#Kelle, A2009|(Kelle, A2009)]], [[Team:Groningen/Literature#Samuel, GN, et al.2009|(Samuel, GN, et al.2009)]], [[Team:Groningen/Literature#Nouri, A, et al.2009|(Nouri, A, et al.2009)]], [[Team:Groningen/Literature#Nicholls, H2008|(Nicholls, H2008)]] . <br />
<br />
==Intellectual property==<br />
Another ethical issue is the intellectual property of DNA. Synthetic biology forces rethinking about what should be patentable [[Team:Groningen/Literature#Samuel, GN, et al.2009|(Samuel, GN, et al.2009)]] ? Living organisms have been patented in the past, the first being a purified form of yeast that was patented by Louis Pasteur. The US patent and trademark office (PTO) has, beside the traditional patent requirements, two addition parts, which biotechnological systems should meet in order to be patentable. First is to show that the organism has little change of developing naturally. Second is that it should be shown that natural selection works against the GMO [[Team:Groningen/Literature#Bhutkar, A2005|(Bhutkar, A2005)]] . The iGEM organisation circumvented this question by using an opensource system in which all Biobricks are documented and freely accessible.<br />
<br />
==Playing God issue==<br />
Another ethical issue, that is most prone in the creating alternative life part of synthetic biology, is the ‘playing God’ issue. This also raises the question what is life? This is an old philosophical question in which different views exists. One is that life is a self-sustained chemical system capable of undergoing Darwinian evolution [[Team:Groningen/Literature#Cleland, CE, et al.2002|(Cleland, CE, et al.2002)]] . Another similar definition states that life is related to possessing metabolic properties, being responsive to the environment, and having the ability to replicate [[Team:Groningen/Literature#Bhutkar, A2005|(Bhutkar, A2005)]] . Another issue which relates to this question is where a line can be drawn between an engineered machine and a living organism. A possibility to make a distinction is when a ‘value’ is assigned to GMOs. Two values can be assigned, instrumental and intrinsic. Instrumental value is being defined by the use it has for humankind, whereas the intrinsic value, independent of the instrumental value, is assigned to any organism being valuable ‘in and of itself’. The question here is: do bacteria posses an intrinsic value? Also this issue becomes more prominent when the field of synthetic biology develops towards using higher organism and even humans.<br />
<br />
==Responsibilities==<br />
What are the responsibilities of the researchers in this field concerning the above-mentioned ethical issues? First of all safety for themselves and co-workers is a responsibility for all researchers. Also these researchers should think about possible implication of the GMO outside the lab. Especially when the organism has a task to perform outside the lab. Once the implications are clear some actions should be taken to try to minimize the risk of negative effects of the GMO for the environment and human health. Another responsibility for the field of synthetic biology lies in the security issues. Self-regulation and also helping the governmental organisation by making effective governmental regulation could be considered as a responsibility of researchers in the field of synthetic biology. Participation in the public debates about synthetic biology could be important for the public opinion on synthetic biology. It should be avoided that the people outside the synthetic biology field are scared away and, without enough insights, feel that synthetic biology is unethical. Other more delicate ethical issues like the difference between life and nonlife and assigning value to the organism is a more complicated issue, how far does the responsibility of the researchers reach in this matter and who else are responsible? In scope of this question it is advisable that both researchers and students are being taught about the risks and ethical aspects of synthetic biology in order to create awareness.<br />
In line with this the question raises what the ethical responsibilities of iGEM participant are? Obviously safety in working with GMOs is important. However also the safety of the GMO should be considered, what are the implications for the environment and human health and how can the risk of possible negative effects be minimized? Security issues are not directly the responsibility of the iGEM participant however it should not be overlooked. It is the responsibility of the iGEM participant to think about the risk of dual-use for their particular project. The iGEM participant could also give more transparency to the field of synthetic biology towards the public by publication of their project plan and results on their wiki-page. It should be encourage that part of the wiki is written for non-participant and even non-scientist in order to allow the public to gain understanding in the possible applications of synthetic biology. Overall responsibility of iGEM participant lies in gaining awareness of the ethical issues concerning synthetic biology and in particular their own project. The teams should try to gain a clear understanding of the ethical issues of their own project and its application.<br />
<br />
=Ethical issues in Heavy metal scavengers with a vertical gasdrive=<br />
<br />
==Introduction==<br />
It is important to consider ethical issues when introducing a new application, especially when genetic modified organisms are involved. In our survey we included an essay question in which the respondents were asked to give their opinion on ethical isues surrouding our project. We wanted to approach the Delphi method for brainstorming in which participant can speak freely and this will open up the discussion.We used the four ethical issues as described by [[Team:Groningen/Literature#Bhutkar, A2005|(Bhutkar, A2005)]] as a guideline. From these answers, and also from the rest of the survey, it appeared that people are most concerend about the safety for the environment and human health.<br />
<br />
==Safety==<br />
Important issues that should be considerend and solved before the bouyant heavy metal scavengers can be used in application are the possible danger for the natural bacterial population, antibiotic resistance that could spread, the concentrated amounts of a heavy metal, unwanted side effects.<br />
Most of these concerns are only applicable when the bacteria end up in the natural environment. This is obviously something that should be prevented in order to keep the system controllable. Keeping the bacteria in a closed water cleaning facility is a controlable environment, however, there is the risk in traditional water cleaning facilities of overflowing. So since there is still a small risk of bacteria ending up in the natural environment this should be considered.<br />
If bacteria end up in the natural environment many risks are present. One of the most mentioned concerns is the transfer of genes from the GMO to another organism. There is no evidence that gene transfer can happen between two different organisms, however, it is possible between two bacteria of the same strain. It does not happen often, it only happens in a stressful environment [[Team:Groningen/Literature#Mindlin, SZ, et al.2002|(Mindlin, SZ, et al.2002, )]], [[Team:Groningen/Literature#Stephenson, JR, et al.1996|(Stephenson, JR, et al.1996)]] . If a GMO end up in a natural environment it could be very stressful so it is possible that genes are transferred. To prevent this it would be best if the bacterium cannot transfer genes and dies. One way to achieve this is implementing a death-gene. This gene start apotheosis and can be transcribed with a delay. So the bacterium can do its job first and after a certain time or at a certain set-of the death-gene starts apotheosis. In this way the GMO is controllable and the possible unwanted side-effects of the GMO in a natural environment or effect because of a mutation are also decreased.<br />
Another safety issue mentioned in the survey is the escape of a antibiotic resistant strain that can spread this resistant among other bacteria. To overcome this risk other selection mechanisms besides antibiotics should be used like a gene that can make glucose out of lactose.<br />
Another concern has to do with the accumulation of toxic metals. People are afraid that this high concentration of metals can poison other organisms that eat the bacterium or a bacterium with the heavy metal stays behind. In order to make sure that a bacterium that accumulates metals also floats to the surface a feedback mechanism between the accumulation device and the gas vesicle devise could be made.<br />
Yet another concern is the removal of the bacteria and the heavy metals. Some people presume that it is very costly to remove the bacteria from the water cleaning facilitation and that it would be very difficult to remove the heavy metals. The removal of the bacteria does not have to be very expensive. It can be done by using a kind of a sieve. The metals can be removed by destruction of the cells. In case of arsenic antimone can be added and the arsenide importer will export all arsenide from the cell. Another concern is how do you know if the GMOs remove enough or even all of the particular heavy metal from the water or sludge? It can be estimated how much of this particular heavy metal a bacterium can take up and a overload of bacteria should be added so you know for sure that all of that particular heavy metal is removed.<br />
Another problem is what if the amount of the heavy metal is lower than expected and therefor not enough metal is accumulated to float? In this case the death gene should make sure that after a certain amount of time or at a certain signal the bacterium starts apotheosis. <br />
There is a possibility that the bacteria do not only take up the selected heavy metal. Bacteria could take up other metals as well, like copper and zinc. These metals do no harm in the drinking water, they are even useful for humans. However the heavy metals would compete with the copper and zinc for the accumulation proteins so most copper and zinc would be removed. Another way to prevent this from happening is over saturating the bacteria with these other metals before letting them clean the water.<br />
<br />
==Security risk==<br />
Security risks are always present. If the concentrated amounts of heavy metal fall into the wrong hands it could be misused. This risk is minimal since water cleaning facilities are not open for the public and the changes are bigger, and the result more catastrophic, if someone puts toxics in the drinking water.<br />
<br />
==Playing God issue==<br />
Most people do not find modifying bacteria as itself morally incorrect. From our survey it appeared that most people do not find it a problem if these modified bacteria are used in a water or sludge cleaning application. <br />
<br />
==Intellectual property==<br />
No other concerns are of interest in this project apart from the basic question can DNA be patented. Most people find this morally incorrect, however, patenting a system or idea which uses modified bacteria should be possible in order to keep up development.<br />
<br />
<br />
{{Team:Groningen/Footer}}</div>JolandaWitteveenhttp://2009.igem.org/Team:Groningen/Ethics/SurveyTeam:Groningen/Ethics/Survey2009-10-21T22:00:36Z<p>JolandaWitteveen: /* Considerations */</p>
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<div>{{Team:Groningen/Header}}<br />
<br />
<br />
<div style="float:left" >{{linkedImage|GroningenPrevious.png|Team:Groningen/Vision}}</div><br />
<div title="Arsie Says UP TO ACCUMULATION" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Parts}}</div><br />
<br />
==Survey==<br />
<br />
===Considerations===<br />
Ethics is an important issue to consider in the new field of synthetic biology. In order to gain more insights in the Dutch public opinion we decided to create a survey. <br />
The survey contains questions about the ethical issues surrounding synthetic biology and also about the ethical issues surrounding our project.<br />
We wanted to reach a mixed public in order to get an idea of the Dutch public opinion on the ethical issues of synthetic biology and our project. We did a convenience sampling so we handed out our survey among family, friends, secondary school pupils and fellow students. We have chosen for this sample construct because it is not expensive and it is easy to apply. We added a link to our survey on our wiki-website. In an attempt to exclude non-Dutch respondents we included the question “do you live or work in the Netherlands?” to our survey. Since we thought that it might make a difference if the participants already knew what synthetic biology was, we included the question “do you know what synthetic biology is?”. Being religious or not was also thought to possibly influence the opinion of the participant about for instance the playing God issue, so a question about religion was included as well. Just as age and gender could make a difference and therefore questions about that were included. <br />
We wanted to approach the Delphi method for brainstorming, also previous described by [[Team:Groningen/Literature#Zwart, SD, et al.2006|(Zwart, SD, et al.2006)]] in the Netherlands and recently used in the EU synth-ethics meeting, to gain more insights in ethics. Central in this method is the fact that participants can give their opinion anonymously and without consequences. The idea behind it is that if someone can speak freely this will open up the discussion. Although we did not organize a discussion, we did try to mimic this by including an essay question in the survey in which we asked the participants to give other ethical and safety issues surrounding our project. Since the survey is anonymously and we also aimed to ask all kinds of users to fill it out this survey approaches the open discussion method.<br />
The questions are based on the four ethical issues of synthetic biology described by [[Team:Groningen/Literature#Bhutkar, A2005|(Bhutkar, A2005)]], safety, security, playing God and intellectual property. All the respondents were shown a cover letter with information about the aim of the survey. The participants could decide to stop at any moment during the survey. After analyzing the data we send a debriefing to the respondents with the conclusion about our survey. For the confidentiality of the respondents we used the program ‘Examine’. In this program all the participants have a random number as ID. Also we have minimizes the number of people who could see or handle the data. The questions consists mostly of theorems, which were translated to a Likert scale afterwards.<br />
<br />
===Results===<br />
The survey was opened from 11-09-2009 until 07-10-2009 (27 days) and was completed by 262 respondents, 147 male and 115 female. <br />
The educational level of the respondents was mixed. As can be seen in figure 1 the different educational groups are all represented. There is a small bias to the educative level university. So the survey is, however, not a completely representative sample of the Dutch population, but it is still a good suggestion for the Dutch public opinion. <br />
<br />
<br />
We consider this trough in the statistical analyses of the data with a non-probability test. We took this into account when we did the statistical analysis and choose, therefore, to do non-probability tests. We have chosen the ‘Mann-Whitney’ test for two independent samples, by questions with more than two independent groups, we use the ‘Kruskal-Wallis H.’ test. <br />
Both procedures are testing equality of population medians among groups. Sometimes if the data looked normal distributed (we have checked this with boxplots), we used an ANOVA two-tailed with the post-hoc ‘Bonferroni’ All test are done with a confidence level of 95% (α=0.05). <br />
<br />
[[Image:Groningen_Figure1Survey.png|400px]]<br />
<br />
Figure 1: Educational level of the respondents of the ethical survey of the iGEM team Groningen 2009 <br />
<br />
<br />
===Inferential statistics=== <br />
<br />
‘’Difference between different levels of education’’<br />
Grouping the university (biology), university (non-biology) and university (engineering) shows that the data can be biased towards the university level educated respondents. <br />
There, however, does not seem to be a difference in response between the different educational levels towards the ethical issues of our project, one exception being the response towards the risk of bio terrorism. The university level educated respondents see less risk of bio terrorism of our project than do the lower level educated respondents. (p=0,001)<br />
There does also seem to be a significant difference in response to the question “how do you feel about bacteria being manipulated for research” between respondents with educational level secondary school and university (biology) (p=0.014, two-way ANOVA with post-hoc bonferoni). University students are more positive towards manipulating bacteria for research then secondary school pupils. This can also be seen when a correlation test is done. The spearman’s rho correlation test was chosen because we use categorical variables and the data is normal distributed. A (weak) correlation was found (R=0,215 and p=0,001) between level of education and openness towards manipulating bacteria. Respondents with a higher level of education are more positive towards manipulating bacteria.<br />
<br />
<br />
''Difference between religious and not religious''<br />
It was predicted that being religious or not could influence the opinion of the respondents towards the playing God issue of our project. No difference, however, could be detected (p=0.96, two tailed Mann-Withney test). Both religious and non-religious respondents did not thought that using GMO’s for water or sludge cleaning is unethical in the sense that researchers are playing God (figure 2).<br />
<br />
<br />
[[Image:Groningen_Figure2Survey.png|400px]]<br />
<br />
Figure 2: Frequency table of the responses towards the question do you think using GMO’s for water or sludge cleaning unethical in the sense that researchers are playing God against being religious or not.<br />
<br />
''Difference between knowing and not knowing what synthetic biology is''<br />
<br />
Half of the respondents (51,1%) did not know what synthetic biology is, 48,9% of the respondents did. It was hypothesized that there would be a difference between respondents that know what synthetic biology is and those who do not, in how they feel about GMO’s being used in application. This, however, did not appear to be the case form our survey (p=0.236, two-tailed Mann-whitney test). <br />
<br />
''Difference between male and female''<br />
<br />
If male and female respondents were compared there appeared to be a difference how they feel about GMO’s being manipulated for research. Here a one-tailed test has been performed because it was presumed that women are, in general, more caring then men. A significant difference between the genders with a p-value of 0,0005 was found showing that women are more thoughtful about the usage of GMO’s for research then man.<br />
<br />
''Difference between older and younger respondents''<br />
<br />
It was also tested if age make a difference in how the participants feel about the ethical issues of our project. One significant difference was found between participants older than 50 and the group aged between 21 and 30. The older group (>50) appeared to be more afraid of misusage of GMO in cleaning water or sludge for bioterrorism than the younger group (p=0,022) <br />
<br />
<br />
===Descriptive statistics===<br />
<br />
No differences were found between other groups so the following results can be considered the same for all groups.<br />
The respondents were asked what they thought of bacteria being manipulated for research. The majority, 89.7%, answered “good” and only 5,8% answered “not good” because it can be dangerous or researchers are playing God. So making GMO’s does not seem to be considered unethical, using these GMO’s in practice was also not considered unethical, 93.5% was positive about it. Safety for the environment and human health was, however, a concern for 24,4% of the respondents. 69,1% of the respondents found, security and usefulness for society, a condition for the usage of GMO’s. The respondents were more critical about the usage of GMO’s for a specific application, in water or sludge cleaning. A majority of the respondents, 50,8%, thought that the usage of GMO’s can be dangerous for the environment or human health so this should be considered before usage. 29% of the respondents trusted the researchers in considering the safety and make the application more safe. Security of GMO’s for applications in the water and sludge cleaning is an issue that concerns 23,7% and they feel that this deserves some thought. Another 36,6% of the respondents also thought that security is an issue, however, there is always the risk of mis usage. The remaining 33,2% of the respondents did not think security is an issue in this application. Another important ethical issue according to Bhutkar [2], the playing God issue, does not seem to be a concern for the usage of GMO’s in water and sludge cleaning according to the respondents of our survey (92,4%). <br />
The fourth ethical issue, the intellectual property, was also considered for using GMO’s in water and sludge cleaning. Although 26,3% of the respondents thought it should be possible to patent the application of the iGEM team Groningen 2009 because it is important for development the majority (57.3%) of the respondents only agreed with patenting the whole system and not the DNA itself. 11.5% of the respondents were against patenting this system in which GMO’s are used (fig 3).<br />
<br />
[[Image:Groningen_Figure3Survey.png|400px]]<br />
<br />
Figure 3: The four ethical issues, as described by bhutkar, considered for the project of the iGEM team Groningen 2009.<br />
<br />
[[Image:Groningen_Figure4Survey.png|400px]]<br />
<br />
Figure 4: This figure shows what the most important ethical issues is in our project according to our respondents. The four issues, safety (A), security (B), intellectual property (C) and playing God (D) are derived from <br />
<br />
The survey included a question, which ethical issue was considered the most important for our project, two answers could be given. In the above figure it can be seen that most respondents, 40%, thought safety is the most important issue. Also in combination with other issues safety is considered to be very important. Also security is thought to be a risk in water and sludge cleaning, by 30% of the participants. As a second choice safety and security (A and B) are the most chosen answers. This shows that intellectual property and playing God play minor roles in the ethics of our project compared to the risks in safety and security. These are clearly the issues that should be taken into account in a possible application of our project.<br />
<br />
===Conclusion===<br />
The results of our survey suggest that in general people in the Netherlands are most concerned about the safety issues, however, they do trust researchers and developers to consider this in case GMO’s are used in application. Also the majority thinks that there is a security risk by using GMO’s for our project in a possible application. Less educated and older people would indicate a higher security risk than a higher educational level and younger people.<br />
<br />
In general manipulation of bacteria for research is considered good. Women, however, are more thoughtful about it than man. People with a higher level education are more open towards using bacteria for research than do lower level educated people. Unexpectedly we found no difference between people who did know what synthetic biology was and those who did not. So it appears that one is not more anxious about something because of a lack of awareness.<br />
Altogether comparing the four ethical issues of Bhutkar, safety, security, playing God and intellectual property, for synthetic biology in general and our project in particular shows that synthetic biological research is considered ok as long as done carefully and safety and security issues are considered.<br />
<br />
<br />
{{Team:Groningen/Footer}}</div>JolandaWitteveenhttp://2009.igem.org/Team:Groningen/Ethics/SurveyTeam:Groningen/Ethics/Survey2009-10-21T22:00:12Z<p>JolandaWitteveen: /* Considerations */</p>
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==Survey==<br />
<br />
===Considerations===<br />
Ethics is an important issue to consider in the new field of synthetic biology. In order to gain more insights in the Dutch public opinion we decided to create a survey. <br />
The survey contains questions about the ethical issues surrounding synthetic biology and also about the ethical issues surrounding our project.<br />
We wanted to reach a mixed public in order to get an idea of the Dutch public opinion on the ethical issues of synthetic biology and our project. We did a convenience sampling so we handed out our survey among family, friends, secondary school pupils and fellow students. We have chosen for this sample construct because it is not expensive and it is easy to apply. We added a link to our survey on our wiki-website. In an attempt to exclude non-Dutch respondents we included the question “do you live or work in the Netherlands?” to our survey. Since we thought that it might make a difference if the participants already knew what synthetic biology was, we included the question “do you know what synthetic biology is?”. Being religious or not was also thought to possibly influence the opinion of the participant about for instance the playing God issue, so a question about religion was included as well. Just as age and gender could make a difference and therefore questions about that were included. <br />
We wanted to approach the Delphi method for brainstorming, also previous described by [[Team:Groningen/Literature#Zwart, SD, et al.2006|(Zwart, SD, et al.2006]] in the Netherlands and recently used in the EU synth-ethics meeting, to gain more insights in ethics. Central in this method is the fact that participants can give their opinion anonymously and without consequences. The idea behind it is that if someone can speak freely this will open up the discussion. Although we did not organize a discussion, we did try to mimic this by including an essay question in the survey in which we asked the participants to give other ethical and safety issues surrounding our project. Since the survey is anonymously and we also aimed to ask all kinds of users to fill it out this survey approaches the open discussion method.<br />
The questions are based on the four ethical issues of synthetic biology described by [[Team:Groningen/Literature#Bhutkar, A2005|(Bhutkar, A2005)]], safety, security, playing God and intellectual property. All the respondents were shown a cover letter with information about the aim of the survey. The participants could decide to stop at any moment during the survey. After analyzing the data we send a debriefing to the respondents with the conclusion about our survey. For the confidentiality of the respondents we used the program ‘Examine’. In this program all the participants have a random number as ID. Also we have minimizes the number of people who could see or handle the data. The questions consists mostly of theorems, which were translated to a Likert scale afterwards.<br />
<br />
===Results===<br />
The survey was opened from 11-09-2009 until 07-10-2009 (27 days) and was completed by 262 respondents, 147 male and 115 female. <br />
The educational level of the respondents was mixed. As can be seen in figure 1 the different educational groups are all represented. There is a small bias to the educative level university. So the survey is, however, not a completely representative sample of the Dutch population, but it is still a good suggestion for the Dutch public opinion. <br />
<br />
<br />
We consider this trough in the statistical analyses of the data with a non-probability test. We took this into account when we did the statistical analysis and choose, therefore, to do non-probability tests. We have chosen the ‘Mann-Whitney’ test for two independent samples, by questions with more than two independent groups, we use the ‘Kruskal-Wallis H.’ test. <br />
Both procedures are testing equality of population medians among groups. Sometimes if the data looked normal distributed (we have checked this with boxplots), we used an ANOVA two-tailed with the post-hoc ‘Bonferroni’ All test are done with a confidence level of 95% (α=0.05). <br />
<br />
[[Image:Groningen_Figure1Survey.png|400px]]<br />
<br />
Figure 1: Educational level of the respondents of the ethical survey of the iGEM team Groningen 2009 <br />
<br />
<br />
===Inferential statistics=== <br />
<br />
‘’Difference between different levels of education’’<br />
Grouping the university (biology), university (non-biology) and university (engineering) shows that the data can be biased towards the university level educated respondents. <br />
There, however, does not seem to be a difference in response between the different educational levels towards the ethical issues of our project, one exception being the response towards the risk of bio terrorism. The university level educated respondents see less risk of bio terrorism of our project than do the lower level educated respondents. (p=0,001)<br />
There does also seem to be a significant difference in response to the question “how do you feel about bacteria being manipulated for research” between respondents with educational level secondary school and university (biology) (p=0.014, two-way ANOVA with post-hoc bonferoni). University students are more positive towards manipulating bacteria for research then secondary school pupils. This can also be seen when a correlation test is done. The spearman’s rho correlation test was chosen because we use categorical variables and the data is normal distributed. A (weak) correlation was found (R=0,215 and p=0,001) between level of education and openness towards manipulating bacteria. Respondents with a higher level of education are more positive towards manipulating bacteria.<br />
<br />
<br />
''Difference between religious and not religious''<br />
It was predicted that being religious or not could influence the opinion of the respondents towards the playing God issue of our project. No difference, however, could be detected (p=0.96, two tailed Mann-Withney test). Both religious and non-religious respondents did not thought that using GMO’s for water or sludge cleaning is unethical in the sense that researchers are playing God (figure 2).<br />
<br />
<br />
[[Image:Groningen_Figure2Survey.png|400px]]<br />
<br />
Figure 2: Frequency table of the responses towards the question do you think using GMO’s for water or sludge cleaning unethical in the sense that researchers are playing God against being religious or not.<br />
<br />
''Difference between knowing and not knowing what synthetic biology is''<br />
<br />
Half of the respondents (51,1%) did not know what synthetic biology is, 48,9% of the respondents did. It was hypothesized that there would be a difference between respondents that know what synthetic biology is and those who do not, in how they feel about GMO’s being used in application. This, however, did not appear to be the case form our survey (p=0.236, two-tailed Mann-whitney test). <br />
<br />
''Difference between male and female''<br />
<br />
If male and female respondents were compared there appeared to be a difference how they feel about GMO’s being manipulated for research. Here a one-tailed test has been performed because it was presumed that women are, in general, more caring then men. A significant difference between the genders with a p-value of 0,0005 was found showing that women are more thoughtful about the usage of GMO’s for research then man.<br />
<br />
''Difference between older and younger respondents''<br />
<br />
It was also tested if age make a difference in how the participants feel about the ethical issues of our project. One significant difference was found between participants older than 50 and the group aged between 21 and 30. The older group (>50) appeared to be more afraid of misusage of GMO in cleaning water or sludge for bioterrorism than the younger group (p=0,022) <br />
<br />
<br />
===Descriptive statistics===<br />
<br />
No differences were found between other groups so the following results can be considered the same for all groups.<br />
The respondents were asked what they thought of bacteria being manipulated for research. The majority, 89.7%, answered “good” and only 5,8% answered “not good” because it can be dangerous or researchers are playing God. So making GMO’s does not seem to be considered unethical, using these GMO’s in practice was also not considered unethical, 93.5% was positive about it. Safety for the environment and human health was, however, a concern for 24,4% of the respondents. 69,1% of the respondents found, security and usefulness for society, a condition for the usage of GMO’s. The respondents were more critical about the usage of GMO’s for a specific application, in water or sludge cleaning. A majority of the respondents, 50,8%, thought that the usage of GMO’s can be dangerous for the environment or human health so this should be considered before usage. 29% of the respondents trusted the researchers in considering the safety and make the application more safe. Security of GMO’s for applications in the water and sludge cleaning is an issue that concerns 23,7% and they feel that this deserves some thought. Another 36,6% of the respondents also thought that security is an issue, however, there is always the risk of mis usage. The remaining 33,2% of the respondents did not think security is an issue in this application. Another important ethical issue according to Bhutkar [2], the playing God issue, does not seem to be a concern for the usage of GMO’s in water and sludge cleaning according to the respondents of our survey (92,4%). <br />
The fourth ethical issue, the intellectual property, was also considered for using GMO’s in water and sludge cleaning. Although 26,3% of the respondents thought it should be possible to patent the application of the iGEM team Groningen 2009 because it is important for development the majority (57.3%) of the respondents only agreed with patenting the whole system and not the DNA itself. 11.5% of the respondents were against patenting this system in which GMO’s are used (fig 3).<br />
<br />
[[Image:Groningen_Figure3Survey.png|400px]]<br />
<br />
Figure 3: The four ethical issues, as described by bhutkar, considered for the project of the iGEM team Groningen 2009.<br />
<br />
[[Image:Groningen_Figure4Survey.png|400px]]<br />
<br />
Figure 4: This figure shows what the most important ethical issues is in our project according to our respondents. The four issues, safety (A), security (B), intellectual property (C) and playing God (D) are derived from <br />
<br />
The survey included a question, which ethical issue was considered the most important for our project, two answers could be given. In the above figure it can be seen that most respondents, 40%, thought safety is the most important issue. Also in combination with other issues safety is considered to be very important. Also security is thought to be a risk in water and sludge cleaning, by 30% of the participants. As a second choice safety and security (A and B) are the most chosen answers. This shows that intellectual property and playing God play minor roles in the ethics of our project compared to the risks in safety and security. These are clearly the issues that should be taken into account in a possible application of our project.<br />
<br />
===Conclusion===<br />
The results of our survey suggest that in general people in the Netherlands are most concerned about the safety issues, however, they do trust researchers and developers to consider this in case GMO’s are used in application. Also the majority thinks that there is a security risk by using GMO’s for our project in a possible application. Less educated and older people would indicate a higher security risk than a higher educational level and younger people.<br />
<br />
In general manipulation of bacteria for research is considered good. Women, however, are more thoughtful about it than man. People with a higher level education are more open towards using bacteria for research than do lower level educated people. Unexpectedly we found no difference between people who did know what synthetic biology was and those who did not. So it appears that one is not more anxious about something because of a lack of awareness.<br />
Altogether comparing the four ethical issues of Bhutkar, safety, security, playing God and intellectual property, for synthetic biology in general and our project in particular shows that synthetic biological research is considered ok as long as done carefully and safety and security issues are considered.<br />
<br />
<br />
{{Team:Groningen/Footer}}</div>JolandaWitteveenhttp://2009.igem.org/Team:Groningen/EthicsTeam:Groningen/Ethics2009-10-21T21:55:51Z<p>JolandaWitteveen: /* Safety */</p>
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= Ethical issues in synthetic biology =<br />
<br />
==Introduction==<br />
Synthetic biology is a relatively new field in biology in which engineering and biology are combined. It can be defined as the engineering of biology: the synthesis of complex, biologically based (or inspired) systems, which display functions that do exist in nature [[Team:Groningen/Literature#Serrano, L2007|(Serrano, L2007)]]. There are a number of approaches to synthetic biology; bioengineering, redesigning life and creating alternative life each with their own ethical concerns [[Team:Groningen/Literature#Deplazes, A2009|(Deplazes, A2009)]], [[Team:Groningen/Literature#Chopra, PK, A2006|(Chopra, PK, A2006)]]. <br />
<br />
==Safety==<br />
An important ethical issue for all disciplines is the safety issue, what are the risks posed by synthetic biology? [[Team:Groningen/Literature#Bhutkar, A2005|Bhutkar 2005]] distinguishes three safety risks. First, the risk of negative environmental impact this includes possible side effects, of genetically modified organism (GMO) that have to perform a task outside the lab, which can affect the environment negatively. Another risk is the risk to contaminate the natural genome pool, which includes genetic exchange between a GMO and a natural organism. The third risk mentioned by Bhutkar is the run-off risk, which includes possible unforeseen effects if a GMO has no controlled lifespan outside the lab. So the safety issue concerns the implication that GMOs could have on human health and the environment [[Team:Groningen/Literature#Kelle, A2009|(Kelle, A2009)]], [[Team:Groningen/Literature#Bhutkar, A2005|(Bhutkar, A2005)]]. Also local [[Team:Groningen/Safety|legislation]] and project specific [[Team:Groningen/Safety|safety]] risks should be considered.<br />
<br />
==Security==<br />
Also security issues are important ethical issues for bioengineering and when defining the difference between bio-safety and bio-security the easiest discrimination is that between mistakes and that of bad intentions. Synthetic biology is a [[Team:Groningen/Glossary#Dual-use|dual-use]] technology; it can be used for good goals or misused and cause harm. Misusage of the techniques and GMO's could result, among other things, in bioweapons and with the threshold for practicing biology getting lower and lower it is creating an unprecedented security problem [[Team:Groningen/Literature#Schmidt2008|(Schmidt, 2008)]]. More and more people will have the possibility to engineer biology and without proper regulatory oversight these socalled [[Team:Groningen/Glossary#Biohacker|biohackers]] can create potential hazards. A list of selected reading about security and possible interventions can be found [http://www.synbioproject.org/topics/synbio101/bibliography/governance/ here], [http://syntheticbiology.org/SB2.0/Biosecurity_resolutions.html here] and [http://www.jcvi.org/cms/research/projects/syngen-options/publications/ here]. Screening of ordered DNA and oligonucleotides for pathogenic agents and to license certain equipment and reagents could make it more controllable. This could be achieved by governmental regulation, however self regulation by the field of synthetic biology will be even better [[Team:Groningen/Literature#Kelle, A2009|(Kelle, A2009)]], [[Team:Groningen/Literature#Samuel, GN, et al.2009|(Samuel, GN, et al.2009)]], [[Team:Groningen/Literature#Nouri, A, et al.2009|(Nouri, A, et al.2009)]], [[Team:Groningen/Literature#Nicholls, H2008|(Nicholls, H2008)]] . <br />
<br />
==Intellectual property==<br />
Another ethical issue is the intellectual property of DNA. Synthetic biology forces rethinking about what should be patentable [[Team:Groningen/Literature#Samuel, GN, et al.2009|(Samuel, GN, et al.2009)]] ? Living organisms have been patented in the past, the first being a purified form of yeast that was patented by Louis Pasteur. The US patent and trademark office (PTO) has, beside the traditional patent requirements, two addition parts, which biotechnological systems should meet in order to be patentable. First is to show that the organism has little change of developing naturally. Second is that it should be shown that natural selection works against the GMO [[Team:Groningen/Literature#Bhutkar, A2005|(Bhutkar, A2005)]] . The iGEM organisation circumvented this question by using an opensource system in which all Biobricks are documented and freely accessible. <br />
<br />
==Playing God issue==<br />
Another ethical issue, that is most prone in the creating alternative life part of synthetic biology, is the ‘playing God’ issue. This also raises the question what is life? This is an old philosophical question in which different views exists. One is that life is a self-sustained chemical system capable of undergoing Darwinian evolution [[Team:Groningen/Literature#Cleland, CE, et al.2002|(Cleland, CE, et al.2002)]] . Another similar definition states that life is related to possessing metabolic properties, being responsive to the environment, and having the ability to replicate [[Team:Groningen/Literature#Bhutkar, A2005|(Bhutkar, A2005)]] . Another issue which relates to this question is where a line can be drawn between an engineered machine and a living organism. A possibility to make a distinction is when a ‘value’ is assigned to GMOs. Two values can be assigned, instrumental and intrinsic. Instrumental value is being defined by the use it has for humankind, whereas the intrinsic value, independent of the instrumental value, is assigned to any organism being valuable ‘in and of itself’. The question here is: do bacteria posses an intrinsic value? Also this issue becomes more prominent when the field of synthetic biology develops towards using higher organism and even humans.<br />
<br />
==Responsibilities==<br />
What are the responsibilities of the researchers in this field concerning the above-mentioned ethical issues? First of all safety for themselves and co-workers is a responsibility for all researchers. Also these researchers should think about possible implication of the GMO outside the lab. Especially when the organism has a task to perform outside the lab. Once the implications are clear some actions should be taken to try to minimize the risk of negative effects of the GMO for the environment and human health. Another responsibility for the field of synthetic biology lies in the security issues. Self-regulation and also helping the governmental organisation by making effective governmental regulation could be considered as a responsibility of researchers in the field of synthetic biology. Participation in the public debates about synthetic biology could be important for the public opinion on synthetic biology. It should be avoided that the people outside the synthetic biology field are scared away and, without enough insights, feel that synthetic biology is unethical. Other more delicate ethical issues like the difference between life and nonlife and assigning value to the organism is a more complicated issue, how far does the responsibility of the researchers reach in this matter and who else are responsible? In scope of this question it is advisable that both researchers and students are being taught about the risks and ethical aspects of synthetic biology in order to create awareness.<br />
In line with this the question raises what the ethical responsibilities of iGEM participant are? Obviously safety in working with GMOs is important. However also the safety of the GMO should be considered, what are the implications for the environment and human health and how can the risk of possible negative effects be minimized? Security issues are not directly the responsibility of the iGEM participant however it should not be overlooked. It is the responsibility of the iGEM participant to think about the risk of dual-use for their particular project. The iGEM participant could also give more transparency to the field of synthetic biology towards the public by publication of their project plan and results on their wiki-page. It should be encourage that part of the wiki is written for non-participant and even non-scientist in order to allow the public to gain understanding in the possible applications of synthetic biology. Overall responsibility of iGEM participant lies in gaining awareness of the ethical issues concerning synthetic biology and in particular their own project. The teams should try to gain a clear understanding of the ethical issues of their own project and its application.<br />
<br />
=Ethical issues in Heavy metal scavengers with a vertical gasdrive=<br />
<br />
==Introduction==<br />
It is important to consider ethical issues when introducing a new application, especially when genetic modified organisms are involved. In our survey we included an essay question in which the respondents were asked to give their opinion on ethical isues surrouding our project. We wanted to approach the Delphi method for brainstorming in which participant can speak freely and this will open up the discussion.We used the four ethical issues as described by [[Team:Groningen/Literature#Bhutkar, A2005|(Bhutkar, A2005)]] as a guideline. From these answers, and also from the rest of the survey, it appeared that people are most concerend about the safety for the environment and human health.<br />
<br />
==Safety==<br />
Important issues that should be considerend and solved before the bouyant heavy metal scavengers can be used in application are the possible danger for the natural bacterial population, antibiotic resistance that could spread, the concentrated amounts of a heavy metal, unwanted side effects.<br />
Most of these concerns are only applicable when the bacteria end up in the natural environment. This is obviously something that should be prevented in order to keep the system controllable. Keeping the bacteria in a closed water cleaning facility is a controlable environment, however, there is the risk in traditional water cleaning facilities of overflowing. So since there is still a small risk of bacteria ending up in the natural environment this should be considered.<br />
If bacteria end up in the natural environment many risks are present. One of the most mentioned concerns is the transfer of genes from the GMO to another organism. There is no evidence that gene transfer can happen between two different organisms, however, it is possible between two bacteria of the same strain. It does not happen often, it only happens in a stressful environment [[Team:Groningen/Literature#Mindlin, SZ, et al.2002|(Mindlin, SZ, et al.2002, )]], [[Team:Groningen/Literature#Stephenson, JR, et al.1996|(Stephenson, JR, et al.1996)]] . If a GMO end up in a natural environment it could be very stressful so it is possible that genes are transferred. To prevent this it would be best if the bacterium cannot transfer genes and dies. One way to achieve this is implementing a death-gene. This gene start apotheosis and can be transcribed with a delay. So the bacterium can do its job first and after a certain time or at a certain set-of the death-gene starts apotheosis. In this way the GMO is controllable and the possible unwanted side-effects of the GMO in a natural environment or effect because of a mutation are also decreased.<br />
Another safety issue mentioned in the survey is the escape of a antibiotic resistant strain that can spread this resistant among other bacteria. To overcome this risk other selection mechanisms besides antibiotics should be used like a gene that can make glucose out of lactose.<br />
Another concern has to do with the accumulation of toxic metals. People are afraid that this high concentration of metals can poison other organisms that eat the bacterium or a bacterium with the heavy metal stays behind. In order to make sure that a bacterium that accumulates metals also floats to the surface a feedback mechanism between the accumulation device and the gas vesicle devise could be made.<br />
Yet another concern is the removal of the bacteria and the heavy metals. Some people presume that it is very costly to remove the bacteria from the water cleaning facilitation and that it would be very difficult to remove the heavy metals. The removal of the bacteria does not have to be very expensive. It can be done by using a kind of a sieve. The metals can be removed by destruction of the cells. In case of arsenic antimone can be added and the arsenide importer will export all arsenide from the cell. Another concern is how do you know if the GMOs remove enough or even all of the particular heavy metal from the water or sludge? It can be estimated how much of this particular heavy metal a bacterium can take up and a overload of bacteria should be added so you know for sure that all of that particular heavy metal is removed.<br />
Another problem is what if the amount of the heavy metal is lower than expected and therefor not enough metal is accumulated to float? In this case the death gene should make sure that after a certain amount of time or at a certain signal the bacterium starts apotheosis. <br />
There is a possibility that the bacteria do not only take up the selected heavy metal. Bacteria could take up other metals as well, like copper and zinc. These metals do no harm in the drinking water, they are even useful for humans. However the heavy metals would compete with the copper and zinc for the accumulation proteins so most copper and zinc would be removed. Another way to prevent this from happening is over saturating the bacteria with these other metals before letting them clean the water.<br />
<br />
==Security risk==<br />
Security risks are always present. If the concentrated amounts of heavy metal fall into the wrong hands it could be misused. This risk is minimal since water cleaning facilities are not open for the public and the changes are bigger, and the result more catastrophic, if someone puts toxics in the drinking water.<br />
<br />
==Playing God issue==<br />
Most people do not find modifying bacteria as itself morally incorrect. From our survey it appeared that most people do not find it a problem if these modified bacteria are used in a water or sludge cleaning application. <br />
<br />
==Intellectual property==<br />
No other concerns are of interest in this project apart from the basic question can DNA be patented. Most people find this morally incorrect, however, patenting a system or idea which uses modified bacteria should be possible in order to keep up development.<br />
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{{Team:Groningen/Footer}}</div>JolandaWitteveenhttp://2009.igem.org/Team:Groningen/EthicsTeam:Groningen/Ethics2009-10-21T21:50:58Z<p>JolandaWitteveen: /* Ethical issues in synthetic biology */</p>
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<br />
[[Category:Team:Groningen]]<br />
<br />
= Ethical issues in synthetic biology =<br />
<br />
==Introduction==<br />
Synthetic biology is a relatively new field in biology in which engineering and biology are combined. It can be defined as the engineering of biology: the synthesis of complex, biologically based (or inspired) systems, which display functions that do exist in nature [[Team:Groningen/Literature#Serrano, L2007|(Serrano, L2007)]]. There are a number of approaches to synthetic biology; bioengineering, redesigning life and creating alternative life each with their own ethical concerns [[Team:Groningen/Literature#Deplazes, A2009|(Deplazes, A2009)]], [[Team:Groningen/Literature#Chopra, PK, A2006|(Chopra, PK, A2006)]]. <br />
<br />
==Safety==<br />
An important ethical issue for all disciplines is the safety issue, what are the risks posed by synthetic biology? [[Team:Groningen/Literature#Bhutkar, A2005|Bhutkar 2005]] distinguishes three safety risks. First, the risk of negative environmental impact this includes possible side effects, of genetically modified organism (GMO) that have to perform a task outside the lab, which can affect the environment negatively. Another risk is the risk to contaminate the natural genome pool, which includes genetic exchange between a GMO and a natural organism. The third risk mentioned by Bhutkar is the run-off risk, which includes possible unforeseen effects if a GMO has no controlled lifespan outside the lab. So the safety issue concerns the implication that GMOs could have on human health and the environment [[Team:Groningen/Literature#Kelle, A2009|(Kelle, A2009)]], [[Team:Groningen/Literature#Bhutkar, A2005|(Bhutkar, A2005)]]. <br />
<br />
==Security==<br />
Also security issues are important ethical issues for bioengineering and when defining the difference between bio-safety and bio-security the easiest discrimination is that between mistakes and that of bad intentions. Synthetic biology is a [[Team:Groningen/Glossary#Dual-use|dual-use]] technology; it can be used for good goals or misused and cause harm. Misusage of the techniques and GMO's could result, among other things, in bioweapons and with the threshold for practicing biology getting lower and lower it is creating an unprecedented security problem [[Team:Groningen/Literature#Schmidt2008|(Schmidt, 2008)]]. More and more people will have the possibility to engineer biology and without proper regulatory oversight these socalled [[Team:Groningen/Glossary#Biohacker|biohackers]] can create potential hazards. A list of selected reading about security and possible interventions can be found [http://www.synbioproject.org/topics/synbio101/bibliography/governance/ here], [http://syntheticbiology.org/SB2.0/Biosecurity_resolutions.html here] and [http://www.jcvi.org/cms/research/projects/syngen-options/publications/ here]. Screening of ordered DNA and oligonucleotides for pathogenic agents and to license certain equipment and reagents could make it more controllable. This could be achieved by governmental regulation, however self regulation by the field of synthetic biology will be even better [[Team:Groningen/Literature#Kelle, A2009|(Kelle, A2009)]], [[Team:Groningen/Literature#Samuel, GN, et al.2009|(Samuel, GN, et al.2009)]], [[Team:Groningen/Literature#Nouri, A, et al.2009|(Nouri, A, et al.2009)]], [[Team:Groningen/Literature#Nicholls, H2008|(Nicholls, H2008)]] . <br />
<br />
==Intellectual property==<br />
Another ethical issue is the intellectual property of DNA. Synthetic biology forces rethinking about what should be patentable [[Team:Groningen/Literature#Samuel, GN, et al.2009|(Samuel, GN, et al.2009)]] ? Living organisms have been patented in the past, the first being a purified form of yeast that was patented by Louis Pasteur. The US patent and trademark office (PTO) has, beside the traditional patent requirements, two addition parts, which biotechnological systems should meet in order to be patentable. First is to show that the organism has little change of developing naturally. Second is that it should be shown that natural selection works against the GMO [[Team:Groningen/Literature#Bhutkar, A2005|(Bhutkar, A2005)]] . The iGEM organisation circumvented this question by using an opensource system in which all Biobricks are documented and freely accessible. <br />
<br />
==Playing God issue==<br />
Another ethical issue, that is most prone in the creating alternative life part of synthetic biology, is the ‘playing God’ issue. This also raises the question what is life? This is an old philosophical question in which different views exists. One is that life is a self-sustained chemical system capable of undergoing Darwinian evolution [[Team:Groningen/Literature#Cleland, CE, et al.2002|(Cleland, CE, et al.2002)]] . Another similar definition states that life is related to possessing metabolic properties, being responsive to the environment, and having the ability to replicate [[Team:Groningen/Literature#Bhutkar, A2005|(Bhutkar, A2005)]] . Another issue which relates to this question is where a line can be drawn between an engineered machine and a living organism. A possibility to make a distinction is when a ‘value’ is assigned to GMOs. Two values can be assigned, instrumental and intrinsic. Instrumental value is being defined by the use it has for humankind, whereas the intrinsic value, independent of the instrumental value, is assigned to any organism being valuable ‘in and of itself’. The question here is: do bacteria posses an intrinsic value? Also this issue becomes more prominent when the field of synthetic biology develops towards using higher organism and even humans.<br />
<br />
==Responsibilities==<br />
What are the responsibilities of the researchers in this field concerning the above-mentioned ethical issues? First of all safety for themselves and co-workers is a responsibility for all researchers. Also these researchers should think about possible implication of the GMO outside the lab. Especially when the organism has a task to perform outside the lab. Once the implications are clear some actions should be taken to try to minimize the risk of negative effects of the GMO for the environment and human health. Another responsibility for the field of synthetic biology lies in the security issues. Self-regulation and also helping the governmental organisation by making effective governmental regulation could be considered as a responsibility of researchers in the field of synthetic biology. Participation in the public debates about synthetic biology could be important for the public opinion on synthetic biology. It should be avoided that the people outside the synthetic biology field are scared away and, without enough insights, feel that synthetic biology is unethical. Other more delicate ethical issues like the difference between life and nonlife and assigning value to the organism is a more complicated issue, how far does the responsibility of the researchers reach in this matter and who else are responsible? In scope of this question it is advisable that both researchers and students are being taught about the risks and ethical aspects of synthetic biology in order to create awareness.<br />
In line with this the question raises what the ethical responsibilities of iGEM participant are? Obviously safety in working with GMOs is important. However also the safety of the GMO should be considered, what are the implications for the environment and human health and how can the risk of possible negative effects be minimized? Security issues are not directly the responsibility of the iGEM participant however it should not be overlooked. It is the responsibility of the iGEM participant to think about the risk of dual-use for their particular project. The iGEM participant could also give more transparency to the field of synthetic biology towards the public by publication of their project plan and results on their wiki-page. It should be encourage that part of the wiki is written for non-participant and even non-scientist in order to allow the public to gain understanding in the possible applications of synthetic biology. Overall responsibility of iGEM participant lies in gaining awareness of the ethical issues concerning synthetic biology and in particular their own project. The teams should try to gain a clear understanding of the ethical issues of their own project and its application.<br />
<br />
=Ethical issues in Heavy metal scavengers with a vertical gasdrive=<br />
<br />
==Introduction==<br />
It is important to consider ethical issues when introducing a new application, especially when genetic modified organisms are involved. In our survey we included an essay question in which the respondents were asked to give their opinion on ethical isues surrouding our project. We wanted to approach the Delphi method for brainstorming in which participant can speak freely and this will open up the discussion.We used the four ethical issues as described by [[Team:Groningen/Literature#Bhutkar, A2005|(Bhutkar, A2005)]] as a guideline. From these answers, and also from the rest of the survey, it appeared that people are most concerend about the safety for the environment and human health.<br />
<br />
==Safety==<br />
Important issues that should be considerend and solved before the bouyant heavy metal scavengers can be used in application are the possible danger for the natural bacterial population, antibiotic resistance that could spread, the concentrated amounts of a heavy metal, unwanted side effects.<br />
Most of these concerns are only applicable when the bacteria end up in the natural environment. This is obviously something that should be prevented in order to keep the system controllable. Keeping the bacteria in a closed water cleaning facility is a controlable environment, however, there is the risk in traditional water cleaning facilities of overflowing. So since there is still a small risk of bacteria ending up in the natural environment this should be considered.<br />
If bacteria end up in the natural environment many risks are present. One of the most mentioned concerns is the transfer of genes from the GMO to another organism. There is no evidence that gene transfer can happen between two different organisms, however, it is possible between two bacteria of the same strain. It does not happen often, it only happens in a stressful environment [[Team:Groningen/Literature#Mindlin, SZ, et al.2002|(Mindlin, SZ, et al.2002, )]], [[Team:Groningen/Literature#Stephenson, JR, et al.1996|(Stephenson, JR, et al.1996)]] . If a GMO end up in a natural environment it could be very stressful so it is possible that genes are transferred. To prevent this it would be best if the bacterium cannot transfer genes and dies. One way to achieve this is implementing a death-gene. This gene start apotheosis and can be transcribed with a delay. So the bacterium can do its job first and after a certain time or at a certain set-of the death-gene starts apotheosis. In this way the GMO is controllable and the possible unwanted side-effects of the GMO in a natural environment or effect because of a mutation are also decreased.<br />
Another safety issue mentioned in the survey is the escape of a antibiotic resistant strain that can spread this resistant among other bacteria. To overcome this risk other selection mechanisms besides antibiotics should be used like a gene that can make glucose out of lactose.<br />
Another concern has to do with the accumulation of toxic metals. People are afraid that this high concentration of metals can poison other organisms that eat the bacterium or a bacterium with the heavy metal stays behind. In order to make sure that a bacterium that accumulates metals also floats to the surface a feedback mechanism between the accumulation device and the gas vesicle devise could be made.<br />
Yet another concern is the removal of the bacteria and the heavy metals. Some people presume that it is very costly to remove the bacteria from the water cleaning facilitation and that it would be very difficult to remove the heavy metals. The removal of the bacteria does not have to be very expensive. It can be done by using a kind of a sieve. The metals can be removed by destruction of the cells. In case of arsenic antimone can be added and the arsenide importer will export all arsenide from the cell. Another concern is how do you know if the GMOs remove enough or even all of the particular heavy metal from the water or sludge? It can be estimated how much of this particular heavy metal a bacterium can take up and a overload of bacteria should be added so you know for sure that all of that particular heavy metal is removed.<br />
Another problem is what if the amount of the heavy metal is lower than expected and therefor not enough metal is accumulated to float? In this case the death gene should make sure that after a certain amount of time or at a certain signal the bacterium starts apotheosis. <br />
There is a possibility that the bacteria do not only take up the selected heavy metal. Bacteria could take up other metals as well, like copper and zinc. These metals do no harm in the drinking water, they are even useful for humans. However the heavy metals would compete with the copper and zinc for the accumulation proteins so most copper and zinc would be removed. Another way to prevent this from happening is over saturating the bacteria with these other metals before letting them clean the water.<br />
<br />
==Security risk==<br />
Security risks are always present. If the concentrated amounts of heavy metal fall into the wrong hands it could be misused. This risk is minimal since water cleaning facilities are not open for the public and the changes are bigger, and the result more catastrophic, if someone puts toxics in the drinking water.<br />
<br />
==Playing God issue==<br />
Most people do not find modifying bacteria as itself morally incorrect. From our survey it appeared that most people do not find it a problem if these modified bacteria are used in a water or sludge cleaning application. <br />
<br />
==Intellectual property==<br />
No other concerns are of interest in this project apart from the basic question can DNA be patented. Most people find this morally incorrect, however, patenting a system or idea which uses modified bacteria should be possible in order to keep up development.<br />
<br />
<br />
{{Team:Groningen/Footer}}</div>JolandaWitteveenhttp://2009.igem.org/Team:Groningen/EthicsTeam:Groningen/Ethics2009-10-21T21:50:00Z<p>JolandaWitteveen: </p>
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<div>{{Team:Groningen/Header}}<br />
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<br />
<div style="float:left" >{{linkedImage|GroningenPrevious.png|Team:Groningen/Safety}}</div><br />
<div title="Arsie Says UP TO ACCUMULATION" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Vision}}</div><br />
<br />
[[Category:Team:Groningen]]<br />
<br />
= Ethical issues in synthetic biology =<br />
<br />
Synthetic biology is a relatively new field in biology in which engineering and biology are combined. It can be defined as the engineering of biology: the synthesis of complex, biologically based (or inspired) systems, which display functions that do exist in nature [[Team:Groningen/Literature#Serrano, L2007|(Serrano, L2007)]]. There are a number of approaches to synthetic biology; bioengineering, redesigning life and creating alternative life each with their own ethical concerns [[Team:Groningen/Literature#Deplazes, A2009|(Deplazes, A2009)]], [[Team:Groningen/Literature#Chopra, PK, A2006|(Chopra, PK, A2006)]]. <br />
<br />
==Safety==<br />
An important ethical issue for all disciplines is the safety issue, what are the risks posed by synthetic biology? [[Team:Groningen/Literature#Bhutkar, A2005|Bhutkar 2005]] distinguishes three safety risks. First, the risk of negative environmental impact this includes possible side effects, of genetically modified organism (GMO) that have to perform a task outside the lab, which can affect the environment negatively. Another risk is the risk to contaminate the natural genome pool, which includes genetic exchange between a GMO and a natural organism. The third risk mentioned by Bhutkar is the run-off risk, which includes possible unforeseen effects if a GMO has no controlled lifespan outside the lab. So the safety issue concerns the implication that GMOs could have on human health and the environment [[Team:Groningen/Literature#Kelle, A2009|(Kelle, A2009)]], [[Team:Groningen/Literature#Bhutkar, A2005|(Bhutkar, A2005)]]. <br />
<br />
==Security==<br />
Also security issues are important ethical issues for bioengineering and when defining the difference between bio-safety and bio-security the easiest discrimination is that between mistakes and that of bad intentions. Synthetic biology is a [[Team:Groningen/Glossary#Dual-use|dual-use]] technology; it can be used for good goals or misused and cause harm. Misusage of the techniques and GMO's could result, among other things, in bioweapons and with the threshold for practicing biology getting lower and lower it is creating an unprecedented security problem [[Team:Groningen/Literature#Schmidt2008|(Schmidt, 2008)]]. More and more people will have the possibility to engineer biology and without proper regulatory oversight these socalled [[Team:Groningen/Glossary#Biohacker|biohackers]] can create potential hazards. A list of selected reading about security and possible interventions can be found [http://www.synbioproject.org/topics/synbio101/bibliography/governance/ here], [http://syntheticbiology.org/SB2.0/Biosecurity_resolutions.html here] and [http://www.jcvi.org/cms/research/projects/syngen-options/publications/ here]. Screening of ordered DNA and oligonucleotides for pathogenic agents and to license certain equipment and reagents could make it more controllable. This could be achieved by governmental regulation, however self regulation by the field of synthetic biology will be even better [[Team:Groningen/Literature#Kelle, A2009|(Kelle, A2009)]], [[Team:Groningen/Literature#Samuel, GN, et al.2009|(Samuel, GN, et al.2009)]], [[Team:Groningen/Literature#Nouri, A, et al.2009|(Nouri, A, et al.2009)]], [[Team:Groningen/Literature#Nicholls, H2008|(Nicholls, H2008)]] . <br />
<br />
==Intellectual property==<br />
Another ethical issue is the intellectual property of DNA. Synthetic biology forces rethinking about what should be patentable [[Team:Groningen/Literature#Samuel, GN, et al.2009|(Samuel, GN, et al.2009)]] ? Living organisms have been patented in the past, the first being a purified form of yeast that was patented by Louis Pasteur. The US patent and trademark office (PTO) has, beside the traditional patent requirements, two addition parts, which biotechnological systems should meet in order to be patentable. First is to show that the organism has little change of developing naturally. Second is that it should be shown that natural selection works against the GMO [[Team:Groningen/Literature#Bhutkar, A2005|(Bhutkar, A2005)]] . The iGEM organisation circumvented this question by using an opensource system in which all Biobricks are documented and freely accessible. <br />
<br />
==Playing God issue==<br />
Another ethical issue, that is most prone in the creating alternative life part of synthetic biology, is the ‘playing God’ issue. This also raises the question what is life? This is an old philosophical question in which different views exists. One is that life is a self-sustained chemical system capable of undergoing Darwinian evolution [[Team:Groningen/Literature#Cleland, CE, et al.2002|(Cleland, CE, et al.2002)]] . Another similar definition states that life is related to possessing metabolic properties, being responsive to the environment, and having the ability to replicate [[Team:Groningen/Literature#Bhutkar, A2005|(Bhutkar, A2005)]] . Another issue which relates to this question is where a line can be drawn between an engineered machine and a living organism. A possibility to make a distinction is when a ‘value’ is assigned to GMOs. Two values can be assigned, instrumental and intrinsic. Instrumental value is being defined by the use it has for humankind, whereas the intrinsic value, independent of the instrumental value, is assigned to any organism being valuable ‘in and of itself’. The question here is: do bacteria posses an intrinsic value? Also this issue becomes more prominent when the field of synthetic biology develops towards using higher organism and even humans.<br />
<br />
==Responsibilities==<br />
What are the responsibilities of the researchers in this field concerning the above-mentioned ethical issues? First of all safety for themselves and co-workers is a responsibility for all researchers. Also these researchers should think about possible implication of the GMO outside the lab. Especially when the organism has a task to perform outside the lab. Once the implications are clear some actions should be taken to try to minimize the risk of negative effects of the GMO for the environment and human health. Another responsibility for the field of synthetic biology lies in the security issues. Self-regulation and also helping the governmental organisation by making effective governmental regulation could be considered as a responsibility of researchers in the field of synthetic biology. Participation in the public debates about synthetic biology could be important for the public opinion on synthetic biology. It should be avoided that the people outside the synthetic biology field are scared away and, without enough insights, feel that synthetic biology is unethical. Other more delicate ethical issues like the difference between life and nonlife and assigning value to the organism is a more complicated issue, how far does the responsibility of the researchers reach in this matter and who else are responsible? In scope of this question it is advisable that both researchers and students are being taught about the risks and ethical aspects of synthetic biology in order to create awareness.<br />
In line with this the question raises what the ethical responsibilities of iGEM participant are? Obviously safety in working with GMOs is important. However also the safety of the GMO should be considered, what are the implications for the environment and human health and how can the risk of possible negative effects be minimized? Security issues are not directly the responsibility of the iGEM participant however it should not be overlooked. It is the responsibility of the iGEM participant to think about the risk of dual-use for their particular project. The iGEM participant could also give more transparency to the field of synthetic biology towards the public by publication of their project plan and results on their wiki-page. It should be encourage that part of the wiki is written for non-participant and even non-scientist in order to allow the public to gain understanding in the possible applications of synthetic biology. Overall responsibility of iGEM participant lies in gaining awareness of the ethical issues concerning synthetic biology and in particular their own project. The teams should try to gain a clear understanding of the ethical issues of their own project and its application.<br />
<br />
=Ethical issues in Heavy metal scavengers with a vertical gasdrive=<br />
<br />
==Introduction==<br />
It is important to consider ethical issues when introducing a new application, especially when genetic modified organisms are involved. In our survey we included an essay question in which the respondents were asked to give their opinion on ethical isues surrouding our project. We wanted to approach the Delphi method for brainstorming in which participant can speak freely and this will open up the discussion.We used the four ethical issues as described by [[Team:Groningen/Literature#Bhutkar, A2005|(Bhutkar, A2005)]] as a guideline. From these answers, and also from the rest of the survey, it appeared that people are most concerend about the safety for the environment and human health.<br />
<br />
==Safety==<br />
Important issues that should be considerend and solved before the bouyant heavy metal scavengers can be used in application are the possible danger for the natural bacterial population, antibiotic resistance that could spread, the concentrated amounts of a heavy metal, unwanted side effects.<br />
Most of these concerns are only applicable when the bacteria end up in the natural environment. This is obviously something that should be prevented in order to keep the system controllable. Keeping the bacteria in a closed water cleaning facility is a controlable environment, however, there is the risk in traditional water cleaning facilities of overflowing. So since there is still a small risk of bacteria ending up in the natural environment this should be considered.<br />
If bacteria end up in the natural environment many risks are present. One of the most mentioned concerns is the transfer of genes from the GMO to another organism. There is no evidence that gene transfer can happen between two different organisms, however, it is possible between two bacteria of the same strain. It does not happen often, it only happens in a stressful environment [[Team:Groningen/Literature#Mindlin, SZ, et al.2002|(Mindlin, SZ, et al.2002, )]], [[Team:Groningen/Literature#Stephenson, JR, et al.1996|(Stephenson, JR, et al.1996)]] . If a GMO end up in a natural environment it could be very stressful so it is possible that genes are transferred. To prevent this it would be best if the bacterium cannot transfer genes and dies. One way to achieve this is implementing a death-gene. This gene start apotheosis and can be transcribed with a delay. So the bacterium can do its job first and after a certain time or at a certain set-of the death-gene starts apotheosis. In this way the GMO is controllable and the possible unwanted side-effects of the GMO in a natural environment or effect because of a mutation are also decreased.<br />
Another safety issue mentioned in the survey is the escape of a antibiotic resistant strain that can spread this resistant among other bacteria. To overcome this risk other selection mechanisms besides antibiotics should be used like a gene that can make glucose out of lactose.<br />
Another concern has to do with the accumulation of toxic metals. People are afraid that this high concentration of metals can poison other organisms that eat the bacterium or a bacterium with the heavy metal stays behind. In order to make sure that a bacterium that accumulates metals also floats to the surface a feedback mechanism between the accumulation device and the gas vesicle devise could be made.<br />
Yet another concern is the removal of the bacteria and the heavy metals. Some people presume that it is very costly to remove the bacteria from the water cleaning facilitation and that it would be very difficult to remove the heavy metals. The removal of the bacteria does not have to be very expensive. It can be done by using a kind of a sieve. The metals can be removed by destruction of the cells. In case of arsenic antimone can be added and the arsenide importer will export all arsenide from the cell. Another concern is how do you know if the GMOs remove enough or even all of the particular heavy metal from the water or sludge? It can be estimated how much of this particular heavy metal a bacterium can take up and a overload of bacteria should be added so you know for sure that all of that particular heavy metal is removed.<br />
Another problem is what if the amount of the heavy metal is lower than expected and therefor not enough metal is accumulated to float? In this case the death gene should make sure that after a certain amount of time or at a certain signal the bacterium starts apotheosis. <br />
There is a possibility that the bacteria do not only take up the selected heavy metal. Bacteria could take up other metals as well, like copper and zinc. These metals do no harm in the drinking water, they are even useful for humans. However the heavy metals would compete with the copper and zinc for the accumulation proteins so most copper and zinc would be removed. Another way to prevent this from happening is over saturating the bacteria with these other metals before letting them clean the water.<br />
<br />
==Security risk==<br />
Security risks are always present. If the concentrated amounts of heavy metal fall into the wrong hands it could be misused. This risk is minimal since water cleaning facilities are not open for the public and the changes are bigger, and the result more catastrophic, if someone puts toxics in the drinking water.<br />
<br />
==Playing God issue==<br />
Most people do not find modifying bacteria as itself morally incorrect. From our survey it appeared that most people do not find it a problem if these modified bacteria are used in a water or sludge cleaning application. <br />
<br />
==Intellectual property==<br />
No other concerns are of interest in this project apart from the basic question can DNA be patented. Most people find this morally incorrect, however, patenting a system or idea which uses modified bacteria should be possible in order to keep up development.<br />
<br />
<br />
{{Team:Groningen/Footer}}</div>JolandaWitteveenhttp://2009.igem.org/Team:Groningen/Project/TransportTeam:Groningen/Project/Transport2009-10-21T21:04:24Z<p>JolandaWitteveen: </p>
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<div style="float:left" >{{linkedImage|GroningenPrevious.png|Team:Groningen/Application}}</div><br />
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<h1>Transport</h1><br />
<b>To isolate heavy metals from the environment we require uptake systems. Several different mechanisms to create such an import system are exist; metal transporters (coupled and uncoupled) and binding proteins in the periplasm. Import systems for several metals were found. We investigated HmtA for copper/zinc uptake. Cloning of the HmtA failed unfortunatly. Citrate coupled transporters, CitH and CitM were also considered as wel as the periplasmic accumulation operon Mer.<br />
Since we chose to focus on arsenic the final device was made with GlpF. GlpF a aquaglycerol porin was found to import not only glycerol but also arsenite and arsenate. This importer was cloned as a BioBrick part and transformed into ''E. coli''. An uptake assay was performed and a metal sensitivity assay, which showed functionality of the GlpF transporter.</b><br />
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==Arsenite uptake via GlpF==<br />
<!--[[Image:GlpF.jpeg|200px|thumb|right|73As(III) and 125Sb(III) uptake into cells of ''E. coli'' is facilitated by the aquaglyceroporin channel GlpF.]]--><br />
<br />
===GlpF===<br />
<br />
====Introduction====<br />
GlpF is an aquaglycerol porin of E.coli which facilitates not only glycerol import, but also arsenic (As) and antimone (Sb) import [[Team:Groningen/Literature#Fu, DX, et al.2000|(Fu, DX, et al.2000]]), [[Team:Groningen/Literature#Meng, YL, et al.2004|(Meng, YL, et al.2004]]), [[Team:Groningen/Literature#Porquet, A, et al.2007|(Porquet, A, et al.2007]]), [[Team:Groningen/Literature#Rosen, BR, et al.2009|(Rosen, BR, et al.2009)]] . It has homologues in other organisms; Fps1p has shown to facilitate arsenic import in yeast and AQP9 is the mammalian homologue [[Team:Groningen/Literature#Porquet, A, et al.2007|(Porquet, A, et al.2007]]), [[Team:Groningen/Literature#Rosen, BR, et al.2009|(Rosen, BR, et al.2009)]] .<br />
The GlpF aquaglycerol porin is a membrane protein with a symmetric arrangement of four independent GlpF channels. One monomer of this tetramer GlpF porin consists of six transmembrane and two half membrane-spanning α-helices that form a right-handed helical bundle around the channel. The channel has a diameter of ~15Å at the periplasmid end, which constricts towards a diameter of ~3.8Å at the beginning of a 28 Å long selective channel that ends at the cytoplasmic end [[Team:Groningen/Literature#Fu, DX, et al.2000|(Fu, DX, et al.2000)]].<br />
The GlpF is a stereospecific channel that is thought to be more selective on molecular size than on chemical structure [[Team:Groningen/Literature#Fu, DX, et al.2000|(Fu, DX, et al.2000]], [[Team:Groningen/Literature#Heller, KB, et al.1980|(Heller, KB, et al.1980)]] . It does allow transport of a variance of non-charged compounds ranging from polyhydric alcohols, glycerol being one of them, arsenic to antimone [[Team:Groningen/Literature#Fu, DX, et al.2000|(Fu, DX, et al.2000]]), [[Team:Groningen/Literature#Meng, YL, et al.2004|(Meng, YL, et al.2004]]), [[Team:Groningen/Literature#Porquet, A, et al.2007|(Porquet, A, et al.2007)]], [[Team:Groningen/Literature#Rosen, BR, et al.2009|(Rosen, BR, et al.2009]]), [[Team:Groningen/Literature#Heller, KB, et al.1980|(Heller, KB, et al.1980)]]. Carbon sugars and ions are shown to be unable to be transported by GlpF [[Team:Groningen/Literature#Heller, KB, et al.1980|(Heller, KB, et al.1980)]]. At physiological pH arsenic and antimone are not present in their ionic state but rather as As(OH)3 and Sb(OH)3 [[Team:Groningen/Literature#Rosen, BR, et al.2009|(Rosen, BR, et al.2009)]]. These elements show a charge distribution similar to glycerol and a smaller but comparable volume. The structural similarities are thought to be the reason for the possibility of these elements to enter the cell by GlpF [[Team:Groningen/Literature#Porquet, A, et al.2007|(Porquet, A, et al.2007)]], GlpF facilitates transport of these compounds down there gradient (inside or outside the cell).<br />
If GlpF behaves as a nonsaturable transporter, a transport rate of 1umol of glycerol is transported per minute per mgr of cell protein [[Team:Groningen/Literature#Heller, KB, et al.1980|(Heller, KB, et al.1980)]].<br />
<br />
====Cloning strategy====<br />
This part has been obtained from the genome of ''E.coli'' 356 in two steps with PCR. First the whole gene was obtained from the genome by using PCR and in the second step an ''EcoR''1 restiction site was removed.<br />
The GlpF PCR product was restricted with ''Xba''I and ''Pst''I and a psB1AC3 vector with a pMed promotor was restricted with ''Spe''I and ''Pst''I. The restriction products were ligated. This resulted in a psB1AC3 vector with a promotor and GlpF.<br />
[[Image:RestictioLigationGlpF.JPG]]<br />
<br />
====Results====<br />
The ability of GlpF (overexpressed under IPTG induction) to transport As(III) was tested by an arsenite uptake [https://2009.igem.org/Team:Groningen/Protocols assay]. Also the full accumulation device (<partinfo>BBa_K190038</partinfo>) was tested using this assay. '''Data and analysis can be found [https://2009.igem.org/Team:Groningen/Project/Accumulation here]. <br />
'''<br />
<br />
[[Image:Growth_WT.gif|310px|left]]<br />
[[Image:Growth GlpF.gif|310px|left]]<br />
[[Image:Growth GlpF fMT.gif|310px|left]]<br />
<br />
The graphs above represent the result of the metal sensitivity [https://2009.igem.org/Team:Groningen/Protocols#Death_assay assay]. The lines in the graphs represent the average optical density of a construct over time. The graph on the left show that increased As(III) levels inhibit growth and, that as more As(III) is added the lower the plateau is. <br />
<br />
The middle graph is from the pLac GlpF construct. The curves are less steep in the log phase compared to WT because of the protein expresion by IPTG induction. In the absence of As(III) the plateau level equals the WT. If arsenite is present the plateaus are lower (OD<sub>600</sub> <0.8) compared to WT. This is due to As(III) uptake by GlpF. <br />
<br />
In the graph on the right we see the curves of low constitutively expressed GlpF and fMT and it shows a similar slope in the log phase compared to pLac GlpF due to protein expression and like WT 0 μM As(III) it has its plateau over OD<sub>600</sub> 0.9. If arsenite is present the plateaus are lower (OD<sub>600</sub> <0.8) compared to WT. This is due to As(III) uptake by GlpF. Here the reduced growth is also an indicator for arsenite uptake. It is difficult to see if fMT has an effect because this assay can not show where the arsenite is and how fMT interferes with the cells detoxificatoin.<br />
<br />
==={{anchor|Modelling}}Modelling uptake GlpF===<br />
<html><br />
<script type="text/javascript" src="/Team:Groningen/Modelling/Model.js?action=raw"></script><br />
<script type="text/javascript" src="/Team:Groningen/Modelling/Arsenic.js?action=raw"></script><br />
</html><br />
<html><style type="text/css"></html><br />
{{InfoBox/Style.css}}<br />
.infoIcon { display: inline; }<br />
<html></style></html><br />
The import of As(III) via GlpF is modelled as a simple import reaction with [[Team:Groningen/Glossary#MichaelisMenten|Michaelis-Menten kinetics]], in part because this makes it easy to specify, but also because we only have very high level data. The following allows a comparison with the data acquired from figure 1B from [[Team:Groningen/Literature#Meng2004|Meng 2004]].<br />
<html><br />
<div style="background:#efe;border:1px solid #9c9;padding:1em;"><br />
<table style="border-collapse:collapse;background:none;"><tr><br />
<td style="border-right:1px solid #9c9;padding-right:1em;"><br />
<dl><br />
<dt>Initial values</dt><br />
<dd><br />
As(III)<sub>ex</sub> = <input type="text" id="As3exInitial" value="9.15164271986822"/> &micro;M<br/><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(10&micro;M &middot; 1mL / 1.092mL)<br />
</dd><br />
<dt>Volumes</dt><br />
<dd><br />
V<sub>total</sub> = <input type="text" id="Vtotal" value="1.1"/> mL<br/><br />
V<sub>cells</sub> = <input type="text" id="Vcells" value="0.0073"/> mL<br/><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(0.1ml &middot; 80mg/mL / 1100mg/mL) </html>{{infoBox|E. coli has a density of approximately 1100mg/mL, see [[Team:Groningen/Project/Vesicle|our gas vesicle page]] for more information.}}<html><br />
</dd><br />
<dt>Kinetic Constants</dt><br />
<dd><br />
<nobr>v5 = <input type="text" id="v5" value="3.1862846729357852"/> &micro;mol/(s&middot;L)</nobr><br/><br />
K5 = <input type="text" id="K5" value="27.71808199428998"/> &micro;M<br/><br />
</dd><br />
</dl><br />
<br />
<button onClick="computeGlpFTransport()">Compute</button><br/><br />
</td><br />
<br />
<td style="padding-left:1em;"><br />
<div id="glpFTransportError" style="color:red"></div><br />
</html>{{graph|Team:Groningen/Graphs/GlpFTransport|id=glpFTransportGraph}}<html><br />
</td><br />
</tr></table><br />
</div><br />
<script type="text/javascript"><br />
<br />
//The graph already initializes itself (and we don't do any other computations).<br />
//addOnloadHook(computeGlpFTransport);<br />
<br />
function computeGlpFTransport() {<br />
document.getElementById('glpFTransportGraph').refresh();<br />
}<br />
</script><br />
</html><br />
<br />
To determine the constants v5 and K5 we performed the following steps:<br />
<br />
# '''Read the wild-type line in figure 1B''' of [[Team:Groningen/Literature#Meng2004|Meng 2004]] by pasting it in a drawing program and aligning/scaling the axes and then manually determining the coordinates of each data point.<br />
# '''Converted to units of concentration''' using the data in Meng 2004 and [http://gchelpdesk.ualberta.ca/CCDB/cgi-bin/STAT_NEW.cgi the CCDB] (assuming that the cells are resting/non-growing), see our [http://spreadsheets.google.com/pub?key=t4gilzCbEaCFAvpEVWUE_zQ Google Docs spreadsheet]. Here we disregarded the fact that the measurements were made by taking out 0.1mL samples, as this does not change the concentrations. Specifically (note that uptake is in nmol/mg):<br />
#* uptake<sub>total</sub> (nmol) = uptake &middot; 8mg &middot; 0.3 {{infoBox|The ratio between dry and wet weight is 0.3 (see the [http://gchelpdesk.ualberta.ca/CCDB/cgi-bin/STAT_NEW.cgi CCDB]).}}<br />
#* As(III)<sub>ex</sub> (&micro;M=nmol/mL) = (10nmol/mL &middot; 1mL - uptake<sub>total</sub>) / (1.1-0.0073)mL {{infoBox|1=The experiment started with 1mL of a 10&micro;M=10nmol/mL solution of As(III). After adding the cells the total volume of the solution was 1.1mL, and 0.0073mL is an estimate of the total volume of cells in the solution, see below.}}<br />
# '''Fit the Michaelis-Menten equation''' to find the constants v5 and K5 in Mathematica (see [http://igemgroningen.googlecode.com/svn/trunk/buoyant/Models/Meng2004%20Figure%201B.nb the Mathematica notebook in SVN]) using the method from [[Team:Groningen/Literature#Goudar1999|Goudar 1999]] (a least squares fit of a closed-form solution of the differential equation).<br />
<br />
{{GraphHeader}}<br />
<br />
<br><br />
<br />
===Missing information/To Do===<br />
*Expression assesment<br />
**Stability<br />
**Level<br />
*Functional assesment<br />
**Uptake speed<br />
**Affinity<br />
**Electrolyte potential generating force<br />
*<del>Q:Eliminate BioBrick restriction sites</del><br />
*<del>Q: What does the ars operon of our <i>E. coli</i> look like? Do we have both ArsA and ArsB? (And what about ArsR and ArsD?)</del> A: We only have ArsRBC, see [[Team:Groningen/BLAST|our BLAST results]].<br />
<br />
<br><br />
<br />
===Additional sources===<br />
<br><br />
* [[Team:Groningen/Literature#Meng2004|Meng 2004]] (As(III) and Sb(III) Uptake by GlpF and Efflux by ArsB in Escherichia coli)<br />
* [[Team:Groningen/Literature#Rosen2009|Rosen 2009]] (Transport pathways for arsenic and selenium: A minireview)<br />
*[[Team:Groningen/Literature#Porquet, A, et al.2007|Porquet, A, et al.2007]] (structural similarity between As(OH)3 and glycerol)<br />
* [[Team:Groningen/Literature#Fu, DX, et al.2000|Fu, DX, et al.2000]] (Structure of the GlpF channel)<br />
*[[Team:Groningen/Literature#Heller, KB, et al.1980|Heller, KB, et al.1980]] (Glycerol transport properties of GlpF)<br />
<br />
==Copper/zinc uptake via HmtA==<br />
<br />
===HmtA===<br />
====Introduction====<br />
HmtA(heavy metal transporter A) from <i>Pseudomonas aeruginosa</i> [http://www.ncbi.nlm.nih.gov/protein/81857196 Q9I147] is a P-type ATPase importer. This membrane protein mediates the uptake of copper (Cu) and zinc (Zn) and was functionally expressed in ''E. coli'' ([http://www.ncbi.nlm.nih.gov/pubmed/19264958 Lewinson 2009]). We want to use this membrane protein to accumulate copper and zinc into the cells. we believe this ATP-driven pump is capable of generating an elevated intracellular concentration of these compounds enabling the harvesting of copper and zinc from the medium.<br />
<br />
====Cloning strategy====<br />
There are several restriction sites to be modified from [https://static.igem.org/mediawiki/2009/8/85/PBAD-HmtA-ClonemanagerFile.zip Lewinson's] pBAD construct. A vector with amp resistance with L-arabinose inducible HmtA-6HIS. The restriction sites have been silently mutated maintaining the amino acid sequence.<br />
We will create these mutations via PCR than digest the old methylated template and clone the product into competent cells.<br />
<br />
====Results==== <br />
[[Image:HmtA_SDS_gel.jpg|200px|thumb|right|[Team:Groningen/Team|HmtA-6HIS on SDS-page]]<br />
So far we have cloned HmtA as a biobrick without EcoRI site in the coding region into the iGEM vector. Unfortunately a mutation occurred at base 103 from the start of the orf. By a point mutation c to t in the first nucleotide of the codon changed arginine 35 to a Cysteine. We do not know the effects but we suspect it might have a great influence due to the very reactive side chain of Cysteine, eventhough it is not in the channel itself based on [http://www.cbs.dtu.dk/services/TMHMM/ TMHMM] predictions which indicate trans membrane helices of a protein. Further cloning is expected to be unsuccessful because the iPTG induced clones grow even slightly better than the empty vector control. This is most likely cause by the missing pLAC-RBS in front of the gene. There was no positive controle with the L-arabinose inducable HmtA-6His in pBAD. We did do expression experiments with the pBAD construct to purified the membrane protein as quality controle. result shown in the figure on the right.<br />
<br />
==Heavy metal uptake coupled to citrate via ''ef''CitH ''bs''CitM==<br />
<br />
Force feeding of the heavy metals into the cell is possible when citrate is the only available carbon source. Citrate in complex with heavy metals can be translocated over the membrane into the cell via citrate transporters.<br />
This can be a very efficient strategy to accumulate vast ammounts of heavy metals.<br />
The two membrane proteins are CitM from ''Bacillus subtilis'' studied by [http://www.ncbi.nlm.nih.gov/pubmed/11053381 B.P Krom]. <i>Bs</i>CitM can transport citrate in complex with Mg<sup>2+</sup>, Ni<sup>2+</sup>, Mn<sup>2+</sup>, Co<sup>2+</sup>, and Zn<sup>2+</sup>. <br />
The other is CitH from ''Enterococcus faecalis'' described by [http://www.ncbi.nlm.nih.gov/pubmed/17042778 V.S Blancato]. <i>Ef</i>CitH catalyzes translocation of the citrate in complex with Ca<sup>2+</sup>, Sr<sup>2+</sup> Mn<sup>2+</sup> Mn<sup>2+</sup> Cd<sup>2+</sup> and Pb<sup>2+</sup>.<br />
<br />
<br />
===Additional sources===<br />
<br />
More information on this topic can be found in:<br />
<br />
Bastiaan Krom. Citrate transporters of <i>Bacillus subtilis</i> PhD thesis. [[http://dissertations.ub.rug.nl/faculties/science/2002/b.p.krom/ Dissertation Groningen]]<br />
<br />
Jessica B. Warner. Regulation and expression of the metal citrate transporter CitM PhD thesis. [[http://dissertations.ub.rug.nl/faculties/science/2002/j.b.warner/ Dissertation Groningen]]<br />
<br />
==Periplasmic accumulation of heavy metals via Mer Operon==<br />
Periplasmic accumulation of heavy metals via Mer proteins enables the harvesting of heavy metals from the medium by binding the cytosolic and periplasmic metals to metallothionein and transporting the metal-protein complex into the periplasm.<br />
The MerR family consists of different proteins for one specific metal (<i>i.e.</i><br />
PbrR (lead), CueR (copper), ZntR (zinc), MerR (mercury), ArsR (arsenic), CadR (cadmium)).<br />
<br />
As the cells die after uptake of Mg (and induction of the Mer transporter), this system is not very well usable for our project. The dead cells will not produce the gas vesicles (it may be used however by having the gas vesicles consitutively expressed), thereby bouyancy may be a problem ([[Team:Groningen/Literature#Pennella2005|Pennella 2005]], [[Team:Groningen/Literature#Kao2008|Kao 2008]]).<br />
<br />
===Missing information/To Do===<br />
*Expression assesment<br />
**Stability<br />
**Level<br />
*Functional assesment<br />
**Uptake speed<br />
**Affinity<br />
**Electrolyte potential generating force<br />
*Eliminate BioBrick restriction sites<br />
<br />
==Planning and requirements==<br />
<br />
* '''Modelling:'''<br />
** Import speed<br />
** Amount <br />
** Max<br />
* '''Lab:'''<br />
** HmtA<br />
*** Zn/Cu alone<br />
*** B-type ATPase (could be use if there is a ATP shortage?)<br />
** CitM (probably not used)<br />
*** Divalent ions<br />
*** Citrate around<br />
*** Citrate can bind metals that are already bound.<br />
** Measurements (both for the "normal" cell and the cell with overexpression of the transporter)<br />
*** Transporter, on/off mechanism, up to what concentration (in the cell) does it still have metal uptake.<br />
*** Measure concentration of metal. difference between begin and end concentrations of metal outside the cell.<br />
*** How fast does it transport metal in/out the cell.<br />
**** Set up tests with (initial) extracellular concentrations of about <sup>1</sup>/<sub>3</sub>K (25% of V<sub>max</sub>), K (50% of V<sub>max</sub>), 3K (75% of V<sub>max</sub>) and 10mM (99.7% of V<sub>max</sub>, corresponding to extremely polluted water), and a control with no arsenic. Obviously, more tests is better. In general a desired fraction of V<sub>max</sub> at the initial concentration can be attained by using an initial concentration of x/(1-x) times K.<br />
**** Determine "final" (steady-state) concentration of As(III) in the solution and in the cells. (Concentration over time is even better!)<br />
**** This means that the total volume of the cells (and the solution) has to be determined. Possibly through looking at the dry weight (without arsenic!).<br />
**** By manipulating the equation for the derivative of As(III) in equilibrium, As(III) can be expressed as a function of As(III)<sub>ex</sub> (given the V and K constants). We can try to fill in the computed V and K constants for GlpF and then use a least squares fit to estimate the V and K constants for ArsB.<br />
**** '''NOTE:''' Interestingly [[Team:Groningen/Literature#Kostal2004|Kostal 2004]] already did an experiment like this with cells that overexpressed ArsR. We're looking at analysing these results under the assumption that overexpressing ArsR only gives a constant factor more accumulation (for 1-100&microM As(III)), but it would be very nice to do this ourselves for unmodified cells to determine whether this is indeed true (and to determine the factor).<br />
<br />
==Export of arsenicum via Ars operon==<br />
<br />
GlpF is the importer of arsenicum. After arsenicum enters the cell, in response the Ars operon produces ArsR. At the same time, ArsB is also produced by Ars operon. This happens because the Ars operon contains three open reading frames: the first is ArsR, second ArsB and the last one is ArsC. ArsB is the exporter of arsenicum. The ars operon is located on the chromosomal DNA of E. coli.<br />
For more information see: [http://biocyc.org/ECOLI/NEW-IMAGE?type=GENE-IN-CHROM-BROWSER&object=EG12235 biocyc].<br />
<br />
[[Image:ArsRBC_operon.PNG|600px]]<br />
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{{Team:Groningen/Project/Footer}}</div>JolandaWitteveenhttp://2009.igem.org/Team:Groningen/ProtocolsTeam:Groningen/Protocols2009-10-21T20:53:00Z<p>JolandaWitteveen: </p>
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<div>{{Team:Groningen/Header}}<br />
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<div style="float:left" >{{linkedImage|GroningenPrevious.png|Team:Groningen/Literature}}</div><br />
<div style="float:right" >[https://igem.org/Judging_Form.cgi?id=190 {{filepath:Next.JPG}}]</div><br />
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[[Category:Team:Groningen]]<br />
[[Category:Protocol]]<br />
[[Category:Escherichia coli]]<br />
<br />
=Protocols=<br />
{|cellpadding="2" cellspacing="2" border="0"<br />
|'''[[Team:Groningen/Protocols#Cloning|<u>Cloning</u>]]'''<br />
|<!--Space--><br />
|'''[[Team:Groningen/Protocols#Quality_control|<u>Quality control</u>]]'''<br />
|<!--Space--><br />
|'''[[Team:Groningen/Protocols#Measurements|<u>Measurements</u>]]''' <br />
|<!--Space--><br />
|'''[[Team:Groningen/Protocols#List_of_solutions|<u>List of solutions</u>]]'''<br />
|<!--Space--><br />
|-<br />
|[[Team:Groningen/Protocols#PCR|PCR]]<br />
|<!--Space--><br />
|[[Team:Groningen/Protocols#Colony_PCR|Colony PCR]]<br />
|<!--Space--><br />
|[[Team:Groningen/Protocols#Fermentation|Fermentation]]<br />
|<!--Space--><br />
|[[Team:Groningen/Protocols#Media|Media]]<br />
|<!--Space--><br />
|-<br />
|[[Team:Groningen/Protocols#Plasmid_Isolation|Plasmid Isolation]]<br />
|<!--Space--><br />
|[[Team:Groningen/Protocols#Restriction_analysis|Restriction analysis]]<br />
|<!--Space--><br />
|[[Team:Groningen/Protocols#Buoyancy_test|Buoyancy test]]<br />
|<!--Space--><br />
|[[Team:Groningen/Protocols#Antibiotics|Antibiotics]]<br />
|<!--Space--><br />
|-<br />
|[[Team:Groningen/Protocols#Restriction|Restriction]]<br />
|<!--Space--><br />
|[[Team:Groningen/Protocols#Membrane_protein_isolation|Membrane protein isolation]]<br />
|<!--Space--><br />
|[[Team:Groningen/Protocols#Metal_uptake_assay_for_E._coliKostal_2004|Metal uptake assay for <i>E. coli</i>]] <br />
|<!--Space--><br />
|[[Team:Groningen/Protocols#Chemicals|Chemicals]]<br />
|<!--Space--><br />
|-<br />
|[[Team:Groningen/Protocols#Annealing_synthetic_oligo.E2.80.99s|Annealing synthetic oligo's]]<br />
|<!--Space--><br />
|<!--Quality control--><br />
|<!--Space--><br />
|[[Team:Groningen/Protocols#Fluorescence_measurement|Fluorescence measurement]]<br />
|<!--Space--><br />
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|-<br />
|[[Team:Groningen/Protocols#Ligation|Ligation]]<br />
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|[[Team:Groningen/Protocols#Death_assay|Death assay]]<br />
|<!--Space--><br />
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|-<br />
|[[Team:Groningen/Protocols#Making_competent_cells|Making competent cells]]<br />
|<!--Space--><br />
|<!--Quality control--><br />
|<!--Space--><br />
|[[Team:Groningen/Protocols#Fluorescence_of_resting_cells_with_J61002-pArsR|Fluorescence of resting cells with J61002-pArsR]]<br />
|<!--Space--><br />
|<!--List of solutions--><br />
|<!--Space--><br />
|-<br />
|[[Team:Groningen/Protocols#Transformation|Transformation]]<br />
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<br />
=Basic Cloning Strategy:=<br />
# Transform ''E. coli'' TOP10 (genotype DH 10B) with GVP (<partinfo>BBa_I750016</partinfo>), a metal ion transporter ([[Team:Groningen/Project/Transport#Copper.2Fzinc_uptake_via_HmtA|HmtA]] and [[Team:Groningen/Project/Transport#Arsenite_uptake_via_GlpF|GlpF]]) and accumulation proteins.<br />
# PCR the restriction sites out and add BioBrick pre- and suffix → Use [http://openwetware.org/wiki/The_BioBricks_Foundation:BBFRFC10 BBFRCF10].<br />
##Primers should be ordered for the different genes.<br />
## Add a RBS (<partinfo>BBa_B0034</partinfo>) in the primer for the BioBrick prefix.<br />
## Add a terminator (<partinfo>BBa_B0014</partinfo>) via cloning. <br />
## For [http://partsregistry.org/wiki/index.php/Part:BBa_I750016 GVP] the RBS is included in the construct, and biobrick suffix is included in the construct. The prefix is missing because of the ''Eco''RI site in the middle of the plasmid!! This may give problems!!<br />
# PCR restriction sites out. '''!!Both PCR reactions for pre/suffix and restriction sites can possibly be done in 3 seperate PCR reactions !!'''<br />
# Test expression / phenotype '''of separate proteins''' (if possible in the vectors they are supplied in). <br />
# Put both systems (GVP and metal import) on a high and low copy number (supplied by "vector group"). This is needed to prevent plasmid / expression incompatibility when both systems are used in one strain. <br />
## The metal transporter and accumulation protein should be cloned behind each other. If possible on a synthetic operon.<br />
## Clone the different systems for Cu, Zn, As, (Hg) in [http://www.partsregistry.org/Assembly:Rolling_assembly parallel]. <br />
# ''(If needed and not already supplied by "vector group")'' [http://www.partsregistry.org/Assembly:Rolling_assembly In parallel clone] metal sensitive promoters in front of a fluorescent protein (GFP) and in front of the gvp cluster.<br />
# ''(If needed and not already supplied by "vector group")'' [http://www.partsregistry.org/Assembly:Rolling_assembly In parallel clone] different promoters in front of the two systems.<br />
## Inducible like [http://partsregistry.org/Part:BBa_I0500 pBad] or [http://partsregistry.org/wiki/index.php?title=Part:BBa_R0010 pLac]<br />
## Constitutive with expected high and low expression yield<br />
## Metal sentitive promoter (only for gvp system)<br />
# Then try to get both systems in one ''E. coli strain'', test different possibilities with the high + low copy nr. vectors<br />
<br />
<br />
==Cloning==<br />
===[http://openwetware.org/wiki/PCR PCR]===<br />
{|cellpadding="2" cellspacing="2" border="0"<br />
|12.5 μL Phusion mastermix<sup>*</sup><BR><br />
1 μL forward primer<BR><br />
1 μL reverse primer<BR><br />
0.5 μL template<BR><br />
10 μL demi water<BR><BR><br />
<sup>*</sup>Phusion master mix contains:<BR><br />
200 μL 5x Phusion HF buffer<BR><br />
8 μL 25 mM dNTP's<BR><br />
282 μL MilliQ water<BR><br />
10 μL Phusion Polymerase<br />
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|<u><b>PCR Reaction</b><BR><br />
</u>Hotstart<BR><br />
95 °C, 2 min.<BR><br />
25 cycles<BR><br />
95 °C, 30 sec.<BR><br />
61 °C, 20 sec.<BR><br />
72 °C, 1.5 min.<BR><br />
End<BR>72 °C, 10 min.<br />
<BR>4 °C, ∞<br />
|}<br />
<br />
===Plasmid isolation===<br />
Usually performed using Miniprep kits like NucleoSpin<SUP><FONT SIZE="-1">®</FONT></SUP> Plasmid, (Machery nagel) or GeneElute™ Plasmid Miniprep Kit (Sigma-Aldrich). This is a consensus protocol.<br />
*Spin down ON Culture in table top centrifuge, 1 min. 13.000 RPM<br />
*Resuspend pellet, RNAse is added to degrade RNA (200 μL)<br />
*Add lysis buffer (200 μL), to lyse the cells and to release their contents<br />
*Add neutralization buffer (350 μL), proteins will denaturate<br />
*Centrifuge in table top centrifuge 10 min. 13.000 RPM<br />
*Add clear lysate in column provided in kit<br />
*Spin down 1 min. 13.000 RPM<br />
*Add wash buffer (usually needs EtOH to be added!) (500 - 750 μL)<br />
*Remove flow-through and spin again to remove residual wash buffer<br />
*Put column in clean 1.5 mL cup and add 15 - 50 μL MilliQ water or Tris buffer (pH=8.0)<sup>*</sup><br />
*Incubate for 1 - 2 min.<br />
*Spin down 1 min. 13.000 RPM<br />
<sup>*</sup>Less volume gives higher concentrations, supplied Tris buffer is claimed to give higher yields<br />
<br />
===Restriction===<br />
*Mix<br />
**1 μL 10x fast digest buffer ([http://www.fermentas.com/ Fermentas]) or correct [http://fermentas.com/techinfo/re/5bufferplussystem.htm#Buffers conventional buffer]<br />
**0.5 μL Enzyme A<sup>*</sup><br />
**0.5 μL Enzyme B<sup>*</sup><br />
**8 μL DNA to be digested<sup>**</sup><br />
*Incubate 0.5 - 1 h @ 37 °C (Fast digest do not require long incubations, when using conventional enzymes 1 h. should be maintained)<br />
*Purify cut plasmid using PCR clean up kit<br />
<sup>*</sup> a combination of two of the following (when using biobrick standard) [http://www.fermentas.com/catalog/re/fastbcui.htm <i>Spe</i>I], [http://www.fermentas.com/catalog/re/fastecori.htm <i>Eco</i>RI], [http://www.fermentas.com/catalog/re/fastpsti.htm <i>Pst</i>I] and/or [http://www.fermentas.com/catalog/re/fastxbai.htm <i>Xba</i>I]<br />
<br><sup>**</sup> When digesting vectors, bring the digested vectors to 1% agarose gel and cut out with scalpel, purify using gel purification kit ([http://www.mn-net.com/Products/NucleicAcidPurification/DNAcleanup/NucleoSpinExtractII/tabid/1452/language/en-US/Default.aspx NucleoSpin<SUP><FONT SIZE="-1">®</FONT></SUP> Extract II, Machery nagel], [http://www.zymoresearch.com/content/zymoclean-gel-dna-recovery-kit-d4001-d4002-d4007-d4007-d4001s Zymoclean™ Gel DNA Recovery Kit] or similar) to an end volume indicated by the kit (End volume determines concentration, variations are possible)<br />
(alternatively, [http://openwetware.org/wiki/Phosphatase_treatment_of_linearized_vector Phosphatase treatment of linearized vector])<br />
<br />
===Annealing synthetic oligo’s===<br />
<u>Phosphorylation of 5' ends & hybridization<SUP><FONT SIZE="-1">[http://openwetware.org/wiki/Silver:_Oligonucleotide_Inserts[1]]</FONT></SUP></u> <br />
*Mix: <br />
**3 μL 100 µM (anti-)sense oligo <br />
**1 μL 10 x PNK (polynucleotide kinase) buffer ([http://www.fermentas.com/catalog/modifyingenzymes/t4polynucleotidekinase.htm Fermentas Buffer A])<sup>**</sup><br />
**2 μL 10mM ATP <sup>**</sup><br />
**1 μL [http://www.fermentas.com/catalog/modifyingenzymes/t4polynucleotidekinase.htm T4 polynucleotide kinase (PNK)] <br />
**3 μL MilliQ<br />
***(for selfcloser control, do not add oligo's. Instead 6 μL MilliQ in total)<br />
*Incubate @ 37 °C for 1.5 hours. <br />
*Mix <br />
**10 μL Sense mixture<br />
**10 μL Anti-sense mixture<br />
**3 μL 0.5 M NaCl <br />
*Place in boiling water for 3 min., and allow the reaction to cool to room temperature.<br />
**Upon reaching room temperature add restricted vector (see for ratio [[Team:Groningen/Protocols#Ligation|Ligation]]<br />
**If kept at low temperature before ligation heat up the annealing mixture up to 65 °C for 1 min. to prevent the formation of multimers<br />
<sup>**</sup>Alternatively T4 DNA Ligase buffer can be used, already containing ATP<br />
===[http://openwetware.org/wiki/DNA_ligation Ligation]===<br />
*Mix<sup>*</sup><br />
**1 μL [http://www.fermentas.com/catalog/modifyingenzymes/t4dnaligase.htm T4 ligase buffer]<br />
**7.5 μL vector (purified from gel)<br />
**1 μL Insert<br />
**0.5 μL [http://www.fermentas.com/catalog/modifyingenzymes/t4dnaligase.htm T4 ligase]<br />
*Incubate<br />
:* 1h RT<br />
:or<br />
:* ON @ 4 °C<br />
<sup>*</sup>This is a consensus, calculations should be performed to have the ligations be done in a 5:1 - 10:1 (Insert:Vector) mass ratio. <br />
<br />
===Making competent cells===<br />
Competent cells: [http://openwetware.org/wiki/TOP10_chemically_competent_cells TOP10] & [http://openwetware.org/wiki/E._coli_genotypes#DB3.1 DB3.1]<br />
*10 mL ON culture is used to inoculate [[Team:Groningen/Protocols#LB.28Agar.29|LB]], 100 μL ON culture per 20 mL<sup>*</sup><br />
*Cultures are grown @ 37 °C until an OD<sub>600</sub> of 0.2 ~ 0.3 is reached.<br />
*Cultures are spinned down 5 min. @ 4000 rpm, 4 °C<br />
*Supernatant is removed and pellet (per 20 mL culture) is resuspended in 5 mL chilled 0.1 M [[Team:Groningen/Protocols#0.1_M_CaCl2|CaCl<sub>2</sub>]]<br />
**Suspension is incubated on ice for 10 min. <br />
*Suspensions are spinned down 5 min. @ 4000 rpm, 4 °C<br />
# Supernatant is removed and pellet is resuspended in 1770 μL chilled 0.1 M [[Team:Groningen/Protocols#0.1_M_CaCl2|CaCl<sub>2</sub>]] and supplemented with 230 μL 87% glycerol prior to making aliquots.<br />
# Cells are divided in 50 μL aliquots<br />
# Cells are snapfrozen in liquid nitrogen and stored @ -80 °C<br />
<sup>*</sup> Cultures should be grown in the ratio 1:5 (medium:air), so 10 mL culture in a 50 mL greiner tube.<br />
<br />
===Transformation===<br />
*Add 10 uL of ligation mixture or 1 uL isolated plasmid to competent cell aliquot<br />
** <b>+ control</b>: 1 μL <partinfo>pSB3K3</partinfo> or <partinfo>pSB1AC3</partinfo> plasmid (no death gene!), <b>- control</b>: 1 μL MilliQ<sup>*</sup><br />
** Alternatively a single cut plasmid can be taken as a ligation control<br />
<sup>*</sup>Alternative - control: 1 μL [http://partsregistry.org/Part:pSB1AC3 pSB1AC3] or [http://partsregistry.org/Part:pSB3K3 pSB3K3] carrying ccdB deathgene<br />
*Incubate on ice for 15 - 30 min.<br />
*Heatshock 45 sec. @ 42 °C or 5 min. 37 °C<br />
*Let cells relax on ice for 1 - 2 min.<br />
*Add [[Team:Groningen/Protocols#LB.28Agar.29|LB]] 200 μL (or 800 μL when spinning cells down, see below)<br />
*Incubate 37 °C, 250 RPM for 1 h<br />
*Plate out on [[Team:Groningen/Protocols#LB.28Agar.29|LB-agar]] + [[Team:Groningen/Protocols#Kanamycin|Kanamycin]] (30 μg/ml for [http://partsregistry.org/Part:pSB3K3 pSB3K3]) or [[Team:Groningen/Protocols#Ampicillin|Ampicillin]] (100 μg/mL for [http://partsregistry.org/Part:pSB1AC3 pSB1AC3])<br />
**Plate out 50 μL & 200 μL (or 100 μL after spinning down and resuspending cells) of cell suspension<br />
*Grow ON @ 37 °C<br />
<br><u>Checking transformations</u><br />
*See if - control is empty for functioning antibiotics and death gene<br />
*See how many colonies on + control for functioning competent cells<br />
*See how many selfclosers and compare to samples (>10x on sample vs. selfcloser)<br />
*If enough transformants, inoculate 3 - 5 colonies in an ON culture<br />
**Alternatively perform colony PCR<br />
<br />
==Quality control==<br />
===Colony PCR===<br />
*Put colony in 1 μL MilliQ water<br />
*Put colony suspension in microwave for 1 min. 1000 W<br />
*Use this as DNA template<br />
*PCR reaction<br />
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{|cellpadding="2" cellspacing="2" border="0"<br />
|21 μL Taq mastermix<sup>*</sup><BR><br />
1 μL forward primer<BR><br />
1 μL reverse primer<BR><br />
1 μL template<BR><br />
1 μL Taq polymerase<BR><BR><br />
<sup>*</sup>Taq master mix contains:<BR><br />
100 μL Taq NH<sub>4</sub><BR><br />
8 μL dNTP's<BR><br />
80 μL MgCl<BR><br />
652 μL MilliQ water<br />
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|<u><b>PCR Reaction<sup>**</sup></b><BR><br />
</u>Hotstart<BR><br />
95 °C, 2 min.<BR><br />
25 cycles<BR><br />
95 °C, 30 sec.<BR><br />
61 °C, 20 sec.<BR><br />
72 °C, 1.5 min.<BR><br />
End<BR>72 °C, 10 min.<br />
<BR>4 °C, ∞<br />
|}<br />
</center><br />
*Put PCR product on agarose gel<br />
<sup>**</sup> Indication, actual reaction program depends on primer set (Temperature of annealing) and the length of the template (Duration of elongation)<br />
<br />
===Restriction analysis===<br />
See also [[Team:Groningen/Protocols#Restriction| Restriction]], however in checking the presence of a multitude of bricks more diverse enzymes can be used. Also the incubation time can be shortened (Pour an agarose gel, wait for it to solidify and put reaction on gel) because it is not required that everything is cut.<br />
<br />
===Membrane protein isolation===<br />
*Use 20 mL of ON culture to start main culture in 1L [[Team:Groningen/Protocols#LB.28Agar.29|LB]] medium containing 50 μg/mL [[Team:Groningen/Protocols#Ampicillin|ampicillin]]<br />
**Incubate 37 °C, 250 RPM until OD<sub>600</sub> = 0.6, (approximately 2.5h of incubation, check the OD<sub>600</sub> every hour)<br />
**Add inducer (e.g. [Team:Groningen/Protocols#IPTG|IPTG]])<br />
**Incubate 1 h, 37 °C, 250 RPM<br />
*Culture Wash.<br />
**Cool culture on ice<br />
**Spin down culture @ 8000 rpm, 10 min, 4°C<br />
**Wash pellet with 40 ml ice-cold 50 mM KPi, pH 7.0<br />
**Spin down culture @ 8000 rpm, 10 min, 4°C<br />
**Resuspend pellet in 12 ml 50 mM KPi pH 7.0<br />
*Homogenization<br />
**Sonication on ice<br />
**9 cycles of 15 sec. sonication, 45 sec. rest<br />
*Separation fractions<br />
**Spin down @ 8000 rpm, 10 min, 4 °C<br />
**Collect supernatant <br />
**Spin down at 90 000 rpm, 25 min, 4 °C<br />
**Resuspend pellet in 1 ml of 50 mM KPi pH 7.0 + 1M NaCl<br />
**Spin down @ 80 000 rpm, 25 min, 4 °C<br />
*Solubilization<br />
**Resuspend in 950 μl of solubilization buffer (50 mM KPi pH 8.0 + 400 mM NaCl + 20% glycerol) and 50 μl of 10% DDM <br />
**Incubate in 4°C with shaking for 30 min.<br />
**Spin down @ 80 000 rpm, 25 min in 4°C<br />
**Collect the supernatant<br />
**(Supernatant can be stored at this stage)<br />
**Add Ni-NTA resin (30 μL [[Team:Groningen/Protocols#Buffer_A|buffer A]], 0.1% DDM (Bis(4-chlorophenyl)methane))<br />
**Incubate ON @ 4 °C<br />
**Spin down, 4 min. 3500 RPM<br />
**Remove supernatant<br />
**was resin with 1 mL [[Team:Groningen/Protocols#Buffer_B|buffer B]], 0.1% DDM<br />
**Spin down, 4 min. 3500 RPM<br />
**Remove supernatant<br />
**Elute protein by [[Team:Groningen/Protocols#Buffer_C|buffer C]], 0.1% DDM (50 μL)<br />
**Add protein loading buffer (with dithiothreitol (DTT))<br />
**Run 12% SDS-PAGE<br />
**Continue to Coomassie staining<br />
<br />
Staining of SDS-PAGE gels with Coomassie Brilliant Blue<br />
*Heat gel in staining solution and shake for 10 min.<br />
*Poor off staining solution and add destain.<br />
*Heat gel in destaining solution and shake.<br />
*Replace destaining solution after 10 min and repeat until ready.<br />
<br />
==Measurements==<br />
===Fermentation===<br />
Performed in 2L autoclavable fermentor with dished bottom vessel stirred fermentor<br />
*Autoclave closed fermentor system <br />
*Inoculate 1.3 L [[Team:Groningen/Protocols#LB.28Agar.29|LB]] (+100 µL [http://www.sigmaaldrich.com/catalog/ProductDetail.do?N4=A6457|SIGMA&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC&lang=en_US%3E Y30 antifoam]) with 20 mL ON culture was used to <br />
**Airflow rate of 1 vvm <br />
**pH @ 7 (by addition of 4 M NaOH or 1 M HCl) <br />
**Temperature @ 37 °C<br />
**Agitation 400 to 800 RPM<sup>*</sup> <br />
**Oxygen concentration >50%<br />
*Take samples every 0.5 to 1 h. to determine optical density at 600 nm <br />
*50 mL samples in every growth phase (pre-exponential, early exponential, exponential, late exponential, steady state) <br />
*Spin samples down 35 min., 1000 RPM and remove supernatant<br />
*Continue [[Team:Groningen/Protocols#Buoyancy_test|Buoyancy test]]<br />
<br />
<sup>*</sup>6-bladed flat disc turbine (Rushton type) impeller (60 mm diameter) at the bottom to disperse the bubbles coming from the sparger underneath and a 3-bladed marine impeller, vortex (60 mm diameter) halfway the broth volume to create an axial flow.<br />
===Buoyancy test===<br />
Continued from cultures (flask or fermentor) after centrifugation<br />
*Resuspend pellet in 1 - 5 mL [[Team:Groningen/Protocols#Saline_solution_.280.15_M.2C_0.9.25_NaCl.29|saline solution]]<br />
*Determine OD<sub>600</sub> <br />
*Dilute suspension to OD<sub>600</sub> 1.5 with [[Team:Groningen/Protocols#Saline_solution_.280.15_M.2C_0.9.25_NaCl.29|saline solution]]<br />
*Put homogeneous suspension in tubes, take care of descent lighting from behind (day light is best)<br />
*Record decrease of buoyancy (matter of hours in fermentation cultures, days in shakeflask cultures)<br />
===Metal uptake assay for <i>E. coli</i><sup><FONT SIZE=-5>[[Team:Groningen/Literature#Kostal2004|Kostal 2004]]</FONT></sup>===<br />
*Grow ON culture of <i>E. coli</i> @ 30 °C <br />
**Use <i>E. coli</i> + control vector, <i>E. coli</i> + [http://partsregistry.org/wiki/index.php?title=Part:BBa_K190023 pArsR]-[http://partsregistry.org/Part:BBa_E1010 RFP], <i>E. coli</i> + [http://partsregistry.org/wiki/index.php/Part:BBa_K190032 pLac-fMT]<br />
*Inoculate day culture 1:50, grow in 1L [[Team:Groningen/Protocols#TB_medium|TB]]-[[Team:Groningen/Protocols#Ampicillin|Amp]] (100ml per time/[As(III)] sample)<br />
**Take OD<sub>600</sub> samples every 1 - 1.5 h of <i>E. coli</i> + [http://partsregistry.org/wiki/index.php/Part:BBa_K190032 pLac-fMT]<br />
**Induce <i>E. coli</i> + [http://partsregistry.org/wiki/index.php/Part:BBa_K190032 pLac-fMT] at OD<sub>600</sub> ~0.6 with 0.5 mM IPTG.<br />
*Harvest the cells @ stationary phase (after ~30 h) by spinning down @ 4000 RPM for 20 min. in Sorval centrifuge. <br />
*Wash 2 times with [[Team:Groningen/Protocols#TB74S_Buffer|TB74S buffer]]<br />
*Resuspend in prewarmed (30 °C) [[Team:Groningen/Protocols#TB74S_Buffer|TB74S buffer]] up to a OD<sub>600</sub> of ~25<br />
**Take a 1 mL sample in small aluminum boxes and dry @ 104 °C for >4 h<br />
**Afterwards measure the dry weight of the sample and calculate the weight/volume of the entire sample.<br />
*For the concentration range:<br />
**Incubate 5 samples (of same time point) for 1h @ 30 °C with 0μM, 10 μM, 20 μM, 50 μM and 100 μM As(III).<br />
*For the concentration range:<br />
**Incubate 5 samples (of same concentration) @ 30 °C with 10 or 100μM As(III) for 0, 10, 20, 40, 60 min.<br />
*Harvest cells by spinning down.<br />
*Wash the cells with [[Team:Groningen/Protocols#TB74S_Buffer|TB74S buffer]]<br />
*Resuspend in 10ml demi water.<br />
*Dry sample @ 65 °C for 2 days.<br />
*Store @ 4 °C or -80 °C<br />
*Determine the amount of As(III) in the cell at different stages and at different uptake concentrations using [http://en.wikipedia.org/wiki/Inductively_coupled_plasma_mass_spectrometry ICP-MS]<br />
<br />
====Analysis of arsenic concentration of ICP-MS====<br />
<br />
*Weigh 0.1g dried <i>E. coli</i> cells.<br />
*Add 5 ml 65% nitric acid.<br />
*For destruction the following microwave program was used:<br />
<BR><center><br />
{|cellpadding="1" cellspacing="1" border="1"<br />
|''' '''<br />
|'''Stage 1'''<br />
|'''Stage 2''' <br />
|-<br />
|Power(max) <br />
|1200 <br />
|1200<br />
|-<br />
|Power(%)<br />
|100 <br />
|100<br />
|-<br />
|Ramp(min) <br />
|15 <br />
|15<br />
|-<br />
|Hold(min) <br />
|0<br />
|30<br />
|-<br />
|Temp(°C) <br />
|140 <br />
|210<br />
|}</center><br />
*Let the samples cool down.<br />
*Dilute the samples by adding demi water up to 50 mL <br />
*If needed, spin down 15 min. @ 4000rpm in a Sorvall centrifuge.<br />
*Measure the arsenic concentration by [http://en.wikipedia.org/wiki/Inductively_coupled_plasma_mass_spectrometry ICP-MS] using both the standard mode (shows interference peak from multi-atomic molecule argon-chloride with the arsenic peak) and the collusion cell technology mode (doesn’t show the interference peak but has a 10x lower resolution than standard mode). <br />
**Use a standard curve between 0 - 10 µg As/L and 0 - 100 µg As/L using a certified 1000 ppm (mg/L) stock<br />
<br />
===Fluorescence measurement===<br />
*Dilute ON culture 1:20 in [[Team:Groningen/Protocols#LB.28Agar.29|LB]]+[[Team:Groningen/Protocols#Ampicillin|Ampicillin]] in 50 mL greiner tube<br />
*Incubate 37 °C, 250 RPM until an OD<sub>600</sub> ~0.5<br />
*Spin down 10 min. 4000 RPM, 4 °C<br />
*Resuspend pellet in [[Team:Groningen/Protocols#LB.28Agar.29|LB]]+[[Team:Groningen/Protocols#Ampicillin|Ampicillin]] (half the volume used before)<br />
*Incubate @ 4 °C, 30 min.<br />
*Load samples onto a 96-wells plate, 250 μL<br />
*Induce by 1.25 μL of the following stock solutions<br />
<br />
{|border="1" <br />
!Metal<br />
!<!--Space--><br />
!<!--Space--><br />
!<!--Space--><br />
!<!--Space--><br />
!<!--Space--><br />
|-<br />
!CuSO<sub>4</sub><br />
|1 M<br />
|100 mM<br />
|10 mM<br />
|1 mM<br />
|0 mM<br />
|-<br />
!ZnSO<sub>4</sub><br />
|1 M<br />
|100 mM<br />
|10 mM<br />
|1 mM<br />
|0 mM<br />
|-<br />
!NaAsO<sub>2<sub><br />
|1 M<br />
|100 mM<br />
|10 mM<br />
|1 mM<br />
|0 mM<br />
|-<br />
!Final con.<br />
!5000 μM<br />
!500 μM<br />
!50 μM<br />
!5 μM<br />
!0 μM<br />
|}<br />
<br />
*Measure the fluorescence and OD<sub>600</sub> every hour.<br />
**RFP is excited @ 580 nm and emission is measured at 609 nm.<br />
*Store the plates between the measurements in a shaking incubator @ 37 °C.<br />
===Fluorescence of resting cells with <partinfo>J61002</partinfo>-[http://partsregistry.org/wiki/index.php/Part:BBa_K190015 pArsR]===<br />
<br />
This protocol was used to be able to correlate the determined fluorescence values with the arsenic concentrations measured by ICP-MS (after an arsenic uptake assay).<br />
<br />
*Cells were used as prepared for the (<partinfo>Bba_K190015</partinfo> ) [[Team:Groningen/Protocols|arsenic uptake assay]]. <br />
*Cultures with an OD<sub>600</sub> of ~25 were induced with 100 µM NaAsO<sub>2</sub> for 60 to 160 min. <br />
*The fluorescence and OD<sub>600</sub> were measured by a plate reader (Tecan, infinite 200, Tecan group, Switzerland). <br />
*Relative promoter units were calculated according to formula 9 from [[Team:Groningen/Literature#Kelly2009|Kelly 2009]].<br />
<br />
===Death assay===<br />
Metal sensitivity assay<sup><FONT Style=-5>[[Team:Groningen/Literature#Lewinson2009|Lewinson 2009]]</FONT></sup> <br />
<br />
Measurement:<br />
<br />
*Grow selected strains ON in [[Team:Groningen/Protocols#LB.28Agar.29|LB medium]] with or without antibiotic<br />
*Induce in culture with inducer (in our case 0.5 mM [[Team:Groningen/Protocols#IPTG|IPTG]])<br />
<br />
Strains used in our tests<br />
{|border="1" <br />
!Test 1:<br />
!Test 2:<br />
|-<br />
|WT (+pSB1AC3) <br />
|WT (+pSB1AC3)<br />
|-<br />
|pLac-HmtA <br />
|pLac-HmtA<br />
|-<br />
|pLac-GlpF <br />
|pLac-GlpF<br />
|-<br />
|pLac-GlpF-fMT <br />
|pLac-GlpF-fMT<br />
|-<br />
|<br />
|plow-GlpF-fMT<br />
|-<br />
|<br />
|pLac-GlpF<br />
|-<br />
|}<br />
<br />
*Measure OD<sub>600</sub> of ON culture and dilute to an OD<sub>600</sub> of 0.05 in [[Team:Groningen/Protocols#LB.28Agar.29|LB]]+[[Team:Groningen/Protocols#Antibiotics|antibiotic]] & inducer ([[Team:Groningen/Protocols#IPTG|IPTG]] in our case). Inducer should be right concentration for use in microtiterplate (because you then dilute culture 150/200=1.33 times)<br />
*Add 150 ul of culture to 96 well microtiter plate (in triplo/quadruplo)<br />
*Add desired concentration of selected metal in 50 μL [[Team:Groningen/Protocols#LB.28Agar.29|LB]]+[[Team:Groningen/Protocols#Antibiotics|antibiotic]]<br />
<br />
Metals used in our test<br />
{|border="1" <br />
!Metal Concentration<br />
|-<br />
|NaAsO<sub>2</sub><br />
|0 μM <br />
|1 μM <br />
|10 μM <br />
|50 μM<br />
|-<br />
|CuSO<sub>4</sub> <br />
|0 μM <br />
|50 μM <br />
|250 μM <br />
|500 μM<br />
|}<br />
<br />
*Measure in Tecan Infinite 200 microplate reader (Tecan Group Ltd., Männedorf, Switzerland) or Tecan microplate *reader. Protocol:<br />
**Measure OD at 600 nm,<br />
**Every 15 minutes for 16-20hrs<br />
**Linear shaking, 6mm<br />
**37°C<br />
<br />
<br />
Analysis:<br />
<br />
*Plot for different strains OD<sub>600</sub> against time<br />
*Plot for different strains, the different metal concentrations against OD<sub>600</sub> at 12 hours<br />
<br />
=List of solutions=<br />
==Media==<br />
===LB(Agar)===<br />
*10 g (Bacto)Trypton<br />
*10 g NaCl<br />
*5 g Yeast extract<br />
*(1.5% Agar, 15 g)<br />
*Dissolve in 1 L demi water<br />
*Autoclave<br />
*Store @ RT (LB) or 60 °C (LB-Agar)<br />
<br />
===TB medium===<br />
* 12 g Bacto-Tryptone<br />
* 24 g Bacto-Yeast Extract<br />
* 4 mL Glycerol [87%]<br />
* Dissolve in 900ml demi water<br />
*Separetely prepare 100 mL Kpi <br />
**0.17 M KH2PO4 (mw=136.09g/mol) (6.94g/300ml)<br />
**0.72 M K2HPO4 (mw=174.18g/mol) (7.62g/300ml)<br />
**dissolve in demi water<br />
*Autoclave and mix<br />
<br />
==Antibiotics==<br />
===[http://openwetware.org/wiki/Ampicillin Ampicillin]===<br />
100 mg/ml Ampicillin (1000x) Stock<br />
* 1 g of Ampicillin sodium salt in 10 mL of demiwater (or 50% EtOH)<br />
* Add NaOH or KOH to allow the Ampicillin to dissolve<br />
* Filter sterilize 0.2 μm filter and aliquot<br />
* Store -20 °C<br />
===[http://openwetware.org/wiki/Chloramphenicol Chloramphenicol]===<br />
35 mg/ml Chloramphenicol (1000x) Stock<br />
* 0.35 g in 10 mL 100% EtOH<br />
* Filter sterilize 0.2 μm filter and aliquot<br />
* Store -20 °C<br />
===[http://openwetware.org/wiki/Kanamycin Kanamycin]===<br />
50 mg/ml Kanamycin (1000x) Stock<br />
* 500 mg in 10 mL demi water<br />
* Filter sterilize 0.2 μm filter and aliquot<br />
* Store -20 °C<br />
<br />
==Chemicals==<br />
===Buffer A===<br />
*10 mM Imidazole <br />
*600 mM NaCl<br />
*50 mM KPi pH 8.0<br />
*10% Glycerol<br />
*0.1% DDM<br />
*Demi water<br />
===Buffer B===<br />
*20 mM Imidazole <br />
*600 mM NaCl<br />
*50 mM KPi pH 8.0<br />
*10% Glycerol<br />
*0.1% DDM<br />
*Demi water<br />
===Buffer C===<br />
*500 mM Imidazole <br />
*600 mM NaCl<br />
*50 mM KPi pH 8.0<br />
*10% Glycerol<br />
*0.1% DDM<br />
*Demi water<br />
===0.1 M CaCl<sub>2</sub>=== <br />
*0.3319 g CaCl<sub>2</sub><br />
*Dissolve in 30 mL demi water<br />
===Destaining solution===<br />
*16 % methanol<br />
*10 % acetic acid<br />
*74 % water <br />
===0.15 M NaCl (Saline solution, 0.9% NaCl)===<br />
*9 g NaCl<br />
*Dissolve in 1 L demi water<br />
===4 M NaOH===<br />
*160 g NaOH<br />
*Dissolve in 1 L demi water<br />
===~1 M HCl===<br />
*500 mL demi water<br />
*500 mL HCl (37%, 11 M)<br />
===[http://openwetware.org/wiki/IPTG 1 M IPTG]===<br />
*2.38 g Isopropyl-beta-D-thiogalactopyranoside (IPTG) in 10 mL demi water.<br />
*Filter sterilize with a 0.22 μm syringe filter.<br />
*Store in 1 mL aliquots at -20 °C.<br />
<br />
===Sodium Arsenite (III)===<br />
*100mM Na-As solution <br />
*filter sterilize<br />
===Staining solution===<br />
*0.25 % Coomassie Brilliant Blue R-250<br />
*50 % methanol<br />
*10 % acetic acid<br />
*40 % water<br />
===TB74S Buffer===<br />
*0.605 g Tris (5mM)<br />
*8.76 g NaCl (150mM)<br />
*Dissolve in 1 L Demi water<br />
*Set pH with HCl to 7.4<br />
===[http://openwetware.org/wiki/TBE 10x TBE buffer]===<br />
*108 g Tris<br />
*55 g Boric acid<br />
*8.3 g EDTA<br />
*Dissolve in 1 L demi water<br />
*Adjust pH to 8.3<br />
<br />
<br />
<br />
{{Team:Groningen/Footer}}</div>JolandaWitteveenhttp://2009.igem.org/Team:Groningen/methodischontwerpenTeam:Groningen/methodischontwerpen2009-10-21T19:18:08Z<p>JolandaWitteveen: /* Design */</p>
<hr />
<div>{{Team:Groningen/Project_Plan/Header}}<br />
<br />
=Design=<br />
A methodical design template was used as a guideline to create a project. The [http://http://doc.utwente.nl/50799/1/rede_Verkerke.pdf design method] used was developed by [http://www.rug.nl/staff/g.j.verkerke/index prof. dr. ir G.J. Vekerke] and [http://www.rug.nl/staff/e.b.van.der.houwen/index ir. E.B.van der Houwen] at the University of Groningen specifically for designing biomedical applications and was taugh to one of our team members. It servered as a good starting point and was a good tool to analyse the problem and designing the project. The list of demands and whishes was very usefull in selection of the best brainstorm ideas.<br />
<br />
{{Team:Groningen/Project_Plan/Footer}}</div>JolandaWitteveenhttp://2009.igem.org/Team:Groningen/methodischontwerpenTeam:Groningen/methodischontwerpen2009-10-21T19:14:50Z<p>JolandaWitteveen: New page: {{Team:Groningen/Project_Plan/Header}} =Design= A methodical design template was used as a guideline to create a project. The [[http://http://doc.utwente.nl/50799/1/rede_Verkerke.pdf desi...</p>
<hr />
<div>{{Team:Groningen/Project_Plan/Header}}<br />
<br />
=Design=<br />
A methodical design template was used as a guideline to create a project. The [[http://http://doc.utwente.nl/50799/1/rede_Verkerke.pdf design method]] used was developed by prof. dr. ir B. Vekerke and dr. W van der Houwen at the University of Groningen specifically for designing biomedical applications and was taugh to one of our team members. It servered as a good starting point and was a good tool to analyse the problem and designing the project. The list of demands and whishes was very usefull in selection of the best brainstorm ideas.<br />
<br />
{{Team:Groningen/Project_Plan/Footer}}</div>JolandaWitteveenhttp://2009.igem.org/Team:Groningen/Project_Plan/Tools_and_DocumentationTeam:Groningen/Project Plan/Tools and Documentation2009-10-21T18:45:05Z<p>JolandaWitteveen: /* Human Practice */</p>
<hr />
<div>{{Team:Groningen/Project_Plan/Header}}<br />
[[Category:Team:Groningen/Disciplines/Configuration_and_Change_Management|Tools and Documentation]]<br />
[[Category:Team:Groningen/Roles/Configuration_Manager|Tools and Documentation]]<br />
[[Category:Team:Groningen/Roles/Facility_Manager|Tools and Documentation]]<br />
<br />
[[Team:Groningen|Our project]] produces [[Team:Groningen/Project_Plan#Deliverables|many artifacts]], this [http://www.upedu.org/upedu/process/artifact/ar_cmpln.htm Configuration Management Plan] (called Tools and Documentation to avoid a lot of jargon) details what tools and processes we use to create these artifacts. Both for dry work and wet work. Do we use Matlab or Mathematica? What programming conventions do we use? Which lab space do we use? When do we use it?<br />
<br />
Basically anyone may change anything at any time for any reason, and the Configuration Manager keeps an eye on how (and how much) the Wiki and the SVN repository are used. He also writes this document (assisted by the Facility Manager(s) and others for lab-specific parts).<br />
<br />
<html><style type="text/css"><br />
.wetwork { color: green; }<br />
</style></html><br />
<br />
==Tools, Environment, and Infrastructure==<br />
===Modelling===<br />
<blockquote><br />
Describe the computing environment and software tools to be used in fulfilling the CM functions throughout the project or product lifecycle.<br />
<br />
Describe the tools and procedures required used to version control configuration items generated throughout the project or product lifecycle.<br />
<br />
Issues involved in setting up the CM environment include:<br />
<br />
*anticipated size of product data<br />
*distribution of the product team<br />
*physical location of servers and client machines<br />
</blockquote><br />
<br />
Most documents are created on the Wiki, which has built-in version control features (a revision history, including comments). For Google Docs the same is true. Our network drive does NOT have version control and should not be used for documents that have to be modified (much). For storing/exchanging Matlab code we will use [http://subversion.tigris.org/ Subversion] in combination with [http://code.google.com/ Google Code]. All team members wishing to alter models/write matlab code should follow the following steps to get set up (focusses on Windows):<br />
<br />
# Download and install Matlab R2009a. (Login and click on My Account, both in the upper-right corner, then click on "My Products / Get Licensed Products and Updates".)<br />
# Download and install TortoiseSVN: http://tortoisesvn.net/downloads<br />
# Checkout the URL https://igemgroningen.googlecode.com/svn/trunk/ using TortoiseSVN by right-clicking in your iGEM directory (in Windows Explorer) and choosing SVN Checkout. Note that you will have to supply the password that can be found on http://code.google.com/hosting/settings.<br />
<br />
Whenever you want to make changes to some of the files in the repository, first update all files by choosing SVN Update from the right-click menu in Windows Explorer (this will download any new changes from others), then make your changes, and finish by committing (again through the right-click menu). Do not forget to supply a commit message explaining the changes.<br />
<br />
Also, in case of conflicts in a Simbiology project you are in trouble (this happens when two people update/change/commit a file at the same time). Unfortunately Mathworks chose to save their projects as one big compressed file, which makes it extremely hard to compare two versions of a project, let alone resolve conflicts. Using [http://winmerge.org/ WinMerge] (with the 7-Zip plugin) these project files can be compared, but you still can't resolve conflicts. So, please stick to one model per project and try to avoid conflicts. If a conflict does occur, contact Jasper for help in resolving it.<br />
<br />
A list of programs that support SBML and there respective capabilities are here [http://sbml.org/SBML_Software_Guide]./SBML_Software_Matrix <br />
<br />
Finally, team members that do '''NOT''' write code can checkout the models using TortoiseSVN in the same way as described above and file issues in the [http://code.google.com/p/igemgroningen/issues/list issue list].<br />
<br />
===Wet Work===<br />
*Tools: <br />
**Ligation ratio: Cloning Tool 6.0 (ask Frans/Sven)<br />
**List of used lab tools (and from whom we got them) can be found on GoogleDocs (in Dutch): Lab benodigdheden<br />
*Environment:<br />
**Lab room D113 in Haren<br />
*Infrastructure:<br />
**Archives: Binders for primer sheets, Articles on the subprojects<br />
**Databases used are [http://openwetware.org/wiki/Main_Page OpenWetWare], [http://www.partsregistry.org/Main_Page Part], [http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2009&group=Groningen Sandbox],[http://www.ncbi.nlm.nih.gov/pubmed/ Pubmed]<br />
<br />
===Human Practice===<br />
A survey was made and executed. The consideration and results can be found [https://2009.igem.org/Team:Groningen/Ethics/Survey here]<br />
<br />
==Documentation==<br />
====Identification Methods====<br />
Private files are stored in a central repository (currently the Y-drive or Google Docs). Agendas and minutes are stored in their respective folders under the date of the meeting in yyyy-mm-dd format to enable easy sorting. Other documents should be named according to the UPEDU standard as much as possible and prefixed by a short abbreviation in brackets (which makes it easy to reference them), for example [PP] for this document.<br />
<br />
All documents that are not necessarily private should be placed on the Wiki under the name of the [http://www.upedu.org/upedu/process/artifact/ovu_arts.htm relevant artifact] (also see the [http://www.upedu.org/upedu/process/artifact/tmpl_cs/ovu_tmplcs.htm list of templates]), always prefixed by Team:Groningen (for example [[Team:Groningen/Vision]]). Images can also be uploaded to the Wiki to accompany texts, but the Wiki should not be used as a general means of exchanging files (for this the Y-drive, Google Docs or Google Groups are more suitable).<br />
<br />
Finally note that any changes to documents on the Wiki should make use of the Summary field below the large edit text box to describe the change (it would be nice if you tick the "This is a minor edit" checkbox if you're making small tweaks, like correcting a spelling mistake). These descriptions can be long, or as short as one word, depending on the situation. Afterwards it should be possible to reasonably easily get an overview of how the document evolved, and to look up specific changes. For documents not on the Wiki a Revision History should be present at the top of the document.<br />
<br />
===={{anchor|DirectoryStructure}} Master of Lists====<br />
{{done}}: Anything about lab stuff (in green!)<br />
<br />
*Our wiki<br />
**[[:Category:Team:Groningen]] contains all our categories (corresponding to some of the UPEDU disciplines).<br />
***[[:Category:Team:Groningen/Disciplines]] contains all our disciplines.<br />
***[[:Category:Team:Groningen/Roles]] contains all our roles.<br />
**[[Team:Groningen]] is our main page, http://www.igemgroningen.com/ redirects to this.<br />
***All our documents are on the Wiki, where possible. Each document is a subpage of the main page, unless it is itself part of another document, then it is a subpage of that document. For example: [[Team:Groningen/Project_Plan]] and [[Team:Groningen/Project_Plan/Risk_List]].<br />
***Note that each document should be preceded by a list of related categories (see this document for example).<br />
**User:... (see, for example, [[User:Verhoeven1981|Michael's user page]])<br />
***Some basic information<br />
***Optionally a photograph<br />
***Categories for each of the roles he/she has.<br />
**Any JavaScript on the Wiki should (as much as possible/desirable) be written in a separate page and included using a script tag with a href attribute referencing the raw version of the page (see [[:Template:Team:Groningen/Header]] for an example), this avoids problems with ampersands, keeps the pages a bit cleaner and makes it possible for browsers to cache long scripts. When it makes sense the page should be a subpage of the page it is used in.<br />
**[https://2009.igem.org/Team:Groningen/Parts Parts list]: <span class="wetwork">List of glycerol stocks in -80 freezer C from MolGen, also the Quality control by PCR and Restriction analysis should be filled in here.</span><br />
<br />
*Subversion repository<br />
**The trunk is at https://igemgroningen.googlecode.com/svn/trunk/<br />
**[https://igemgroningen.googlecode.com/svn/trunk/glucose/ /trunk/glucose/]<br />
***The models for the glucose sensing idea (which we're not pursuing any further).<br />
**[https://igemgroningen.googlecode.com/svn/trunk/buoyant/ /trunk/buoyant/]<br />
***[[Team:Groningen/Modelling|Models]] (mostly .sbproj files) for the [[Team:Groningen/Vision|buoyant bacteria]].<br />
<br />
*Google Docs<br />
**Unfortunately Google Docs does not allow sharing of a directory structure.<br />
**Contact Information for 3rd parties.<br />
**Minutes and agendas.<br />
**Availability of team members.<br />
**Balance sheet: estimated costs for tickets, hotel, lab etc and estimated sponsoring.<br />
**<span class="wetwork">Primers: list with ordering date and concentration of the primers when they did arrive (also for oligo's).<br />
**<span class="wetwork">Chemicals / Materials taken from MolGen: list with all chemicals, materials, enzymes taken from MolGen<br />
**<span class="wetwork">Fridge/Freezer Stock list: list with different sheets for general stocks in the fridge and freeze, additionally a sheet for -20 freezer stocks of all labworkers.<br />
**<span class="wetwork">Lab tools: list of all the stuff (machines, pipettes, etc) we use from microbiology and practicum pool (Labbenodigdheden).<br />
*Y-drive<br />
**The transfer documents.<br />
**Literature?<br />
**Miscellaneous files<br />
{{Team:Groningen/Project_Plan/Footer}}</div>JolandaWitteveenhttp://2009.igem.org/Team:Groningen/Project_Plan/Tools_and_DocumentationTeam:Groningen/Project Plan/Tools and Documentation2009-10-21T18:43:32Z<p>JolandaWitteveen: /* Wet Work */</p>
<hr />
<div>{{Team:Groningen/Project_Plan/Header}}<br />
[[Category:Team:Groningen/Disciplines/Configuration_and_Change_Management|Tools and Documentation]]<br />
[[Category:Team:Groningen/Roles/Configuration_Manager|Tools and Documentation]]<br />
[[Category:Team:Groningen/Roles/Facility_Manager|Tools and Documentation]]<br />
<br />
[[Team:Groningen|Our project]] produces [[Team:Groningen/Project_Plan#Deliverables|many artifacts]], this [http://www.upedu.org/upedu/process/artifact/ar_cmpln.htm Configuration Management Plan] (called Tools and Documentation to avoid a lot of jargon) details what tools and processes we use to create these artifacts. Both for dry work and wet work. Do we use Matlab or Mathematica? What programming conventions do we use? Which lab space do we use? When do we use it?<br />
<br />
Basically anyone may change anything at any time for any reason, and the Configuration Manager keeps an eye on how (and how much) the Wiki and the SVN repository are used. He also writes this document (assisted by the Facility Manager(s) and others for lab-specific parts).<br />
<br />
<html><style type="text/css"><br />
.wetwork { color: green; }<br />
</style></html><br />
<br />
==Tools, Environment, and Infrastructure==<br />
===Modelling===<br />
<blockquote><br />
Describe the computing environment and software tools to be used in fulfilling the CM functions throughout the project or product lifecycle.<br />
<br />
Describe the tools and procedures required used to version control configuration items generated throughout the project or product lifecycle.<br />
<br />
Issues involved in setting up the CM environment include:<br />
<br />
*anticipated size of product data<br />
*distribution of the product team<br />
*physical location of servers and client machines<br />
</blockquote><br />
<br />
Most documents are created on the Wiki, which has built-in version control features (a revision history, including comments). For Google Docs the same is true. Our network drive does NOT have version control and should not be used for documents that have to be modified (much). For storing/exchanging Matlab code we will use [http://subversion.tigris.org/ Subversion] in combination with [http://code.google.com/ Google Code]. All team members wishing to alter models/write matlab code should follow the following steps to get set up (focusses on Windows):<br />
<br />
# Download and install Matlab R2009a. (Login and click on My Account, both in the upper-right corner, then click on "My Products / Get Licensed Products and Updates".)<br />
# Download and install TortoiseSVN: http://tortoisesvn.net/downloads<br />
# Checkout the URL https://igemgroningen.googlecode.com/svn/trunk/ using TortoiseSVN by right-clicking in your iGEM directory (in Windows Explorer) and choosing SVN Checkout. Note that you will have to supply the password that can be found on http://code.google.com/hosting/settings.<br />
<br />
Whenever you want to make changes to some of the files in the repository, first update all files by choosing SVN Update from the right-click menu in Windows Explorer (this will download any new changes from others), then make your changes, and finish by committing (again through the right-click menu). Do not forget to supply a commit message explaining the changes.<br />
<br />
Also, in case of conflicts in a Simbiology project you are in trouble (this happens when two people update/change/commit a file at the same time). Unfortunately Mathworks chose to save their projects as one big compressed file, which makes it extremely hard to compare two versions of a project, let alone resolve conflicts. Using [http://winmerge.org/ WinMerge] (with the 7-Zip plugin) these project files can be compared, but you still can't resolve conflicts. So, please stick to one model per project and try to avoid conflicts. If a conflict does occur, contact Jasper for help in resolving it.<br />
<br />
A list of programs that support SBML and there respective capabilities are here [http://sbml.org/SBML_Software_Guide]./SBML_Software_Matrix <br />
<br />
Finally, team members that do '''NOT''' write code can checkout the models using TortoiseSVN in the same way as described above and file issues in the [http://code.google.com/p/igemgroningen/issues/list issue list].<br />
<br />
===Wet Work===<br />
*Tools: <br />
**Ligation ratio: Cloning Tool 6.0 (ask Frans/Sven)<br />
**List of used lab tools (and from whom we got them) can be found on GoogleDocs (in Dutch): Lab benodigdheden<br />
*Environment:<br />
**Lab room D113 in Haren<br />
*Infrastructure:<br />
**Archives: Binders for primer sheets, Articles on the subprojects<br />
**Databases used are [http://openwetware.org/wiki/Main_Page OpenWetWare], [http://www.partsregistry.org/Main_Page Part], [http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2009&group=Groningen Sandbox],[http://www.ncbi.nlm.nih.gov/pubmed/ Pubmed]<br />
<br />
===Human Practice===<br />
[https://gmw01.housing.rug.nl/cgi-bin/inferentie.pl?qst_id=5744 Survey]<br />
<br />
==Documentation==<br />
====Identification Methods====<br />
Private files are stored in a central repository (currently the Y-drive or Google Docs). Agendas and minutes are stored in their respective folders under the date of the meeting in yyyy-mm-dd format to enable easy sorting. Other documents should be named according to the UPEDU standard as much as possible and prefixed by a short abbreviation in brackets (which makes it easy to reference them), for example [PP] for this document.<br />
<br />
All documents that are not necessarily private should be placed on the Wiki under the name of the [http://www.upedu.org/upedu/process/artifact/ovu_arts.htm relevant artifact] (also see the [http://www.upedu.org/upedu/process/artifact/tmpl_cs/ovu_tmplcs.htm list of templates]), always prefixed by Team:Groningen (for example [[Team:Groningen/Vision]]). Images can also be uploaded to the Wiki to accompany texts, but the Wiki should not be used as a general means of exchanging files (for this the Y-drive, Google Docs or Google Groups are more suitable).<br />
<br />
Finally note that any changes to documents on the Wiki should make use of the Summary field below the large edit text box to describe the change (it would be nice if you tick the "This is a minor edit" checkbox if you're making small tweaks, like correcting a spelling mistake). These descriptions can be long, or as short as one word, depending on the situation. Afterwards it should be possible to reasonably easily get an overview of how the document evolved, and to look up specific changes. For documents not on the Wiki a Revision History should be present at the top of the document.<br />
<br />
===={{anchor|DirectoryStructure}} Master of Lists====<br />
{{done}}: Anything about lab stuff (in green!)<br />
<br />
*Our wiki<br />
**[[:Category:Team:Groningen]] contains all our categories (corresponding to some of the UPEDU disciplines).<br />
***[[:Category:Team:Groningen/Disciplines]] contains all our disciplines.<br />
***[[:Category:Team:Groningen/Roles]] contains all our roles.<br />
**[[Team:Groningen]] is our main page, http://www.igemgroningen.com/ redirects to this.<br />
***All our documents are on the Wiki, where possible. Each document is a subpage of the main page, unless it is itself part of another document, then it is a subpage of that document. For example: [[Team:Groningen/Project_Plan]] and [[Team:Groningen/Project_Plan/Risk_List]].<br />
***Note that each document should be preceded by a list of related categories (see this document for example).<br />
**User:... (see, for example, [[User:Verhoeven1981|Michael's user page]])<br />
***Some basic information<br />
***Optionally a photograph<br />
***Categories for each of the roles he/she has.<br />
**Any JavaScript on the Wiki should (as much as possible/desirable) be written in a separate page and included using a script tag with a href attribute referencing the raw version of the page (see [[:Template:Team:Groningen/Header]] for an example), this avoids problems with ampersands, keeps the pages a bit cleaner and makes it possible for browsers to cache long scripts. When it makes sense the page should be a subpage of the page it is used in.<br />
**[https://2009.igem.org/Team:Groningen/Parts Parts list]: <span class="wetwork">List of glycerol stocks in -80 freezer C from MolGen, also the Quality control by PCR and Restriction analysis should be filled in here.</span><br />
<br />
*Subversion repository<br />
**The trunk is at https://igemgroningen.googlecode.com/svn/trunk/<br />
**[https://igemgroningen.googlecode.com/svn/trunk/glucose/ /trunk/glucose/]<br />
***The models for the glucose sensing idea (which we're not pursuing any further).<br />
**[https://igemgroningen.googlecode.com/svn/trunk/buoyant/ /trunk/buoyant/]<br />
***[[Team:Groningen/Modelling|Models]] (mostly .sbproj files) for the [[Team:Groningen/Vision|buoyant bacteria]].<br />
<br />
*Google Docs<br />
**Unfortunately Google Docs does not allow sharing of a directory structure.<br />
**Contact Information for 3rd parties.<br />
**Minutes and agendas.<br />
**Availability of team members.<br />
**Balance sheet: estimated costs for tickets, hotel, lab etc and estimated sponsoring.<br />
**<span class="wetwork">Primers: list with ordering date and concentration of the primers when they did arrive (also for oligo's).<br />
**<span class="wetwork">Chemicals / Materials taken from MolGen: list with all chemicals, materials, enzymes taken from MolGen<br />
**<span class="wetwork">Fridge/Freezer Stock list: list with different sheets for general stocks in the fridge and freeze, additionally a sheet for -20 freezer stocks of all labworkers.<br />
**<span class="wetwork">Lab tools: list of all the stuff (machines, pipettes, etc) we use from microbiology and practicum pool (Labbenodigdheden).<br />
*Y-drive<br />
**The transfer documents.<br />
**Literature?<br />
**Miscellaneous files<br />
{{Team:Groningen/Project_Plan/Footer}}</div>JolandaWitteveenhttp://2009.igem.org/Team:Groningen/Project_Plan/Tools_and_DocumentationTeam:Groningen/Project Plan/Tools and Documentation2009-10-21T18:42:55Z<p>JolandaWitteveen: /* Wet Work */</p>
<hr />
<div>{{Team:Groningen/Project_Plan/Header}}<br />
[[Category:Team:Groningen/Disciplines/Configuration_and_Change_Management|Tools and Documentation]]<br />
[[Category:Team:Groningen/Roles/Configuration_Manager|Tools and Documentation]]<br />
[[Category:Team:Groningen/Roles/Facility_Manager|Tools and Documentation]]<br />
<br />
[[Team:Groningen|Our project]] produces [[Team:Groningen/Project_Plan#Deliverables|many artifacts]], this [http://www.upedu.org/upedu/process/artifact/ar_cmpln.htm Configuration Management Plan] (called Tools and Documentation to avoid a lot of jargon) details what tools and processes we use to create these artifacts. Both for dry work and wet work. Do we use Matlab or Mathematica? What programming conventions do we use? Which lab space do we use? When do we use it?<br />
<br />
Basically anyone may change anything at any time for any reason, and the Configuration Manager keeps an eye on how (and how much) the Wiki and the SVN repository are used. He also writes this document (assisted by the Facility Manager(s) and others for lab-specific parts).<br />
<br />
<html><style type="text/css"><br />
.wetwork { color: green; }<br />
</style></html><br />
<br />
==Tools, Environment, and Infrastructure==<br />
===Modelling===<br />
<blockquote><br />
Describe the computing environment and software tools to be used in fulfilling the CM functions throughout the project or product lifecycle.<br />
<br />
Describe the tools and procedures required used to version control configuration items generated throughout the project or product lifecycle.<br />
<br />
Issues involved in setting up the CM environment include:<br />
<br />
*anticipated size of product data<br />
*distribution of the product team<br />
*physical location of servers and client machines<br />
</blockquote><br />
<br />
Most documents are created on the Wiki, which has built-in version control features (a revision history, including comments). For Google Docs the same is true. Our network drive does NOT have version control and should not be used for documents that have to be modified (much). For storing/exchanging Matlab code we will use [http://subversion.tigris.org/ Subversion] in combination with [http://code.google.com/ Google Code]. All team members wishing to alter models/write matlab code should follow the following steps to get set up (focusses on Windows):<br />
<br />
# Download and install Matlab R2009a. (Login and click on My Account, both in the upper-right corner, then click on "My Products / Get Licensed Products and Updates".)<br />
# Download and install TortoiseSVN: http://tortoisesvn.net/downloads<br />
# Checkout the URL https://igemgroningen.googlecode.com/svn/trunk/ using TortoiseSVN by right-clicking in your iGEM directory (in Windows Explorer) and choosing SVN Checkout. Note that you will have to supply the password that can be found on http://code.google.com/hosting/settings.<br />
<br />
Whenever you want to make changes to some of the files in the repository, first update all files by choosing SVN Update from the right-click menu in Windows Explorer (this will download any new changes from others), then make your changes, and finish by committing (again through the right-click menu). Do not forget to supply a commit message explaining the changes.<br />
<br />
Also, in case of conflicts in a Simbiology project you are in trouble (this happens when two people update/change/commit a file at the same time). Unfortunately Mathworks chose to save their projects as one big compressed file, which makes it extremely hard to compare two versions of a project, let alone resolve conflicts. Using [http://winmerge.org/ WinMerge] (with the 7-Zip plugin) these project files can be compared, but you still can't resolve conflicts. So, please stick to one model per project and try to avoid conflicts. If a conflict does occur, contact Jasper for help in resolving it.<br />
<br />
A list of programs that support SBML and there respective capabilities are here [http://sbml.org/SBML_Software_Guide]./SBML_Software_Matrix <br />
<br />
Finally, team members that do '''NOT''' write code can checkout the models using TortoiseSVN in the same way as described above and file issues in the [http://code.google.com/p/igemgroningen/issues/list issue list].<br />
<br />
===Wet Work===<br />
*Tools: <br />
**<span class="wetwork">Ligation ratio: Cloning Tool 6.0 (ask Frans/Sven)<br />
**<span class="wetwork">List of used lab tools (and from whom we got them) can be found on GoogleDocs (in Dutch): Lab benodigdheden<br />
*Environment:<br />
**<span class="wetwork">Lab room D113 in Haren<br />
*Infrastructure:<br />
**<span class="wetwork">Archives: Binders for primer sheets, Articles on the subprojects<br />
**<span class="wetwork">Databases used are [http://openwetware.org/wiki/Main_Page OpenWetWare], [http://www.partsregistry.org/Main_Page Part], [http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2009&group=Groningen Sandbox],[http://www.ncbi.nlm.nih.gov/pubmed/ Pubmed]<br />
<br />
===Human Practice===<br />
[https://gmw01.housing.rug.nl/cgi-bin/inferentie.pl?qst_id=5744 Survey]<br />
<br />
==Documentation==<br />
====Identification Methods====<br />
Private files are stored in a central repository (currently the Y-drive or Google Docs). Agendas and minutes are stored in their respective folders under the date of the meeting in yyyy-mm-dd format to enable easy sorting. Other documents should be named according to the UPEDU standard as much as possible and prefixed by a short abbreviation in brackets (which makes it easy to reference them), for example [PP] for this document.<br />
<br />
All documents that are not necessarily private should be placed on the Wiki under the name of the [http://www.upedu.org/upedu/process/artifact/ovu_arts.htm relevant artifact] (also see the [http://www.upedu.org/upedu/process/artifact/tmpl_cs/ovu_tmplcs.htm list of templates]), always prefixed by Team:Groningen (for example [[Team:Groningen/Vision]]). Images can also be uploaded to the Wiki to accompany texts, but the Wiki should not be used as a general means of exchanging files (for this the Y-drive, Google Docs or Google Groups are more suitable).<br />
<br />
Finally note that any changes to documents on the Wiki should make use of the Summary field below the large edit text box to describe the change (it would be nice if you tick the "This is a minor edit" checkbox if you're making small tweaks, like correcting a spelling mistake). These descriptions can be long, or as short as one word, depending on the situation. Afterwards it should be possible to reasonably easily get an overview of how the document evolved, and to look up specific changes. For documents not on the Wiki a Revision History should be present at the top of the document.<br />
<br />
===={{anchor|DirectoryStructure}} Master of Lists====<br />
{{done}}: Anything about lab stuff (in green!)<br />
<br />
*Our wiki<br />
**[[:Category:Team:Groningen]] contains all our categories (corresponding to some of the UPEDU disciplines).<br />
***[[:Category:Team:Groningen/Disciplines]] contains all our disciplines.<br />
***[[:Category:Team:Groningen/Roles]] contains all our roles.<br />
**[[Team:Groningen]] is our main page, http://www.igemgroningen.com/ redirects to this.<br />
***All our documents are on the Wiki, where possible. Each document is a subpage of the main page, unless it is itself part of another document, then it is a subpage of that document. For example: [[Team:Groningen/Project_Plan]] and [[Team:Groningen/Project_Plan/Risk_List]].<br />
***Note that each document should be preceded by a list of related categories (see this document for example).<br />
**User:... (see, for example, [[User:Verhoeven1981|Michael's user page]])<br />
***Some basic information<br />
***Optionally a photograph<br />
***Categories for each of the roles he/she has.<br />
**Any JavaScript on the Wiki should (as much as possible/desirable) be written in a separate page and included using a script tag with a href attribute referencing the raw version of the page (see [[:Template:Team:Groningen/Header]] for an example), this avoids problems with ampersands, keeps the pages a bit cleaner and makes it possible for browsers to cache long scripts. When it makes sense the page should be a subpage of the page it is used in.<br />
**[https://2009.igem.org/Team:Groningen/Parts Parts list]: <span class="wetwork">List of glycerol stocks in -80 freezer C from MolGen, also the Quality control by PCR and Restriction analysis should be filled in here.</span><br />
<br />
*Subversion repository<br />
**The trunk is at https://igemgroningen.googlecode.com/svn/trunk/<br />
**[https://igemgroningen.googlecode.com/svn/trunk/glucose/ /trunk/glucose/]<br />
***The models for the glucose sensing idea (which we're not pursuing any further).<br />
**[https://igemgroningen.googlecode.com/svn/trunk/buoyant/ /trunk/buoyant/]<br />
***[[Team:Groningen/Modelling|Models]] (mostly .sbproj files) for the [[Team:Groningen/Vision|buoyant bacteria]].<br />
<br />
*Google Docs<br />
**Unfortunately Google Docs does not allow sharing of a directory structure.<br />
**Contact Information for 3rd parties.<br />
**Minutes and agendas.<br />
**Availability of team members.<br />
**Balance sheet: estimated costs for tickets, hotel, lab etc and estimated sponsoring.<br />
**<span class="wetwork">Primers: list with ordering date and concentration of the primers when they did arrive (also for oligo's).<br />
**<span class="wetwork">Chemicals / Materials taken from MolGen: list with all chemicals, materials, enzymes taken from MolGen<br />
**<span class="wetwork">Fridge/Freezer Stock list: list with different sheets for general stocks in the fridge and freeze, additionally a sheet for -20 freezer stocks of all labworkers.<br />
**<span class="wetwork">Lab tools: list of all the stuff (machines, pipettes, etc) we use from microbiology and practicum pool (Labbenodigdheden).<br />
*Y-drive<br />
**The transfer documents.<br />
**Literature?<br />
**Miscellaneous files<br />
{{Team:Groningen/Project_Plan/Footer}}</div>JolandaWitteveenhttp://2009.igem.org/Team:Groningen/Project_PlanTeam:Groningen/Project Plan2009-10-21T18:40:56Z<p>JolandaWitteveen: /* Risk Management - NEW */</p>
<hr />
<div>{{Team:Groningen/Project_Plan/Header}}<br />
[[Category:Team:Groningen/Disciplines/Project_Management|Project Plan]]<br />
[[Category:Team:Groningen/Roles/Project_Manager|Project Plan]]<br />
<br />
<html><style type="text/css"><br />
.wetwork { color: green; }<br />
</style></html><br />
<br />
The project plan (known in [http://www.upedu.org/ UPEDU] as "[http://www.upedu.org/upedu/process/artifact/ar_sdp.htm Software Development Plan]") is meant to hold all information necessary for the management of the project. This includes things like the planning, role assignments, information on resources, etc. This particular project plan applies to the 2009 iGEM project at the University of Groningen.<br />
<br />
=={{anchor|ProjectOverview}} Project Overview==<br />
===Project Purpose, Scope, and Objectives===<br />
The purpose of the 2009 iGEM Groningen project is [https://2009.igem.org/Judging/Judging_Criteria to have a great summer, and have fun attending the Jamboree].<br />
<br />
===Assumptions and Constraints===<br />
This plan assumes that we can raise enough funds to acquire all the needed materials, and that the university will supply the necessary facilities (like lab space). These will have to be organized before the start of the summer, as both iGEM and our individual schedules require us to do most of the work during the summer. We assume that we can collect enough data during the summer labwork and modelling to be presented during the iGEM Jamboree in oct/nov 2009. <br />
<br />
Our assumptions and constraints were defined in a list of wishes and demands as followed:<br />
<br />
<b><u>Demands</u></b><br />
:*Labwork is feasible in 3 month with 8 students.<br />
:*Modelling feasible in 3 months with 3 students.<br />
:*Everyone has to agree with the idea<br />
:*Financially feasible with a budget of 31.170,00 euro of which 8000 euros used for labwork.<br />
:*A new concept, not yet done before in this way, either the iGEM or in the synthetic biology<br />
:*Meets the criteria of iGEM<br />
:*Materials have to be attainable, either via the RuG or able to order in.<br />
:*Parameters for modelling have to be known, or available or attainable.<br />
:*Knowledge about the genes that we are using has to be available.<br />
<br />
<b><u>Wishes</u></b><br />
:*Not too complicated, not too many genes or gene clusters<br />
:*Has to have an application<br />
:*Using BioBricks that are easy to obtain, preferable available at the RuG.<br />
:*Preferably knowledge on the host, genes and/or end products has to be available at the RuG<br />
:*Some students in the team have experience working with the host.<br />
:*It is possible to find sponsoring for this project.<br />
<br />
==={{anchor|Deliverables}} Project Deliverables===<br />
During the course of this project the following will be delivered:<br />
<br />
*An initial idea for a new "machine" in the iGEM sense, including a preliminary feasibility study.<br />
*A detailed specification of the requirements of this machine.<br />
*A detailed design of this "machine", including an analysis of the usage scenarios and results of simulations of computer models of (parts of) this "machine".<br />
*The actual "machine" (consisting of cells)<br />
*A presentation for the jamboree in November.<br />
*A poster for the jamboree in November<br />
*A report of our findings<br />
<br />
===Evolution of the Project Plan===<br />
The project plan will be set up during the [[Team:Groningen/Project_Plan/Inception|Inception]] phase and after each iteration the [[#ProjectPlan|planning]] is updated to reflect the actual progress and give updated estimates. Other parts of the project plan are not scheduled to be updated after the Inception and will only be updated to fix errors, clarify the existing text or to reflect a change in circumstances.<br />
<br />
==Project Organization==<br />
This is covered by our [[Team:Groningen/Team|team page]] and Google Docs for contact information (for privacy reasons).<br />
<br />
=={{anchor|ManagementProcess}} Management Process==<br />
===Project Estimates===<br />
The estimated costs for the project are as presented in the balance sheet (GoogleDocs). The estimated time for the project is 3-4 hours a week per person from march-july. Full-time (about 40 hours a week per person) during the summer (july-september). From september untill the jamboree on 30 okt another 3-4 hours a week per person (turned out to be a lot more full-time for most people).<br />
<br />
==={{anchor|ProjectPlan}} Project Plan===<br />
This project recognizes the following [http://www.upedu.org/upedu/process/itrwkfls/iwf_iwfs.htm phases and milestones] (only broad descriptions of the milestones are given here):<br />
<br />
*[[Team:Groningen/Project_Plan/Inception|Inception]]: until 18 may, milestone: initial idea, including preliminary feasibility study.<br />
*[[Team:Groningen/Project_Plan/Elaboration|Elaboration]]: until the summer (1st of july), milestone: initial design and modelling.<br />
*[[Team:Groningen/Project_Plan/Construction|Construction]]: during the summer (1 july-30 august), milestone: the actual "product".<br />
*[[Team:Groningen/Project_Plan/Transition|Transition]]: after the summer (30 august-31 dec), milestone: the jamboree presentation and documentation for the next team.<br />
<br />
Note that during the elaboration there should probably already be some lab work (if at all possible) to assist in modelling, and during the summer the modelling work will probably continue. Detailed planning of each of the iterations should be documented in iteration plans.<br />
<br />
{| border="1"<br />
!Iteration<br />
!End date<br />
!Objectives<br />
|-<br />
|[[Team:Groningen/Project_Plan/Inception/1|Inception 1]]<br />
|2009-04-20<br />
|Narrow down ideas to about three ideas that should be investigated further.<br />
|-<br />
|[[Team:Groningen/Project_Plan/Inception/2|Inception 2]]<br />
|2009-05-18<br />
|Choose idea we're going to work on. The most important requirements should be identified.<br />
|-<br />
|[[Team:Groningen/Project_Plan/Elaboration/1|Elaboration 1]]<br />
|2009-06-01<br />
|The initial design should be made, the requirements refined. Some initial model prototypes should be made to gain experience in modelling.<br />
|-<br />
|[[Team:Groningen/Project_Plan/Elaboration/2|Elaboration 2]]<br />
|2009-06-30<br />
|Models of our design should be made and verified, the design refined. And if possible some things may have to be verified and or tried out in the lab.<br />
|-<br />
|[[Team:Groningen/Project_Plan/Construction/1|Construction 1]]<br />
|2009-07-21<br />
|All necessary equipment and materials should be known and in the lab.<br />
|-<br />
|[[Team:Groningen/Project_Plan/Construction/2|Construction 2]]<br />
|2009-08-11 <br />
|A checkup if the system works, including all parts, should be able to be done.<br />
|-<br />
|[[Team:Groningen/Project_Plan/Construction/3|Construction 3]]<br />
|2009-09-01<br />
|Everything (in the lab) finished :)<br />
|-<br />
|[[Team:Groningen/Project_Plan/Transition/1|Transition 1]]<br />
|15-10-09<br />
|Presentation for the jamboree and the wiki should be (made and) polished.<br />
|-<br />
|[[Team:Groningen/Project_Plan/Transition/2|Transition 2]]<br />
|31-12-09<br />
|Our documentation should be prepared for and transferred to the next team.<br />
|}<br />
<br />
===Project Monitoring and Control===<br />
<br />
====Requirements Management====<br />
The requirements for this system are captured in [[Team:Groningen/Vision|the Vision document]].<br />
<br />
====Quality Control====<br />
The '''Quality of the constructs''' developed by Labworkers should be conform the following quality control regulations<br />
<br />
After transformation of the cells with Your Favorite Construct:<br />
*Pick a few colonies.<br />
**Grow o/n culture of a single colony.<br />
**Continue with miniprep on o/n culture.<br />
**Check insert length by PCR and restriction analysis, as written in document: Quality control (Overdrachts document iGEM 2008).<br />
***For restriction analysis try to find a restriction enzyme which makes a single cut in the vector and a single cut in the insert, or a double digestion with one which cuts in the vector and one which cuts in the insert.<br />
*Streak positive colony with inoculation eye out for single colonies.<br />
**Pick a single colony.<br />
**Grow o/n culture and make glycerol stock (put -80 position on [https://2009.igem.org/Team:Groningen/Parts Parts List]) next day. <br />
**Miniprep and continue with cloning / checking insert length.<br />
<br />
====Reporting and Measurement====<br />
During the non-summer period (march-july, sept-oct) a meeting will be held on a weekly basis. During the meeting the progression of the project, finance, contact with other iGEM teams and organisation subjects will be discussed. Every other week the advisors are invited for this meeting. During the summer period (july-sept) a meeting will also be held on a weekly basis with the whole team.<br />
<br />
====Risk Management ====<br />
Risks will be identified in Inception Phase using the steps identified in the RUP for Small Projects activity “Identify and Assess Risks”. Project risk is evaluated at least once per iteration and documented in this table (See [[Team:Groningen/Project_Plan/Risk_List|Risk List]]).<br />
<br />
===={{anchor|ConfigurationManagement}} Configuration Management====<br />
See [[Team:Groningen/Project_Plan/Tools and Documentation|Tools and Documentation]].<br />
<br />
=={{anchor|Process_Guidelines}}{{anchor|UPEDU}} Process Guidelines==<br />
This project attempts to follow a standardized process for software development ([http://www.upedu.org/ UPEDU]), adapting it for use with iGEM. Where possible roles, activities, etc. are simply copied from UPEDU (and ultimately RUP). To cope with the difference between software development and genetic engineering we make the following changes:<br />
<br />
*A new role "[[:Category:Team:Groningen/Roles/Modeller|Modeller]]" for someone who models the design on a computer and attempts to verify the design before it is implemented in the lab.<br />
*The "[[:Category:Team:Groningen/Roles/Implementer|Implementer]]" role is changed to reflect lab work instead of programming.<br />
*We also added some roles to take care of things like keeping minutes, doing PR and so on.<br />
<br />
In practice we ignored most of UPEDU, because of a lack of experience with such approaches within the team (and a lack of time to make up for it), making it inefficient to use, but also because some features proved hard to implement, we specifically had trouble with:<br />
<br />
*'''Using iterative development.''' In software development it is possible to make small changes quickly, allowing iterations as short as one or two weeks (much longer iterations would not have suited the time scale of iGEM). However, in synthetic biology it is more efficient to work on many things at once over a longer time frame (simply because each small change necessarily takes a certain amount of time). In practice this meant that a more waterfall-esque development process was used, where the construction phase consisted mostly of preparing everything for testing and analysis, leaving all testing, analysis and combination of results for the transition phase.<br />
*'''Making a clear separation between requirements(, architecture), design, implementation and tests.''' With software it is possible to create pretty much anything you can define, so one starts by defining what the system should do (at least in part), then figures out how this could be implemented in an abstract way and only then actually implements it (tests can usually be developed in parallel). In part this approach can and should(!) be adopted in synthetic biology, but some things do make it hard to do this rigorously. For example, there is hardly any standard way to abstractly talk about system components yet. Hopefully parts and "devices" might help with this in the near future.<br />
<br />
What did work for us to varying degrees:<br />
<br />
*'''Identifying risks''' early on in the project. Without UPEDU we probably would not have done this and this could have led to severe problems. Luckily not that many of the risks actually materialized, but we did implement some of the mitigation strategies, leading to less nasty surprises. For example, we made an overview of everyone's availability early on in the project and tried to prevent having only one person work on a single part as much as possible.<br />
*'''Using a standardized [[Team:Groningen/methodischontwerpen|design process]].''' Not specifically UPEDU, but it fits well within the framework and it helped us to structure our project selection process.<br />
*'''Recognizing different phases''' in our development. This especially helped in the beginning by giving at least some structure to our planning and providing a vocabulary to talk about such things.<br />
*'''Using iterations.''' Although we had definite problems in our use of iterations they did give some structure to the construction phase for example.<br />
*Describing our '''Tools and Documentation'''. Although not always used consistently it did help us keep an overview what was stored where.<br />
*Having specific '''Roles'''. Not all roles were used (consistently), but it did provide us with at least some structure to deal with eleven team members (it is not easy to do this "free-style").<br />
<br />
=={{anchor|Annexes}} Annexes==<br />
During the inception phase ideas were generated and selected using a [[Team:Groningen/methodischontwerpen|design template]]. For selection of the ideas a [[Team:Groningen/Project_Plan#Assumptions_and_Constraints|list of demands and wishes]] was made.<br />
<br />
{{Team:Groningen/Project_Plan/Footer}}</div>JolandaWitteveenhttp://2009.igem.org/Team:Groningen/Project_PlanTeam:Groningen/Project Plan2009-10-21T18:40:30Z<p>JolandaWitteveen: /* Reporting and Measurement */</p>
<hr />
<div>{{Team:Groningen/Project_Plan/Header}}<br />
[[Category:Team:Groningen/Disciplines/Project_Management|Project Plan]]<br />
[[Category:Team:Groningen/Roles/Project_Manager|Project Plan]]<br />
<br />
<html><style type="text/css"><br />
.wetwork { color: green; }<br />
</style></html><br />
<br />
The project plan (known in [http://www.upedu.org/ UPEDU] as "[http://www.upedu.org/upedu/process/artifact/ar_sdp.htm Software Development Plan]") is meant to hold all information necessary for the management of the project. This includes things like the planning, role assignments, information on resources, etc. This particular project plan applies to the 2009 iGEM project at the University of Groningen.<br />
<br />
=={{anchor|ProjectOverview}} Project Overview==<br />
===Project Purpose, Scope, and Objectives===<br />
The purpose of the 2009 iGEM Groningen project is [https://2009.igem.org/Judging/Judging_Criteria to have a great summer, and have fun attending the Jamboree].<br />
<br />
===Assumptions and Constraints===<br />
This plan assumes that we can raise enough funds to acquire all the needed materials, and that the university will supply the necessary facilities (like lab space). These will have to be organized before the start of the summer, as both iGEM and our individual schedules require us to do most of the work during the summer. We assume that we can collect enough data during the summer labwork and modelling to be presented during the iGEM Jamboree in oct/nov 2009. <br />
<br />
Our assumptions and constraints were defined in a list of wishes and demands as followed:<br />
<br />
<b><u>Demands</u></b><br />
:*Labwork is feasible in 3 month with 8 students.<br />
:*Modelling feasible in 3 months with 3 students.<br />
:*Everyone has to agree with the idea<br />
:*Financially feasible with a budget of 31.170,00 euro of which 8000 euros used for labwork.<br />
:*A new concept, not yet done before in this way, either the iGEM or in the synthetic biology<br />
:*Meets the criteria of iGEM<br />
:*Materials have to be attainable, either via the RuG or able to order in.<br />
:*Parameters for modelling have to be known, or available or attainable.<br />
:*Knowledge about the genes that we are using has to be available.<br />
<br />
<b><u>Wishes</u></b><br />
:*Not too complicated, not too many genes or gene clusters<br />
:*Has to have an application<br />
:*Using BioBricks that are easy to obtain, preferable available at the RuG.<br />
:*Preferably knowledge on the host, genes and/or end products has to be available at the RuG<br />
:*Some students in the team have experience working with the host.<br />
:*It is possible to find sponsoring for this project.<br />
<br />
==={{anchor|Deliverables}} Project Deliverables===<br />
During the course of this project the following will be delivered:<br />
<br />
*An initial idea for a new "machine" in the iGEM sense, including a preliminary feasibility study.<br />
*A detailed specification of the requirements of this machine.<br />
*A detailed design of this "machine", including an analysis of the usage scenarios and results of simulations of computer models of (parts of) this "machine".<br />
*The actual "machine" (consisting of cells)<br />
*A presentation for the jamboree in November.<br />
*A poster for the jamboree in November<br />
*A report of our findings<br />
<br />
===Evolution of the Project Plan===<br />
The project plan will be set up during the [[Team:Groningen/Project_Plan/Inception|Inception]] phase and after each iteration the [[#ProjectPlan|planning]] is updated to reflect the actual progress and give updated estimates. Other parts of the project plan are not scheduled to be updated after the Inception and will only be updated to fix errors, clarify the existing text or to reflect a change in circumstances.<br />
<br />
==Project Organization==<br />
This is covered by our [[Team:Groningen/Team|team page]] and Google Docs for contact information (for privacy reasons).<br />
<br />
=={{anchor|ManagementProcess}} Management Process==<br />
===Project Estimates===<br />
The estimated costs for the project are as presented in the balance sheet (GoogleDocs). The estimated time for the project is 3-4 hours a week per person from march-july. Full-time (about 40 hours a week per person) during the summer (july-september). From september untill the jamboree on 30 okt another 3-4 hours a week per person (turned out to be a lot more full-time for most people).<br />
<br />
==={{anchor|ProjectPlan}} Project Plan===<br />
This project recognizes the following [http://www.upedu.org/upedu/process/itrwkfls/iwf_iwfs.htm phases and milestones] (only broad descriptions of the milestones are given here):<br />
<br />
*[[Team:Groningen/Project_Plan/Inception|Inception]]: until 18 may, milestone: initial idea, including preliminary feasibility study.<br />
*[[Team:Groningen/Project_Plan/Elaboration|Elaboration]]: until the summer (1st of july), milestone: initial design and modelling.<br />
*[[Team:Groningen/Project_Plan/Construction|Construction]]: during the summer (1 july-30 august), milestone: the actual "product".<br />
*[[Team:Groningen/Project_Plan/Transition|Transition]]: after the summer (30 august-31 dec), milestone: the jamboree presentation and documentation for the next team.<br />
<br />
Note that during the elaboration there should probably already be some lab work (if at all possible) to assist in modelling, and during the summer the modelling work will probably continue. Detailed planning of each of the iterations should be documented in iteration plans.<br />
<br />
{| border="1"<br />
!Iteration<br />
!End date<br />
!Objectives<br />
|-<br />
|[[Team:Groningen/Project_Plan/Inception/1|Inception 1]]<br />
|2009-04-20<br />
|Narrow down ideas to about three ideas that should be investigated further.<br />
|-<br />
|[[Team:Groningen/Project_Plan/Inception/2|Inception 2]]<br />
|2009-05-18<br />
|Choose idea we're going to work on. The most important requirements should be identified.<br />
|-<br />
|[[Team:Groningen/Project_Plan/Elaboration/1|Elaboration 1]]<br />
|2009-06-01<br />
|The initial design should be made, the requirements refined. Some initial model prototypes should be made to gain experience in modelling.<br />
|-<br />
|[[Team:Groningen/Project_Plan/Elaboration/2|Elaboration 2]]<br />
|2009-06-30<br />
|Models of our design should be made and verified, the design refined. And if possible some things may have to be verified and or tried out in the lab.<br />
|-<br />
|[[Team:Groningen/Project_Plan/Construction/1|Construction 1]]<br />
|2009-07-21<br />
|All necessary equipment and materials should be known and in the lab.<br />
|-<br />
|[[Team:Groningen/Project_Plan/Construction/2|Construction 2]]<br />
|2009-08-11 <br />
|A checkup if the system works, including all parts, should be able to be done.<br />
|-<br />
|[[Team:Groningen/Project_Plan/Construction/3|Construction 3]]<br />
|2009-09-01<br />
|Everything (in the lab) finished :)<br />
|-<br />
|[[Team:Groningen/Project_Plan/Transition/1|Transition 1]]<br />
|15-10-09<br />
|Presentation for the jamboree and the wiki should be (made and) polished.<br />
|-<br />
|[[Team:Groningen/Project_Plan/Transition/2|Transition 2]]<br />
|31-12-09<br />
|Our documentation should be prepared for and transferred to the next team.<br />
|}<br />
<br />
===Project Monitoring and Control===<br />
<br />
====Requirements Management====<br />
The requirements for this system are captured in [[Team:Groningen/Vision|the Vision document]].<br />
<br />
====Quality Control====<br />
The '''Quality of the constructs''' developed by Labworkers should be conform the following quality control regulations<br />
<br />
After transformation of the cells with Your Favorite Construct:<br />
*Pick a few colonies.<br />
**Grow o/n culture of a single colony.<br />
**Continue with miniprep on o/n culture.<br />
**Check insert length by PCR and restriction analysis, as written in document: Quality control (Overdrachts document iGEM 2008).<br />
***For restriction analysis try to find a restriction enzyme which makes a single cut in the vector and a single cut in the insert, or a double digestion with one which cuts in the vector and one which cuts in the insert.<br />
*Streak positive colony with inoculation eye out for single colonies.<br />
**Pick a single colony.<br />
**Grow o/n culture and make glycerol stock (put -80 position on [https://2009.igem.org/Team:Groningen/Parts Parts List]) next day. <br />
**Miniprep and continue with cloning / checking insert length.<br />
<br />
====Reporting and Measurement====<br />
During the non-summer period (march-july, sept-oct) a meeting will be held on a weekly basis. During the meeting the progression of the project, finance, contact with other iGEM teams and organisation subjects will be discussed. Every other week the advisors are invited for this meeting. During the summer period (july-sept) a meeting will also be held on a weekly basis with the whole team.<br />
<br />
====Risk Management - NEW====<br />
Risks will be identified in Inception Phase using the steps identified in the RUP for Small Projects activity “Identify and Assess Risks”. Project risk is evaluated at least once per iteration and documented in this table (See [[Team:Groningen/Project_Plan/Risk_List|Risk List]]).<br />
<br />
===={{anchor|ConfigurationManagement}} Configuration Management====<br />
See [[Team:Groningen/Project_Plan/Tools and Documentation|Tools and Documentation]].<br />
<br />
=={{anchor|Process_Guidelines}}{{anchor|UPEDU}} Process Guidelines==<br />
This project attempts to follow a standardized process for software development ([http://www.upedu.org/ UPEDU]), adapting it for use with iGEM. Where possible roles, activities, etc. are simply copied from UPEDU (and ultimately RUP). To cope with the difference between software development and genetic engineering we make the following changes:<br />
<br />
*A new role "[[:Category:Team:Groningen/Roles/Modeller|Modeller]]" for someone who models the design on a computer and attempts to verify the design before it is implemented in the lab.<br />
*The "[[:Category:Team:Groningen/Roles/Implementer|Implementer]]" role is changed to reflect lab work instead of programming.<br />
*We also added some roles to take care of things like keeping minutes, doing PR and so on.<br />
<br />
In practice we ignored most of UPEDU, because of a lack of experience with such approaches within the team (and a lack of time to make up for it), making it inefficient to use, but also because some features proved hard to implement, we specifically had trouble with:<br />
<br />
*'''Using iterative development.''' In software development it is possible to make small changes quickly, allowing iterations as short as one or two weeks (much longer iterations would not have suited the time scale of iGEM). However, in synthetic biology it is more efficient to work on many things at once over a longer time frame (simply because each small change necessarily takes a certain amount of time). In practice this meant that a more waterfall-esque development process was used, where the construction phase consisted mostly of preparing everything for testing and analysis, leaving all testing, analysis and combination of results for the transition phase.<br />
*'''Making a clear separation between requirements(, architecture), design, implementation and tests.''' With software it is possible to create pretty much anything you can define, so one starts by defining what the system should do (at least in part), then figures out how this could be implemented in an abstract way and only then actually implements it (tests can usually be developed in parallel). In part this approach can and should(!) be adopted in synthetic biology, but some things do make it hard to do this rigorously. For example, there is hardly any standard way to abstractly talk about system components yet. Hopefully parts and "devices" might help with this in the near future.<br />
<br />
What did work for us to varying degrees:<br />
<br />
*'''Identifying risks''' early on in the project. Without UPEDU we probably would not have done this and this could have led to severe problems. Luckily not that many of the risks actually materialized, but we did implement some of the mitigation strategies, leading to less nasty surprises. For example, we made an overview of everyone's availability early on in the project and tried to prevent having only one person work on a single part as much as possible.<br />
*'''Using a standardized [[Team:Groningen/methodischontwerpen|design process]].''' Not specifically UPEDU, but it fits well within the framework and it helped us to structure our project selection process.<br />
*'''Recognizing different phases''' in our development. This especially helped in the beginning by giving at least some structure to our planning and providing a vocabulary to talk about such things.<br />
*'''Using iterations.''' Although we had definite problems in our use of iterations they did give some structure to the construction phase for example.<br />
*Describing our '''Tools and Documentation'''. Although not always used consistently it did help us keep an overview what was stored where.<br />
*Having specific '''Roles'''. Not all roles were used (consistently), but it did provide us with at least some structure to deal with eleven team members (it is not easy to do this "free-style").<br />
<br />
=={{anchor|Annexes}} Annexes==<br />
During the inception phase ideas were generated and selected using a [[Team:Groningen/methodischontwerpen|design template]]. For selection of the ideas a [[Team:Groningen/Project_Plan#Assumptions_and_Constraints|list of demands and wishes]] was made.<br />
<br />
{{Team:Groningen/Project_Plan/Footer}}</div>JolandaWitteveenhttp://2009.igem.org/Team:Groningen/Project_PlanTeam:Groningen/Project Plan2009-10-21T18:36:45Z<p>JolandaWitteveen: /* {{anchor|ProjectPlan}} Project Plan */</p>
<hr />
<div>{{Team:Groningen/Project_Plan/Header}}<br />
[[Category:Team:Groningen/Disciplines/Project_Management|Project Plan]]<br />
[[Category:Team:Groningen/Roles/Project_Manager|Project Plan]]<br />
<br />
<html><style type="text/css"><br />
.wetwork { color: green; }<br />
</style></html><br />
<br />
The project plan (known in [http://www.upedu.org/ UPEDU] as "[http://www.upedu.org/upedu/process/artifact/ar_sdp.htm Software Development Plan]") is meant to hold all information necessary for the management of the project. This includes things like the planning, role assignments, information on resources, etc. This particular project plan applies to the 2009 iGEM project at the University of Groningen.<br />
<br />
=={{anchor|ProjectOverview}} Project Overview==<br />
===Project Purpose, Scope, and Objectives===<br />
The purpose of the 2009 iGEM Groningen project is [https://2009.igem.org/Judging/Judging_Criteria to have a great summer, and have fun attending the Jamboree].<br />
<br />
===Assumptions and Constraints===<br />
This plan assumes that we can raise enough funds to acquire all the needed materials, and that the university will supply the necessary facilities (like lab space). These will have to be organized before the start of the summer, as both iGEM and our individual schedules require us to do most of the work during the summer. We assume that we can collect enough data during the summer labwork and modelling to be presented during the iGEM Jamboree in oct/nov 2009. <br />
<br />
Our assumptions and constraints were defined in a list of wishes and demands as followed:<br />
<br />
<b><u>Demands</u></b><br />
:*Labwork is feasible in 3 month with 8 students.<br />
:*Modelling feasible in 3 months with 3 students.<br />
:*Everyone has to agree with the idea<br />
:*Financially feasible with a budget of 31.170,00 euro of which 8000 euros used for labwork.<br />
:*A new concept, not yet done before in this way, either the iGEM or in the synthetic biology<br />
:*Meets the criteria of iGEM<br />
:*Materials have to be attainable, either via the RuG or able to order in.<br />
:*Parameters for modelling have to be known, or available or attainable.<br />
:*Knowledge about the genes that we are using has to be available.<br />
<br />
<b><u>Wishes</u></b><br />
:*Not too complicated, not too many genes or gene clusters<br />
:*Has to have an application<br />
:*Using BioBricks that are easy to obtain, preferable available at the RuG.<br />
:*Preferably knowledge on the host, genes and/or end products has to be available at the RuG<br />
:*Some students in the team have experience working with the host.<br />
:*It is possible to find sponsoring for this project.<br />
<br />
==={{anchor|Deliverables}} Project Deliverables===<br />
During the course of this project the following will be delivered:<br />
<br />
*An initial idea for a new "machine" in the iGEM sense, including a preliminary feasibility study.<br />
*A detailed specification of the requirements of this machine.<br />
*A detailed design of this "machine", including an analysis of the usage scenarios and results of simulations of computer models of (parts of) this "machine".<br />
*The actual "machine" (consisting of cells)<br />
*A presentation for the jamboree in November.<br />
*A poster for the jamboree in November<br />
*A report of our findings<br />
<br />
===Evolution of the Project Plan===<br />
The project plan will be set up during the [[Team:Groningen/Project_Plan/Inception|Inception]] phase and after each iteration the [[#ProjectPlan|planning]] is updated to reflect the actual progress and give updated estimates. Other parts of the project plan are not scheduled to be updated after the Inception and will only be updated to fix errors, clarify the existing text or to reflect a change in circumstances.<br />
<br />
==Project Organization==<br />
This is covered by our [[Team:Groningen/Team|team page]] and Google Docs for contact information (for privacy reasons).<br />
<br />
=={{anchor|ManagementProcess}} Management Process==<br />
===Project Estimates===<br />
The estimated costs for the project are as presented in the balance sheet (GoogleDocs). The estimated time for the project is 3-4 hours a week per person from march-july. Full-time (about 40 hours a week per person) during the summer (july-september). From september untill the jamboree on 30 okt another 3-4 hours a week per person (turned out to be a lot more full-time for most people).<br />
<br />
==={{anchor|ProjectPlan}} Project Plan===<br />
This project recognizes the following [http://www.upedu.org/upedu/process/itrwkfls/iwf_iwfs.htm phases and milestones] (only broad descriptions of the milestones are given here):<br />
<br />
*[[Team:Groningen/Project_Plan/Inception|Inception]]: until 18 may, milestone: initial idea, including preliminary feasibility study.<br />
*[[Team:Groningen/Project_Plan/Elaboration|Elaboration]]: until the summer (1st of july), milestone: initial design and modelling.<br />
*[[Team:Groningen/Project_Plan/Construction|Construction]]: during the summer (1 july-30 august), milestone: the actual "product".<br />
*[[Team:Groningen/Project_Plan/Transition|Transition]]: after the summer (30 august-31 dec), milestone: the jamboree presentation and documentation for the next team.<br />
<br />
Note that during the elaboration there should probably already be some lab work (if at all possible) to assist in modelling, and during the summer the modelling work will probably continue. Detailed planning of each of the iterations should be documented in iteration plans.<br />
<br />
{| border="1"<br />
!Iteration<br />
!End date<br />
!Objectives<br />
|-<br />
|[[Team:Groningen/Project_Plan/Inception/1|Inception 1]]<br />
|2009-04-20<br />
|Narrow down ideas to about three ideas that should be investigated further.<br />
|-<br />
|[[Team:Groningen/Project_Plan/Inception/2|Inception 2]]<br />
|2009-05-18<br />
|Choose idea we're going to work on. The most important requirements should be identified.<br />
|-<br />
|[[Team:Groningen/Project_Plan/Elaboration/1|Elaboration 1]]<br />
|2009-06-01<br />
|The initial design should be made, the requirements refined. Some initial model prototypes should be made to gain experience in modelling.<br />
|-<br />
|[[Team:Groningen/Project_Plan/Elaboration/2|Elaboration 2]]<br />
|2009-06-30<br />
|Models of our design should be made and verified, the design refined. And if possible some things may have to be verified and or tried out in the lab.<br />
|-<br />
|[[Team:Groningen/Project_Plan/Construction/1|Construction 1]]<br />
|2009-07-21<br />
|All necessary equipment and materials should be known and in the lab.<br />
|-<br />
|[[Team:Groningen/Project_Plan/Construction/2|Construction 2]]<br />
|2009-08-11 <br />
|A checkup if the system works, including all parts, should be able to be done.<br />
|-<br />
|[[Team:Groningen/Project_Plan/Construction/3|Construction 3]]<br />
|2009-09-01<br />
|Everything (in the lab) finished :)<br />
|-<br />
|[[Team:Groningen/Project_Plan/Transition/1|Transition 1]]<br />
|15-10-09<br />
|Presentation for the jamboree and the wiki should be (made and) polished.<br />
|-<br />
|[[Team:Groningen/Project_Plan/Transition/2|Transition 2]]<br />
|31-12-09<br />
|Our documentation should be prepared for and transferred to the next team.<br />
|}<br />
<br />
===Project Monitoring and Control===<br />
<br />
====Requirements Management====<br />
The requirements for this system are captured in [[Team:Groningen/Vision|the Vision document]].<br />
<br />
====Quality Control====<br />
The '''Quality of the constructs''' developed by Labworkers should be conform the following quality control regulations<br />
<br />
After transformation of the cells with Your Favorite Construct:<br />
*Pick a few colonies.<br />
**Grow o/n culture of a single colony.<br />
**Continue with miniprep on o/n culture.<br />
**Check insert length by PCR and restriction analysis, as written in document: Quality control (Overdrachts document iGEM 2008).<br />
***For restriction analysis try to find a restriction enzyme which makes a single cut in the vector and a single cut in the insert, or a double digestion with one which cuts in the vector and one which cuts in the insert.<br />
*Streak positive colony with inoculation eye out for single colonies.<br />
**Pick a single colony.<br />
**Grow o/n culture and make glycerol stock (put -80 position on [https://2009.igem.org/Team:Groningen/Parts Parts List]) next day. <br />
**Miniprep and continue with cloning / checking insert length.<br />
<br />
====Reporting and Measurement====<br />
During the non-summer period (march-july, sept-oct) a meeting will be held on a weekly basis. During the meeting the progression of the project, finance, contact with other iGEM teams and organisation subjects will be discussed. Every other week the advisors are invited for this meeting. During the summer period (july-sept) a meeting will also be held on a weekly basis with the whole team. {{todo|A short summary of these meetings will be available on the wiki Notebook.}}<br />
<br />
====Risk Management - NEW====<br />
Risks will be identified in Inception Phase using the steps identified in the RUP for Small Projects activity “Identify and Assess Risks”. Project risk is evaluated at least once per iteration and documented in this table (See [[Team:Groningen/Project_Plan/Risk_List|Risk List]]).<br />
<br />
===={{anchor|ConfigurationManagement}} Configuration Management====<br />
See [[Team:Groningen/Project_Plan/Tools and Documentation|Tools and Documentation]].<br />
<br />
=={{anchor|Process_Guidelines}}{{anchor|UPEDU}} Process Guidelines==<br />
This project attempts to follow a standardized process for software development ([http://www.upedu.org/ UPEDU]), adapting it for use with iGEM. Where possible roles, activities, etc. are simply copied from UPEDU (and ultimately RUP). To cope with the difference between software development and genetic engineering we make the following changes:<br />
<br />
*A new role "[[:Category:Team:Groningen/Roles/Modeller|Modeller]]" for someone who models the design on a computer and attempts to verify the design before it is implemented in the lab.<br />
*The "[[:Category:Team:Groningen/Roles/Implementer|Implementer]]" role is changed to reflect lab work instead of programming.<br />
*We also added some roles to take care of things like keeping minutes, doing PR and so on.<br />
<br />
In practice we ignored most of UPEDU, because of a lack of experience with such approaches within the team (and a lack of time to make up for it), making it inefficient to use, but also because some features proved hard to implement, we specifically had trouble with:<br />
<br />
*'''Using iterative development.''' In software development it is possible to make small changes quickly, allowing iterations as short as one or two weeks (much longer iterations would not have suited the time scale of iGEM). However, in synthetic biology it is more efficient to work on many things at once over a longer time frame (simply because each small change necessarily takes a certain amount of time). In practice this meant that a more waterfall-esque development process was used, where the construction phase consisted mostly of preparing everything for testing and analysis, leaving all testing, analysis and combination of results for the transition phase.<br />
*'''Making a clear separation between requirements(, architecture), design, implementation and tests.''' With software it is possible to create pretty much anything you can define, so one starts by defining what the system should do (at least in part), then figures out how this could be implemented in an abstract way and only then actually implements it (tests can usually be developed in parallel). In part this approach can and should(!) be adopted in synthetic biology, but some things do make it hard to do this rigorously. For example, there is hardly any standard way to abstractly talk about system components yet. Hopefully parts and "devices" might help with this in the near future.<br />
<br />
What did work for us to varying degrees:<br />
<br />
*'''Identifying risks''' early on in the project. Without UPEDU we probably would not have done this and this could have led to severe problems. Luckily not that many of the risks actually materialized, but we did implement some of the mitigation strategies, leading to less nasty surprises. For example, we made an overview of everyone's availability early on in the project and tried to prevent having only one person work on a single part as much as possible.<br />
*'''Using a standardized [[Team:Groningen/methodischontwerpen|design process]].''' Not specifically UPEDU, but it fits well within the framework and it helped us to structure our project selection process.<br />
*'''Recognizing different phases''' in our development. This especially helped in the beginning by giving at least some structure to our planning and providing a vocabulary to talk about such things.<br />
*'''Using iterations.''' Although we had definite problems in our use of iterations they did give some structure to the construction phase for example.<br />
*Describing our '''Tools and Documentation'''. Although not always used consistently it did help us keep an overview what was stored where.<br />
*Having specific '''Roles'''. Not all roles were used (consistently), but it did provide us with at least some structure to deal with eleven team members (it is not easy to do this "free-style").<br />
<br />
=={{anchor|Annexes}} Annexes==<br />
During the inception phase ideas were generated and selected using a [[Team:Groningen/methodischontwerpen|design template]]. For selection of the ideas a [[Team:Groningen/Project_Plan#Assumptions_and_Constraints|list of demands and wishes]] was made.<br />
<br />
{{Team:Groningen/Project_Plan/Footer}}</div>JolandaWitteveenhttp://2009.igem.org/Team:Groningen/Project_Plan/Inception/1Team:Groningen/Project Plan/Inception/12009-10-21T18:34:01Z<p>JolandaWitteveen: New page: {{Team:Groningen/Project_Plan/Header}} <br> <br> <br> =Iteration 1= At this point in time we were in such an early stage that we have no structured planning for this iteration. <br> <br> ...</p>
<hr />
<div>{{Team:Groningen/Project_Plan/Header}}<br />
<br><br />
<br><br />
<br><br />
=Iteration 1=<br />
At this point in time we were in such an early stage that we have no structured planning for this iteration. <br />
<br><br />
<br><br />
<br><br />
<br><br />
{{Team:Groningen/Footer}}</div>JolandaWitteveenhttp://2009.igem.org/Team:Groningen/Project_PlanTeam:Groningen/Project Plan2009-10-21T18:27:15Z<p>JolandaWitteveen: /* {{anchor|ProjectPlan}} Project Plan */</p>
<hr />
<div>{{Team:Groningen/Project_Plan/Header}}<br />
[[Category:Team:Groningen/Disciplines/Project_Management|Project Plan]]<br />
[[Category:Team:Groningen/Roles/Project_Manager|Project Plan]]<br />
<br />
<html><style type="text/css"><br />
.wetwork { color: green; }<br />
</style></html><br />
<br />
The project plan (known in [http://www.upedu.org/ UPEDU] as "[http://www.upedu.org/upedu/process/artifact/ar_sdp.htm Software Development Plan]") is meant to hold all information necessary for the management of the project. This includes things like the planning, role assignments, information on resources, etc. This particular project plan applies to the 2009 iGEM project at the University of Groningen.<br />
<br />
=={{anchor|ProjectOverview}} Project Overview==<br />
===Project Purpose, Scope, and Objectives===<br />
The purpose of the 2009 iGEM Groningen project is [https://2009.igem.org/Judging/Judging_Criteria to have a great summer, and have fun attending the Jamboree].<br />
<br />
===Assumptions and Constraints===<br />
This plan assumes that we can raise enough funds to acquire all the needed materials, and that the university will supply the necessary facilities (like lab space). These will have to be organized before the start of the summer, as both iGEM and our individual schedules require us to do most of the work during the summer. We assume that we can collect enough data during the summer labwork and modelling to be presented during the iGEM Jamboree in oct/nov 2009. <br />
<br />
Our assumptions and constraints were defined in a list of wishes and demands as followed:<br />
<br />
<b><u>Demands</u></b><br />
:*Labwork is feasible in 3 month with 8 students.<br />
:*Modelling feasible in 3 months with 3 students.<br />
:*Everyone has to agree with the idea<br />
:*Financially feasible with a budget of 31.170,00 euro of which 8000 euros used for labwork.<br />
:*A new concept, not yet done before in this way, either the iGEM or in the synthetic biology<br />
:*Meets the criteria of iGEM<br />
:*Materials have to be attainable, either via the RuG or able to order in.<br />
:*Parameters for modelling have to be known, or available or attainable.<br />
:*Knowledge about the genes that we are using has to be available.<br />
<br />
<b><u>Wishes</u></b><br />
:*Not too complicated, not too many genes or gene clusters<br />
:*Has to have an application<br />
:*Using BioBricks that are easy to obtain, preferable available at the RuG.<br />
:*Preferably knowledge on the host, genes and/or end products has to be available at the RuG<br />
:*Some students in the team have experience working with the host.<br />
:*It is possible to find sponsoring for this project.<br />
<br />
==={{anchor|Deliverables}} Project Deliverables===<br />
During the course of this project the following will be delivered:<br />
<br />
*An initial idea for a new "machine" in the iGEM sense, including a preliminary feasibility study.<br />
*A detailed specification of the requirements of this machine.<br />
*A detailed design of this "machine", including an analysis of the usage scenarios and results of simulations of computer models of (parts of) this "machine".<br />
*The actual "machine" (consisting of cells)<br />
*A presentation for the jamboree in November.<br />
*A poster for the jamboree in November<br />
*A report of our findings<br />
<br />
===Evolution of the Project Plan===<br />
The project plan will be set up during the [[Team:Groningen/Project_Plan/Inception|Inception]] phase and after each iteration the [[#ProjectPlan|planning]] is updated to reflect the actual progress and give updated estimates. Other parts of the project plan are not scheduled to be updated after the Inception and will only be updated to fix errors, clarify the existing text or to reflect a change in circumstances.<br />
<br />
==Project Organization==<br />
This is covered by our [[Team:Groningen/Team|team page]] and Google Docs for contact information (for privacy reasons).<br />
<br />
=={{anchor|ManagementProcess}} Management Process==<br />
===Project Estimates===<br />
The estimated costs for the project are as presented in the balance sheet (GoogleDocs). The estimated time for the project is 3-4 hours a week per person from march-july. Full-time (about 40 hours a week per person) during the summer (july-september). From september untill the jamboree on 30 okt another 3-4 hours a week per person (turned out to be a lot more full-time for most people).<br />
<br />
==={{anchor|ProjectPlan}} Project Plan===<br />
This project recognizes the following [http://www.upedu.org/upedu/process/itrwkfls/iwf_iwfs.htm phases and milestones] (only broad descriptions of the milestones are given here):<br />
<br />
*[[Team:Groningen/Project_Plan/Inception|Inception]]: until 18 may, milestone: initial idea, including preliminary feasibility study.<br />
*[[Team:Groningen/Project_Plan/Elaboration|Elaboration]]: until the summer (1st of july), milestone: initial design and modelling.<br />
*[[Team:Groningen/Project_Plan/Construction|Construction]]: during the summer (1 july-30 august), milestone: the actual "product".<br />
*[[Team:Groningen/Project_Plan/Transition|Transition]]: after the summer (30 august-30 okt), milestone: the jamboree presentation (and documentation for the next team)<br />
<br />
Note that during the elaboration there should probably already be some lab work (if at all possible) to assist in modelling, and during the summer the modelling work will probably continue. Detailed planning of each of the iterations should be documented in iteration plans.<br />
<br />
{| border="1"<br />
!Iteration<br />
!End date<br />
!Objectives<br />
|-<br />
|[[Team:Groningen/Project_Plan/Inception/1|Inception 1]]<br />
|2009-04-20<br />
|Narrow down ideas to about three ideas that should be investigated further.<br />
|-<br />
|[[Team:Groningen/Project_Plan/Inception/2|Inception 2]]<br />
|2009-05-18<br />
|Choose idea we're going to work on. The most important requirements should be identified.<br />
|-<br />
|[[Team:Groningen/Project_Plan/Elaboration/1|Elaboration 1]]<br />
|2009-06-01<br />
|The initial design should be made, the requirements refined. Some initial model prototypes should be made to gain experience in modelling.<br />
|-<br />
|[[Team:Groningen/Project_Plan/Elaboration/2|Elaboration 2]]<br />
|2009-06-30<br />
|Models of our design should be made and verified, the design refined. And if possible some things may have to be verified and or tried out in the lab.<br />
|-<br />
|[[Team:Groningen/Project_Plan/Construction/1|Construction 1]]<br />
|2009-07-21<br />
|All necessary equipment and materials should be known and in the lab.<br />
|-<br />
|[[Team:Groningen/Project_Plan/Construction/2|Construction 2]]<br />
|2009-08-11 <br />
|A checkup if the system works, including all parts, should be able to be done.<br />
|-<br />
|[[Team:Groningen/Project_Plan/Construction/3|Construction 3]]<br />
|2009-09-01<br />
|Everything (in the lab) finished :)<br />
|-<br />
|[[Team:Groningen/Project_Plan/Transition/1|Transition 1]]<br />
|15-10-09<br />
|Presentation for the jamboree and the wiki should be (made and) polished.<br />
|-<br />
|[[Team:Groningen/Project_Plan/Transition/2|Transition 2]]<br />
|31-12-09<br />
|Our documentation should be prepared for and transferred to the next team.<br />
|}<br />
<br />
===Project Monitoring and Control===<br />
<br />
====Requirements Management====<br />
The requirements for this system are captured in [[Team:Groningen/Vision|the Vision document]].<br />
<br />
====Quality Control====<br />
The '''Quality of the constructs''' developed by Labworkers should be conform the following quality control regulations<br />
<br />
After transformation of the cells with Your Favorite Construct:<br />
*Pick a few colonies.<br />
**Grow o/n culture of a single colony.<br />
**Continue with miniprep on o/n culture.<br />
**Check insert length by PCR and restriction analysis, as written in document: Quality control (Overdrachts document iGEM 2008).<br />
***For restriction analysis try to find a restriction enzyme which makes a single cut in the vector and a single cut in the insert, or a double digestion with one which cuts in the vector and one which cuts in the insert.<br />
*Streak positive colony with inoculation eye out for single colonies.<br />
**Pick a single colony.<br />
**Grow o/n culture and make glycerol stock (put -80 position on [https://2009.igem.org/Team:Groningen/Parts Parts List]) next day. <br />
**Miniprep and continue with cloning / checking insert length.<br />
<br />
====Reporting and Measurement====<br />
During the non-summer period (march-july, sept-oct) a meeting will be held on a weekly basis. During the meeting the progression of the project, finance, contact with other iGEM teams and organisation subjects will be discussed. Every other week the advisors are invited for this meeting. During the summer period (july-sept) a meeting will also be held on a weekly basis with the whole team. {{todo|A short summary of these meetings will be available on the wiki Notebook.}}<br />
<br />
====Risk Management - NEW====<br />
Risks will be identified in Inception Phase using the steps identified in the RUP for Small Projects activity “Identify and Assess Risks”. Project risk is evaluated at least once per iteration and documented in this table (See [[Team:Groningen/Project_Plan/Risk_List|Risk List]]).<br />
<br />
===={{anchor|ConfigurationManagement}} Configuration Management====<br />
See [[Team:Groningen/Project_Plan/Tools and Documentation|Tools and Documentation]].<br />
<br />
=={{anchor|Process_Guidelines}}{{anchor|UPEDU}} Process Guidelines==<br />
This project attempts to follow a standardized process for software development ([http://www.upedu.org/ UPEDU]), adapting it for use with iGEM. Where possible roles, activities, etc. are simply copied from UPEDU (and ultimately RUP). To cope with the difference between software development and genetic engineering we make the following changes:<br />
<br />
*A new role "[[:Category:Team:Groningen/Roles/Modeller|Modeller]]" for someone who models the design on a computer and attempts to verify the design before it is implemented in the lab.<br />
*The "[[:Category:Team:Groningen/Roles/Implementer|Implementer]]" role is changed to reflect lab work instead of programming.<br />
*We also added some roles to take care of things like keeping minutes, doing PR and so on.<br />
<br />
In practice we ignored most of UPEDU, because of a lack of experience with such approaches within the team (and a lack of time to make up for it), making it inefficient to use, but also because some features proved hard to implement, we specifically had trouble with:<br />
<br />
*'''Using iterative development.''' In software development it is possible to make small changes quickly, allowing iterations as short as one or two weeks (much longer iterations would not have suited the time scale of iGEM). However, in synthetic biology it is more efficient to work on many things at once over a longer time frame (simply because each small change necessarily takes a certain amount of time). In practice this meant that a more waterfall-esque development process was used, where the construction phase consisted mostly of preparing everything for testing and analysis, leaving all testing, analysis and combination of results for the transition phase.<br />
*'''Making a clear separation between requirements(, architecture), design, implementation and tests.''' With software it is possible to create pretty much anything you can define, so one starts by defining what the system should do (at least in part), then figures out how this could be implemented in an abstract way and only then actually implements it (tests can usually be developed in parallel). In part this approach can and should(!) be adopted in synthetic biology, but some things do make it hard to do this rigorously. For example, there is hardly any standard way to abstractly talk about system components yet. Hopefully parts and "devices" might help with this in the near future.<br />
<br />
What did work for us to varying degrees:<br />
<br />
*'''Identifying risks''' early on in the project. Without UPEDU we probably would not have done this and this could have led to severe problems. Luckily not that many of the risks actually materialized, but we did implement some of the mitigation strategies, leading to less nasty surprises. For example, we made an overview of everyone's availability early on in the project and tried to prevent having only one person work on a single part as much as possible.<br />
*'''Using a standardized [[Team:Groningen/methodischontwerpen|design process]].''' Not specifically UPEDU, but it fits well within the framework and it helped us to structure our project selection process.<br />
*'''Recognizing different phases''' in our development. This especially helped in the beginning by giving at least some structure to our planning and providing a vocabulary to talk about such things.<br />
*'''Using iterations.''' Although we had definite problems in our use of iterations they did give some structure to the construction phase for example.<br />
*Describing our '''Tools and Documentation'''. Although not always used consistently it did help us keep an overview what was stored where.<br />
*Having specific '''Roles'''. Not all roles were used (consistently), but it did provide us with at least some structure to deal with eleven team members (it is not easy to do this "free-style").<br />
<br />
=={{anchor|Annexes}} Annexes==<br />
During the inception phase ideas were generated and selected using a [[Team:Groningen/methodischontwerpen|design template]]. For selection of the ideas a [[Team:Groningen/Project_Plan#Assumptions_and_Constraints|list of demands and wishes]] was made.<br />
<br />
{{Team:Groningen/Project_Plan/Footer}}</div>JolandaWitteveenhttp://2009.igem.org/Team:Groningen/Project_PlanTeam:Groningen/Project Plan2009-10-21T18:24:57Z<p>JolandaWitteveen: /* {{anchor|Deliverables}} Project Deliverables */</p>
<hr />
<div>{{Team:Groningen/Project_Plan/Header}}<br />
[[Category:Team:Groningen/Disciplines/Project_Management|Project Plan]]<br />
[[Category:Team:Groningen/Roles/Project_Manager|Project Plan]]<br />
<br />
<html><style type="text/css"><br />
.wetwork { color: green; }<br />
</style></html><br />
<br />
The project plan (known in [http://www.upedu.org/ UPEDU] as "[http://www.upedu.org/upedu/process/artifact/ar_sdp.htm Software Development Plan]") is meant to hold all information necessary for the management of the project. This includes things like the planning, role assignments, information on resources, etc. This particular project plan applies to the 2009 iGEM project at the University of Groningen.<br />
<br />
=={{anchor|ProjectOverview}} Project Overview==<br />
===Project Purpose, Scope, and Objectives===<br />
The purpose of the 2009 iGEM Groningen project is [https://2009.igem.org/Judging/Judging_Criteria to have a great summer, and have fun attending the Jamboree].<br />
<br />
===Assumptions and Constraints===<br />
This plan assumes that we can raise enough funds to acquire all the needed materials, and that the university will supply the necessary facilities (like lab space). These will have to be organized before the start of the summer, as both iGEM and our individual schedules require us to do most of the work during the summer. We assume that we can collect enough data during the summer labwork and modelling to be presented during the iGEM Jamboree in oct/nov 2009. <br />
<br />
Our assumptions and constraints were defined in a list of wishes and demands as followed:<br />
<br />
<b><u>Demands</u></b><br />
:*Labwork is feasible in 3 month with 8 students.<br />
:*Modelling feasible in 3 months with 3 students.<br />
:*Everyone has to agree with the idea<br />
:*Financially feasible with a budget of 31.170,00 euro of which 8000 euros used for labwork.<br />
:*A new concept, not yet done before in this way, either the iGEM or in the synthetic biology<br />
:*Meets the criteria of iGEM<br />
:*Materials have to be attainable, either via the RuG or able to order in.<br />
:*Parameters for modelling have to be known, or available or attainable.<br />
:*Knowledge about the genes that we are using has to be available.<br />
<br />
<b><u>Wishes</u></b><br />
:*Not too complicated, not too many genes or gene clusters<br />
:*Has to have an application<br />
:*Using BioBricks that are easy to obtain, preferable available at the RuG.<br />
:*Preferably knowledge on the host, genes and/or end products has to be available at the RuG<br />
:*Some students in the team have experience working with the host.<br />
:*It is possible to find sponsoring for this project.<br />
<br />
==={{anchor|Deliverables}} Project Deliverables===<br />
During the course of this project the following will be delivered:<br />
<br />
*An initial idea for a new "machine" in the iGEM sense, including a preliminary feasibility study.<br />
*A detailed specification of the requirements of this machine.<br />
*A detailed design of this "machine", including an analysis of the usage scenarios and results of simulations of computer models of (parts of) this "machine".<br />
*The actual "machine" (consisting of cells)<br />
*A presentation for the jamboree in November.<br />
*A poster for the jamboree in November<br />
*A report of our findings<br />
<br />
===Evolution of the Project Plan===<br />
The project plan will be set up during the [[Team:Groningen/Project_Plan/Inception|Inception]] phase and after each iteration the [[#ProjectPlan|planning]] is updated to reflect the actual progress and give updated estimates. Other parts of the project plan are not scheduled to be updated after the Inception and will only be updated to fix errors, clarify the existing text or to reflect a change in circumstances.<br />
<br />
==Project Organization==<br />
This is covered by our [[Team:Groningen/Team|team page]] and Google Docs for contact information (for privacy reasons).<br />
<br />
=={{anchor|ManagementProcess}} Management Process==<br />
===Project Estimates===<br />
The estimated costs for the project are as presented in the balance sheet (GoogleDocs). The estimated time for the project is 3-4 hours a week per person from march-july. Full-time (about 40 hours a week per person) during the summer (july-september). From september untill the jamboree on 30 okt another 3-4 hours a week per person (turned out to be a lot more full-time for most people).<br />
<br />
==={{anchor|ProjectPlan}} Project Plan===<br />
This project recognizes the following [http://www.upedu.org/upedu/process/itrwkfls/iwf_iwfs.htm phases and milestones] (only broad descriptions of the milestones are given here):<br />
<br />
*[[Team:Groningen/Project_Plan/Inception|Inception]]: until 18 may, milestone: initial idea, including preliminary feasibility study.<br />
*[[Team:Groningen/Project_Plan/Elaboration|Elaboration]]: until the summer (1st of july), milestone: initial design and modelling.<br />
*[[Team:Groningen/Project_Plan/Construction|Construction]]: during the summer (1 july-30 august), milestone: the actual "product".<br />
*[[Team:Groningen/Project_Plan/Transition|Transition]]: after the summer (30 august-30 okt), milestone: the jamboree presentation (and documentation for the next team?)<br />
<br />
Note that during the elaboration there should probably already be some lab work (if at all possible) to assist in modelling, and during the summer the modelling (and design?) work will probably continue. Detailed planning of each of the iterations is (should be) documented in iteration plans.<br />
<br />
{| border="1"<br />
!Iteration<br />
!End date<br />
!Objectives<br />
|-<br />
|[[Team:Groningen/Project_Plan/Inception/1|Inception 1]]<br />
|2009-04-20<br />
|Narrow down ideas to about three ideas that should be investigated further.<br />
|-<br />
|[[Team:Groningen/Project_Plan/Inception/2|Inception 2]]<br />
|2009-05-18<br />
|Choose idea we're going to work on. The most important requirements should be identified.<br />
|-<br />
|[[Team:Groningen/Project_Plan/Elaboration/1|Elaboration 1]]<br />
|2009-06-01<br />
|The initial design should be made, the requirements refined. Some initial model prototypes should be made to gain experience in modelling.<br />
|-<br />
|[[Team:Groningen/Project_Plan/Elaboration/2|Elaboration 2]]<br />
|2009-06-30<br />
|Models of our design should be made and verified, the design refined. And if possible some things may have to be verified and or tried out in the lab.<br />
|-<br />
|[[Team:Groningen/Project_Plan/Construction/1|Construction 1]]<br />
|2009-07-21<br />
|All necessary equipment and materials should be known and in the lab.<br />
|-<br />
|[[Team:Groningen/Project_Plan/Construction/2|Construction 2]]<br />
|2009-08-11 <br />
|A checkup if the system works, including all parts, should be able to be done.<br />
|-<br />
|[[Team:Groningen/Project_Plan/Construction/3|Construction 3]]<br />
|2009-09-01<br />
|Everything (in the lab) finished :)<br />
|-<br />
|[[Team:Groningen/Project_Plan/Transition/1|Transition 1]]<br />
|15-10-09<br />
|Presentation for the jamboree and the wiki should be (made and) polished.<br />
|-<br />
|[[Team:Groningen/Project_Plan/Transition/2|Transition 2]]<br />
|31-12-09<br />
|Our documentation should be prepared for and transferred to the next team.<br />
|}<br />
<br />
===Project Monitoring and Control===<br />
<br />
====Requirements Management====<br />
The requirements for this system are captured in [[Team:Groningen/Vision|the Vision document]].<br />
<br />
====Quality Control====<br />
The '''Quality of the constructs''' developed by Labworkers should be conform the following quality control regulations<br />
<br />
After transformation of the cells with Your Favorite Construct:<br />
*Pick a few colonies.<br />
**Grow o/n culture of a single colony.<br />
**Continue with miniprep on o/n culture.<br />
**Check insert length by PCR and restriction analysis, as written in document: Quality control (Overdrachts document iGEM 2008).<br />
***For restriction analysis try to find a restriction enzyme which makes a single cut in the vector and a single cut in the insert, or a double digestion with one which cuts in the vector and one which cuts in the insert.<br />
*Streak positive colony with inoculation eye out for single colonies.<br />
**Pick a single colony.<br />
**Grow o/n culture and make glycerol stock (put -80 position on [https://2009.igem.org/Team:Groningen/Parts Parts List]) next day. <br />
**Miniprep and continue with cloning / checking insert length.<br />
<br />
====Reporting and Measurement====<br />
During the non-summer period (march-july, sept-oct) a meeting will be held on a weekly basis. During the meeting the progression of the project, finance, contact with other iGEM teams and organisation subjects will be discussed. Every other week the advisors are invited for this meeting. During the summer period (july-sept) a meeting will also be held on a weekly basis with the whole team. {{todo|A short summary of these meetings will be available on the wiki Notebook.}}<br />
<br />
====Risk Management - NEW====<br />
Risks will be identified in Inception Phase using the steps identified in the RUP for Small Projects activity “Identify and Assess Risks”. Project risk is evaluated at least once per iteration and documented in this table (See [[Team:Groningen/Project_Plan/Risk_List|Risk List]]).<br />
<br />
===={{anchor|ConfigurationManagement}} Configuration Management====<br />
See [[Team:Groningen/Project_Plan/Tools and Documentation|Tools and Documentation]].<br />
<br />
=={{anchor|Process_Guidelines}}{{anchor|UPEDU}} Process Guidelines==<br />
This project attempts to follow a standardized process for software development ([http://www.upedu.org/ UPEDU]), adapting it for use with iGEM. Where possible roles, activities, etc. are simply copied from UPEDU (and ultimately RUP). To cope with the difference between software development and genetic engineering we make the following changes:<br />
<br />
*A new role "[[:Category:Team:Groningen/Roles/Modeller|Modeller]]" for someone who models the design on a computer and attempts to verify the design before it is implemented in the lab.<br />
*The "[[:Category:Team:Groningen/Roles/Implementer|Implementer]]" role is changed to reflect lab work instead of programming.<br />
*We also added some roles to take care of things like keeping minutes, doing PR and so on.<br />
<br />
In practice we ignored most of UPEDU, because of a lack of experience with such approaches within the team (and a lack of time to make up for it), making it inefficient to use, but also because some features proved hard to implement, we specifically had trouble with:<br />
<br />
*'''Using iterative development.''' In software development it is possible to make small changes quickly, allowing iterations as short as one or two weeks (much longer iterations would not have suited the time scale of iGEM). However, in synthetic biology it is more efficient to work on many things at once over a longer time frame (simply because each small change necessarily takes a certain amount of time). In practice this meant that a more waterfall-esque development process was used, where the construction phase consisted mostly of preparing everything for testing and analysis, leaving all testing, analysis and combination of results for the transition phase.<br />
*'''Making a clear separation between requirements(, architecture), design, implementation and tests.''' With software it is possible to create pretty much anything you can define, so one starts by defining what the system should do (at least in part), then figures out how this could be implemented in an abstract way and only then actually implements it (tests can usually be developed in parallel). In part this approach can and should(!) be adopted in synthetic biology, but some things do make it hard to do this rigorously. For example, there is hardly any standard way to abstractly talk about system components yet. Hopefully parts and "devices" might help with this in the near future.<br />
<br />
What did work for us to varying degrees:<br />
<br />
*'''Identifying risks''' early on in the project. Without UPEDU we probably would not have done this and this could have led to severe problems. Luckily not that many of the risks actually materialized, but we did implement some of the mitigation strategies, leading to less nasty surprises. For example, we made an overview of everyone's availability early on in the project and tried to prevent having only one person work on a single part as much as possible.<br />
*'''Using a standardized [[Team:Groningen/methodischontwerpen|design process]].''' Not specifically UPEDU, but it fits well within the framework and it helped us to structure our project selection process.<br />
*'''Recognizing different phases''' in our development. This especially helped in the beginning by giving at least some structure to our planning and providing a vocabulary to talk about such things.<br />
*'''Using iterations.''' Although we had definite problems in our use of iterations they did give some structure to the construction phase for example.<br />
*Describing our '''Tools and Documentation'''. Although not always used consistently it did help us keep an overview what was stored where.<br />
*Having specific '''Roles'''. Not all roles were used (consistently), but it did provide us with at least some structure to deal with eleven team members (it is not easy to do this "free-style").<br />
<br />
=={{anchor|Annexes}} Annexes==<br />
During the inception phase ideas were generated and selected using a [[Team:Groningen/methodischontwerpen|design template]]. For selection of the ideas a [[Team:Groningen/Project_Plan#Assumptions_and_Constraints|list of demands and wishes]] was made.<br />
<br />
{{Team:Groningen/Project_Plan/Footer}}</div>JolandaWitteveenhttp://2009.igem.org/Team:Groningen/HumanPracticeTeam:Groningen/HumanPractice2009-10-21T18:21:06Z<p>JolandaWitteveen: </p>
<hr />
<div>{{Team:Groningen/Header}}<br />
[[Category:Team:Groningen]]<br />
<br />
{|style="clear:both"<br />
|<html><style type="text/css"><br />
.intro { margin-left:0px; margin-top:10px; padding:10px; border-left:solid 5px #FFF6D5; border-right:solid 5px #FFF6D5; text-align:justify;background:#FFFFE5; }<br />
</style></html><br />
<div class="intro"><br />
=Human Practice=<br />
<br />
Ethical issues are important in the new field of synthetic biology. In literature the four issues of Bhutkar are often used. These four issues, [[Team:Groningen/Ethics#safety|safety]], [[Team:Groningen/Ethics#security|security]], [[Team:Groningen/Ethics#PlayingGod| the playing God issue]] and [[Team:Groningen/Ethics#intellectual property| intellectual property]] are also used as guidelines in this project.<br />
In order to gain more knowledge about the opinion of the Dutch people we gave out a [[Team:Groningen/Ethics/Survey|survey]]. This survey was put on our website and handed out to friends and family, a convenience sampling. We did try to reach a mixed public.<br />
From our survey it appeared that most people do not mind that bacteria are modified for research nor for our project. Some concerns to exist, mostly about safety risks, 40%, but also about security risks, 30%. Women are more concerned about using genetically modified organisms (GMO’s) than do men, also younger and less educated people tend to be more concerned about the security issues. <br />
Overall, most people do approve the usage of modified bacteria for our project, however they expect the researcher and developers to consider the safety risks very carefully.<br />
[[Team:Groningen/Ethics|Ethical issues surrounding our project]] also concern the Dutch. Most people find the risk of bacteria ending up in the natural environment and maybe transferring genes to other bacteria or other unwanted side-effects, the most prominent ethical risks of our project. Also the concentrated amounts of heavy metal in side the bacteria concerns the public. Adding a death gene to the bacteria makes it more controllable and careful removal of the bacteria decreases the risks of the concentrated amounts of heavy metals.<br />
From our survey we found that our project is morally correct, however, safety issues should be considered carefully.<br />
Read more about the ethical issues of synthetic biology and our project and the results of our survey [[Team:Groningen/Ethics|here]].<br />
</div><br />
<br><br><br><br />
</div><br />
|}<br />
<br />
{| cellpadding="45"<br />
|{{linkedImage|Eye_Vision.png|Team:Groningen/Vision}}<br />
|{{linkedImage|Hardhat_Safety.png|Team:Groningen/Safety}}<br />
|{{linkedImage|Human_Practice_Balance.png|Team:Groningen/Ethics}}<br />
|{{linkedImage|Balloon_Communication.png|Team:Groningen/Ethics/Survey}}<br />
|}<br />
<br />
{{Team:Groningen/Footer}}</div>JolandaWitteveenhttp://2009.igem.org/Team:Groningen/HumanPracticeTeam:Groningen/HumanPractice2009-10-21T18:17:09Z<p>JolandaWitteveen: </p>
<hr />
<div>{{Team:Groningen/Header}}<br />
[[Category:Team:Groningen]]<br />
=Human Practice=<br />
<br />
Ethical issues are important in the new field of synthetic biology. In literature the four issues of Bhutkar are often used. These four issues, [[Team:Groningen/Ethics#safety|safety]], [[Team:Groningen/Ethics#security|security]], [[Team:Groningen/Ethics#PlayingGod| the playing God issue]] and [[Team:Groningen/Ethics#intellectual property| intellectual property]] are also used as guidelines in this project.<br />
In order to gain more knowledge about the opinion of the Dutch people we gave out a [[Team:Groningen/Ethics/Survey|survey]]. This survey was put on our website and handed out to friends and family, a convenience sampling. We did try to reach a mixed public.<br />
From our survey it appeared that most people do not mind that bacteria are modified for research nor for our project. Some concerns to exist, mostly about safety risks, 40%, but also about security risks, 30%. Women are more concerned about using genetically modified organisms (GMO’s) than do men, also younger and less educated people tend to be more concerned about the security issues. <br />
Overall, most people do approve the usage of modified bacteria for our project, however they expect the researcher and developers to consider the safety risks very carefully.<br />
[[Team:Groningen/Ethics|Ethical issues surrounding our project]] also concern the Dutch. Most people find the risk of bacteria ending up in the natural environment and maybe transferring genes to other bacteria or other unwanted side-effects, the most prominent ethical risks of our project. Also the concentrated amounts of heavy metal in side the bacteria concerns the public. Adding a death gene to the bacteria makes it more controllable and careful removal of the bacteria decreases the risks of the concentrated amounts of heavy metals.<br />
From our survey we found that our project is morally correct, however, safety issues should be considered carefully.<br />
Read more about the ethical issues of synthetic biology and our project and the results of our survey [[Team:Groningen/Ethics|here]].<br />
<br><br />
<br><br />
<br><br />
<br />
{| cellpadding="45"<br />
|{{linkedImage|Eye_Vision.png|Team:Groningen/Vision}}<br />
|{{linkedImage|Hardhat_Safety.png|Team:Groningen/Safety}}<br />
|{{linkedImage|Human_Practice_Balance.png|Team:Groningen/Ethics}}<br />
|{{linkedImage|Balloon_Communication.png|Team:Groningen/Ethics/Survey}}<br />
|}<br />
<br />
{{Team:Groningen/Footer}}</div>JolandaWitteveenhttp://2009.igem.org/Team:Groningen/HumanPracticeTeam:Groningen/HumanPractice2009-10-21T18:10:50Z<p>JolandaWitteveen: </p>
<hr />
<div>{{Team:Groningen/Header}}<br />
[[Category:Team:Groningen]]<br />
=Human Practice=<br />
<br />
Ethical issues are important in the new field of synthetic biology. In literature the four issues of Bhutkar are often used. These four issues, [[Team:Groningen/Ethics#safety|safety]], [[Team:Groningen/Ethics#security|security]], [[Team:Groningen/Ethics#PlayingGod| the playing God issue]] and [[Team:Groningen/Ethics#intellectual property| intellectual property]] are also used as guidelines in this project.<br />
In order to gain more knowledge about the opinion of the Dutch people we gave out a [[Team:Groningen/Ethics/Survey|survey]]. This survey was put on our website and handed out to friends and family, a convenience sampling. We did try to reach a mixed public.<br />
From our survey it appeared that most people do not mind that bacteria are modified for research nor for our project. Some concerns to exist, mostly about safety risks, 40%, but also about security risks, 30%. Women are more concerned about using genetically modified organisms (GMO’s) than do men, also younger and less educated people tend to be more concerned about the security issues. <br />
Overall, most people do approve the usage of modified bacteria for our project, however they expect the researcher and developers to consider the safety risks very carefully.<br />
[[Team:Groningen/Ethics|Ethical issues surrounding our project]] also concern the Dutch. Most people find the risk of bacteria ending up in the natural environment and maybe transferring genes to other bacteria or other unwanted side-effects, the most prominent ethical risks of our project. Also the concentrated amounts of heavy metal in side the bacteria concerns the public. Adding a death gene to the bacteria makes it more controllable and careful removal of the bacteria decreases the risks of the concentrated amounts of heavy metals.<br />
From our survey we found that our project is morally correct, however, safety issues should be considered carefully.<br />
Read more about the ethical issues of synthetic biology and our project and the results of our survey [[Team:Groningen/Ethics|here]].<br />
<br><br />
<br><br />
<br><br />
<br />
{| cellpadding="45"<br />
|{{linkedImage|Eye_Vision.png|Team:Groningen/Vision}}<br />
|{{linkedImage|Hardhat_Safety.png|Team:Groningen/Safety}}<br />
|{{linkedImage|Human_Practice_Balance.png|Team:Groningen/Ethics}}<br />
|{{linkedImage|Balloon_Communication.png|right|100px|Team:Groningen/Ethics/Survey}}<br />
|}<br />
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{{Team:Groningen/Footer}}</div>JolandaWitteveenhttp://2009.igem.org/Team:Groningen/HumanPracticeTeam:Groningen/HumanPractice2009-10-21T18:09:43Z<p>JolandaWitteveen: </p>
<hr />
<div>{{Team:Groningen/Header}}<br />
[[Category:Team:Groningen]]<br />
=Human Practice=<br />
<br />
Ethical issues are important in the new field of synthetic biology. In literature the four issues of Bhutkar are often used. These four issues, [[Team:Groningen/Ethics#safety|safety]], [[Team:Groningen/Ethics#security|security]], [[Team:Groningen/Ethics#PlayingGod| the playing God issue]] and [[Team:Groningen/Ethics#intellectual property| intellectual property]] are also used as guidelines in this project.<br />
In order to gain more knowledge about the opinion of the Dutch people we gave out a [[Team:Groningen/Ethics/Survey|survey]]. This survey was put on our website and handed out to friends and family, a convenience sampling. We did try to reach a mixed public.<br />
From our survey it appeared that most people do not mind that bacteria are modified for research nor for our project. Some concerns to exist, mostly about safety risks, 40%, but also about security risks, 30%. Women are more concerned about using genetically modified organisms (GMO’s) than do men, also younger and less educated people tend to be more concerned about the security issues. <br />
Overall, most people do approve the usage of modified bacteria for our project, however they expect the researcher and developers to consider the safety risks very carefully.<br />
[[Team:Groningen/Ethics|Ethical issues surrounding our project]] also concern the Dutch. Most people find the risk of bacteria ending up in the natural environment and maybe transferring genes to other bacteria or other unwanted side-effects, the most prominent ethical risks of our project. Also the concentrated amounts of heavy metal in side the bacteria concerns the public. Adding a death gene to the bacteria makes it more controllable and careful removal of the bacteria decreases the risks of the concentrated amounts of heavy metals.<br />
From our survey we found that our project is morally correct, however, safety issues should be considered carefully.<br />
Read more about the ethical issues of synthetic biology and our project and the results of our survey [[Team:Groningen/Ethics|here]].<br />
<br><br />
<br><br />
<br><br />
<br />
{| cellpadding="20"<br />
|{{linkedImage|Eye_Vision.png|Team:Groningen/Vision}}<br />
|{{linkedImage|Hardhat_Safety.png|Team:Groningen/Safety}}<br />
|{{linkedImage|Human_Practice_Balance.png|Team:Groningen/Ethics}}<br />
|{{linkedImage|Balloon_Communication.png|right|100px|Team:Groningen/Ethics/Survey}}<br />
|}<br />
<br />
{{Team:Groningen/Footer}}</div>JolandaWitteveenhttp://2009.igem.org/Team:Groningen/HumanPracticeTeam:Groningen/HumanPractice2009-10-21T18:08:51Z<p>JolandaWitteveen: </p>
<hr />
<div>{{Team:Groningen/Header}}<br />
[[Category:Team:Groningen]]<br />
=Human Practice=<br />
<br />
Ethical issues are important in the new field of synthetic biology. In literature the four issues of Bhutkar are often used. These four issues, [[Team:Groningen/Ethics#safety|safety]], [[Team:Groningen/Ethics#security|security]], [[Team:Groningen/Ethics#PlayingGod| the playing God issue]] and [[Team:Groningen/Ethics#intellectual property| intellectual property]] are also used as guidelines in this project.<br />
In order to gain more knowledge about the opinion of the Dutch people we gave out a [[Team:Groningen/Ethics/Survey|survey]]. This survey was put on our website and handed out to friends and family, a convenience sampling. We did try to reach a mixed public.<br />
From our survey it appeared that most people do not mind that bacteria are modified for research nor for our project. Some concerns to exist, mostly about safety risks, 40%, but also about security risks, 30%. Women are more concerned about using genetically modified organisms (GMO’s) than do men, also younger and less educated people tend to be more concerned about the security issues. <br />
Overall, most people do approve the usage of modified bacteria for our project, however they expect the researcher and developers to consider the safety risks very carefully.<br />
[[Team:Groningen/Ethics|Ethical issues surrounding our project]] also concern the Dutch. Most people find the risk of bacteria ending up in the natural environment and maybe transferring genes to other bacteria or other unwanted side-effects, the most prominent ethical risks of our project. Also the concentrated amounts of heavy metal in side the bacteria concerns the public. Adding a death gene to the bacteria makes it more controllable and careful removal of the bacteria decreases the risks of the concentrated amounts of heavy metals.<br />
From our survey we found that our project is morally correct, however, safety issues should be considered carefully.<br />
Read more about the ethical issues of synthetic biology and our project and the results of our survey [[Team:Groningen/Ethics|here]].<br />
<br><br />
<br><br />
<br><br />
<br />
{|<br />
|{{linkedImage|Eye_Vision.png|Team:Groningen/Vision|100px}}<br />
|-<br />
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|-<br />
|{{linkedImage|Human_Practice_Balance.png|Team:Groningen/Ethics}}<br />
|-<br />
|{{linkedImage|Balloon_Communication.png|right|100px|Team:Groningen/Ethics/Survey}}<br />
|}<br />
<br />
{{Team:Groningen/Footer}}</div>JolandaWitteveenhttp://2009.igem.org/Team:Groningen/HumanPracticeTeam:Groningen/HumanPractice2009-10-21T18:08:29Z<p>JolandaWitteveen: </p>
<hr />
<div>{{Team:Groningen/Header}}<br />
[[Category:Team:Groningen]]<br />
=Human Practice=<br />
<br />
Ethical issues are important in the new field of synthetic biology. In literature the four issues of Bhutkar are often used. These four issues, [[Team:Groningen/Ethics#safety|safety]], [[Team:Groningen/Ethics#security|security]], [[Team:Groningen/Ethics#PlayingGod| the playing God issue]] and [[Team:Groningen/Ethics#intellectual property| intellectual property]] are also used as guidelines in this project.<br />
In order to gain more knowledge about the opinion of the Dutch people we gave out a [[Team:Groningen/Ethics/Survey|survey]]. This survey was put on our website and handed out to friends and family, a convenience sampling. We did try to reach a mixed public.<br />
From our survey it appeared that most people do not mind that bacteria are modified for research nor for our project. Some concerns to exist, mostly about safety risks, 40%, but also about security risks, 30%. Women are more concerned about using genetically modified organisms (GMO’s) than do men, also younger and less educated people tend to be more concerned about the security issues. <br />
Overall, most people do approve the usage of modified bacteria for our project, however they expect the researcher and developers to consider the safety risks very carefully.<br />
[[Team:Groningen/Ethics|Ethical issues surrounding our project]] also concern the Dutch. Most people find the risk of bacteria ending up in the natural environment and maybe transferring genes to other bacteria or other unwanted side-effects, the most prominent ethical risks of our project. Also the concentrated amounts of heavy metal in side the bacteria concerns the public. Adding a death gene to the bacteria makes it more controllable and careful removal of the bacteria decreases the risks of the concentrated amounts of heavy metals.<br />
From our survey we found that our project is morally correct, however, safety issues should be considered carefully.<br />
Read more about the ethical issues of synthetic biology and our project and the results of our survey [[Team:Groningen/Ethics|here]].<br />
<br><br />
<br><br />
<br><br />
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{|<br />
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|{{linkedImage|Human_Practice_Balance.png|Team:Groningen/Ethics}}<br />
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|{{linkedImage|Balloon_Communication.png|right|100px|Team:Groningen/Ethics/Survey}}<br />
|}<br />
<br />
{{Team:Groningen/Footer}}</div>JolandaWitteveenhttp://2009.igem.org/File:Human_Practice_Balance.pngFile:Human Practice Balance.png2009-10-21T18:07:19Z<p>JolandaWitteveen: uploaded a new version of "Image:Human Practice Balance.png"</p>
<hr />
<div></div>JolandaWitteveenhttp://2009.igem.org/File:Eye_Vision.pngFile:Eye Vision.png2009-10-21T18:03:56Z<p>JolandaWitteveen: uploaded a new version of "Image:Eye Vision.png"</p>
<hr />
<div></div>JolandaWitteveenhttp://2009.igem.org/File:Balloon_Communication.pngFile:Balloon Communication.png2009-10-21T18:01:35Z<p>JolandaWitteveen: uploaded a new version of "Image:Balloon Communication.png"</p>
<hr />
<div></div>JolandaWitteveenhttp://2009.igem.org/File:Hardhat_Safety.pngFile:Hardhat Safety.png2009-10-21T17:57:12Z<p>JolandaWitteveen: uploaded a new version of "Image:Hardhat Safety.png"</p>
<hr />
<div></div>JolandaWitteveenhttp://2009.igem.org/File:Hardhat_Safety.pngFile:Hardhat Safety.png2009-10-21T17:55:47Z<p>JolandaWitteveen: uploaded a new version of "Image:Hardhat Safety.png"</p>
<hr />
<div></div>JolandaWitteveenhttp://2009.igem.org/Team:Groningen/HumanPracticeTeam:Groningen/HumanPractice2009-10-21T17:46:23Z<p>JolandaWitteveen: </p>
<hr />
<div>{{Team:Groningen/Header}}<br />
[[Category:Team:Groningen]]<br />
=Human Practice=<br />
<br />
Ethical issues are important in the new field of synthetic biology. In literature the four issues of Bhutkar are often used. These four issues, [[Team:Groningen/Ethics#safety|safety]], [[Team:Groningen/Ethics#security|security]], [[Team:Groningen/Ethics#PlayingGod| the playing God issue]] and [[Team:Groningen/Ethics#intellectual property| intellectual property]] are also used as guidelines in this project.<br />
In order to gain more knowledge about the opinion of the Dutch people we gave out a [[Team:Groningen/Ethics/Survey|survey]]. This survey was put on our website and handed out to friends and family, a convenience sampling. We did try to reach a mixed public.<br />
From our survey it appeared that most people do not mind that bacteria are modified for research nor for our project. Some concerns to exist, mostly about safety risks, 40%, but also about security risks, 30%. Women are more concerned about using genetically modified organisms (GMO’s) than do men, also younger and less educated people tend to be more concerned about the security issues. <br />
Overall, most people do approve the usage of modified bacteria for our project, however they expect the researcher and developers to consider the safety risks very carefully.<br />
[[Team:Groningen/Ethics|Ethical issues surrounding our project]] also concern the Dutch. Most people find the risk of bacteria ending up in the natural environment and maybe transferring genes to other bacteria or other unwanted side-effects, the most prominent ethical risks of our project. Also the concentrated amounts of heavy metal in side the bacteria concerns the public. Adding a death gene to the bacteria makes it more controllable and careful removal of the bacteria decreases the risks of the concentrated amounts of heavy metals.<br />
From our survey we found that our project is morally correct, however, safety issues should be considered carefully.<br />
Read more about the ethical issues of synthetic biology and our project and the results of our survey [[Team:Groningen/Ethics|here]].<br />
<br />
{|<br />
|{{linkedImage|Eye_Vision.png|Team:Groningen/Vision|100px}}<br />
|{{linkedImage|Hardhat_Safety.png|center|100px|Team:Groningen/Safety}}<br />
|{{linkedImage|Human_Practice_Balance.png|center|100px|Team:Groningen/Ethics}}<br />
|{{linkedImage|Balloon_Communication.png|right|100px|Team:Groningen/Ethics/Survey}}<br />
|}<br />
<br />
{{Team:Groningen/Footer}}</div>JolandaWitteveenhttp://2009.igem.org/Team:Groningen/Project/TransportTeam:Groningen/Project/Transport2009-10-21T16:49:21Z<p>JolandaWitteveen: </p>
<hr />
<div>{{Team:Groningen/Project/Header|}}<br />
<div title="Arsie Says UP TO ACCUMULATION" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Project/Accumulation}}</div><br />
<br />
{| style="clear:both"<br />
|<html><style type="text/css"><br />
.intro { margin-left:0px; margin-top:10px; padding:10px; border-left:solid 5px #FFF6D5; border-right:solid 5px #FFF6D5; text-align:justify;background:#FFFFE5; }<br />
</style></html><br />
<div class="intro"><br />
<h2>Transport</h2><br />
'''To isolate heavy metals from the environment we require uptake systems. So far we found several different mechanisms to create such a system. We investigated three kinds of metal uptake:'''<br />
*Metal transporters, Membrane proteins that transport the metal from the environment (<i>i.e.</i> wastewater) to the cytoplasm<br />
**Uncoupled<br />
**Coupled with 'helper' compounds<br />
*Metal binding proteins in the periplasm<br />
<br />
'''We have investigated several systems to determine which are suitable for the final design. The following systems are under consideration:'''<br />
<br />
*Arsenite uptake via GlpF<br />
*Copper/zinc uptake via HmtA<br />
*Heavy metal uptake coupled to citrate via ''ef''CitH ''bs''CitM <br />
*Periplasmic accumulation of heavy metals via Mer Operon.<br />
<br />
'''We chose to focus on GlpF and HmtA, the final device was made with GlpF for arsenate purification. '''<br />
<br><br><br><br />
</div><br />
|}<br />
<br />
<br />
<br />
==Arsenite uptake via GlpF==<br />
<!--[[Image:GlpF.jpeg|200px|thumb|right|73As(III) and 125Sb(III) uptake into cells of E. coli is facilitated by the aquaglyceroporin channel GlpF.]]--><br />
<br />
===GlpF===<br />
<br />
====Introduction====<br />
GlpF is an aquaglycerol porin of E.coli which facilitates not only glycerol import, but also arsenic (As) and antimone (Sb) import [[Team:Groningen/Literature#Fu, DX, et al.2000|(Fu, DX, et al.2000]]), [[Team:Groningen/Literature#Meng, YL, et al.2004|(Meng, YL, et al.2004]]), [[Team:Groningen/Literature#Porquet, A, et al.2007|(Porquet, A, et al.2007]]), [[Team:Groningen/Literature#Rosen, BR, et al.2009|(Rosen, BR, et al.2009)]] . It has homologues in other organisms; Fps1p has shown to facilitate arsenic import in yeast and AQP9 is the mammalian homologue [[Team:Groningen/Literature#Porquet, A, et al.2007|(Porquet, A, et al.2007]]), [[Team:Groningen/Literature#Rosen, BR, et al.2009|(Rosen, BR, et al.2009)]] .<br />
The GlpF aquaglycerol porin is a membrane protein with a symmetric arrangement of four independent GlpF channels. One monomer of this tetramer GlpF porin consists of six transmembrane and two half membrane-spanning α-helices that form a right-handed helical bundle around the channel. The channel has a diameter of ~15Å at the periplasmid end, which constricts towards a diameter of ~3.8Å at the beginning of a 28 Å long selective channel that ends at the cytoplasmic end [[Team:Groningen/Literature#Fu, DX, et al.2000|(Fu, DX, et al.2000)]].<br />
The GlpF is a stereospecific channel that is thought to be more selective on molecular size than on chemical structure [[Team:Groningen/Literature#Fu, DX, et al.2000|(Fu, DX, et al.2000]], [[Team:Groningen/Literature#Heller, KB, et al.1980|(Heller, KB, et al.1980)]] . It does allow transport of a variance of non-charged compounds ranging from polyhydric alcohols, glycerol being one of them, arsenic to antimone [[Team:Groningen/Literature#Fu, DX, et al.2000|(Fu, DX, et al.2000]]), [[Team:Groningen/Literature#Meng, YL, et al.2004|(Meng, YL, et al.2004]]), [[Team:Groningen/Literature#Porquet, A, et al.2007|(Porquet, A, et al.2007)]], [[Team:Groningen/Literature#Rosen, BR, et al.2009|(Rosen, BR, et al.2009]]), [[Team:Groningen/Literature#Heller, KB, et al.1980|(Heller, KB, et al.1980)]]. Carbon sugars and ions are shown to be unable to be transported by GlpF [[Team:Groningen/Literature#Heller, KB, et al.1980|(Heller, KB, et al.1980)]]. At physiological pH arsenic and antimone are not present in their ionic state but rather as As(OH)3 and Sb(OH)3 [[Team:Groningen/Literature#Rosen, BR, et al.2009|(Rosen, BR, et al.2009)]]. These elements show a charge distribution similar to glycerol and a smaller but comparable volume. The structural similarities are thought to be the reason for the possibility of these elements to enter the cell by GlpF [[Team:Groningen/Literature#Porquet, A, et al.2007|(Porquet, A, et al.2007)]], GlpF facilitates transport of these compounds down there gradient (inside or outside the cell).<br />
If GlpF behaves as a nonsaturable transporter, a transport rate of 1umol of glycerol is transported per minute per mgr of cell protein [[Team:Groningen/Literature#Heller, KB, et al.1980|(Heller, KB, et al.1980)]]. The transport rate of GlpF for arsenic is estimated to be……<br />
<br />
====Cloning strategy====<br />
This part has been obtained from the genome of ''E.coli'' 356 in two steps with PCR. First the whole gene was obtained from the genome by using PCR and in the second step an ''EcoR''1 restiction site was removed.<br />
The GlpF PCR product was restricted with ''Xba''I and ''Pst''I and a psB1AC3 vector with a pMed promotor was restricted with ''Spe''I and ''Pst''I. The restriction products were ligated. This resulted in a psB1AC3 vector with a promotor and GlpF.<br />
[[Image:RestictioLigationGlpF.JPG]]<br />
<br />
====Results====<br />
The ability of GlpF (overexpressed under lactose induction) to transport As(III) was tested by an arsenite uptake [https://2009.igem.org/Team:Groningen/Protocols assay]. Also the full accumulation device (<partinfo>BBa_K190038</partinfo>) was tested using this assay. '''Data and analysis can be found [https://2009.igem.org/Team:Groningen/Project/Accumulation here]. <br />
'''<br />
The expression of GlpF was not tested by SDS-PAGE or protein purification.<br />
<br />
===Modelling uptake GlpF===<br />
<html><br />
<script type="text/javascript" src="/Team:Groningen/Modelling/Model.js?action=raw"></script><br />
<script type="text/javascript" src="/Team:Groningen/Modelling/Arsenic.js?action=raw"></script><br />
</html><br />
<html><style type="text/css"></html><br />
{{InfoBox/Style.css}}<br />
.infoIcon { display: inline; }<br />
<html></style></html><br />
The import of As(III) via GlpF is modelled as a simple import reaction with [[Team:Groningen/Glossary#MichaelisMenten|Michaelis-Menten kinetics]], in part because this makes it easy to specify, but also because we only have very high level data. The following allows a comparison with the data acquired from figure 1B from [[Team:Groningen/Literature#Meng2004|Meng 2004]].<br />
<html><br />
<div style="background:#efe;border:1px solid #9c9;padding:1em;"><br />
<table style="border-collapse:collapse;background:none;"><tr><br />
<td style="border-right:1px solid #9c9;padding-right:1em;"><br />
<dl><br />
<dt>Initial values</dt><br />
<dd><br />
As(III)<sub>ex</sub> = <input type="text" id="As3exInitial" value="9.15164271986822"/> &micro;M<br/><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(10&micro;M &middot; 1mL / 1.092mL)<br />
</dd><br />
<dt>Volumes</dt><br />
<dd><br />
V<sub>total</sub> = <input type="text" id="Vtotal" value="1.1"/> mL<br/><br />
V<sub>cells</sub> = <input type="text" id="Vcells" value="0.0073"/> mL<br/><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(0.1ml &middot; 80mg/mL / 1100mg/mL) </html>{{infoBox|E. coli has a density of approximately 1100mg/mL, see [[Team:Groningen/Project/Vesicle|our gas vesicle page]] for more information.}}<html><br />
</dd><br />
<dt>Kinetic Constants</dt><br />
<dd><br />
<nobr>v5 = <input type="text" id="v5" value="3.1862846729357852"/> &micro;mol/(s&middot;L)</nobr><br/><br />
K5 = <input type="text" id="K5" value="27.71808199428998"/> &micro;M<br/><br />
</dd><br />
</dl><br />
<br />
<button onClick="computeGlpFTransport()">Compute</button><br/><br />
</td><br />
<br />
<td style="padding-left:1em;"><br />
<div id="glpFTransportError" style="color:red"></div><br />
</html>{{graph|Team:Groningen/Graphs/GlpFTransport|id=glpFTransportGraph}}<html><br />
</td><br />
</tr></table><br />
</div><br />
<script type="text/javascript"><br />
<br />
//The graph already initializes itself (and we don't do any other computations).<br />
//addOnloadHook(computeGlpFTransport);<br />
<br />
function computeGlpFTransport() {<br />
document.getElementById('glpFTransportGraph').refresh();<br />
}<br />
</script><br />
</html><br />
<br />
To determine the constants v5 and K5 we performed the following steps:<br />
<br />
# '''Read the wild-type line in figure 1B''' of [[Team:Groningen/Literature#Meng2004|Meng 2004]] by pasting it in a drawing program and aligning/scaling the axes and then manually determining the coordinates of each data point.<br />
# '''Converted to units of concentration''' using the data in Meng 2004 and [http://gchelpdesk.ualberta.ca/CCDB/cgi-bin/STAT_NEW.cgi the CCDB] (assuming that the cells are resting/non-growing), see our [http://spreadsheets.google.com/pub?key=t4gilzCbEaCFAvpEVWUE_zQ Google Docs spreadsheet]. Here we disregarded the fact that the measurements were made by taking out 0.1mL samples, as this does not change the concentrations. Specifically (note that uptake is in nmol/mg):<br />
#* uptake<sub>total</sub> (nmol) = uptake &middot; 8mg &middot; 0.3 {{infoBox|The ratio between dry and wet weight is 0.3 (see the [http://gchelpdesk.ualberta.ca/CCDB/cgi-bin/STAT_NEW.cgi CCDB]).}}<br />
#* As(III)<sub>ex</sub> (&micro;M=nmol/mL) = (10nmol/mL &middot; 1mL - uptake<sub>total</sub>) / (1.1-0.0073)mL {{infoBox|1=The experiment started with 1mL of a 10&micro;M=10nmol/mL solution of As(III). After adding the cells the total volume of the solution was 1.1mL, and 0.0073mL is an estimate of the total volume of cells in the solution, see below.}}<br />
# '''Fit the Michaelis-Menten equation''' to find the constants v5 and K5 in Mathematica (see [http://igemgroningen.googlecode.com/svn/trunk/buoyant/Models/Meng2004%20Figure%201B.nb the Mathematica notebook in SVN]) using the method from [[Team:Groningen/Literature#Goudar1999|Goudar 1999]] (a least squares fit of a closed-form solution of the differential equation).<br />
<br />
{{GraphHeader}}<br />
<br />
<br><br />
<br />
===Missing information/To Do===<br />
*Expression assesment<br />
**Stability<br />
**Level<br />
*Functional assesment<br />
**Uptake speed<br />
**Affinity<br />
**Electrolyte potential generating force<br />
*<del>Q:Eliminate BioBrick restriction sites</del><br />
*<del>Q: What does the ars operon of our <i>E. coli</i> look like? Do we have both ArsA and ArsB? (And what about ArsR and ArsD?)</del> A: We only have ArsRBC, see [[Team:Groningen/BLAST|our BLAST results]].<br />
<br />
<br><br />
<br />
===Additional sources===<br />
<br><br />
* [[Team:Groningen/Literature#Meng2004|Meng 2004]] (As(III) and Sb(III) Uptake by GlpF and Efflux by ArsB in Escherichia coli)<br />
* [[Team:Groningen/Literature#Rosen2009|Rosen 2009]] (Transport pathways for arsenic and selenium: A minireview)<br />
*[[Team:Groningen/Literature#Porquet, A, et al.2007|Porquet, A, et al.2007]] (structural similarity between As(OH)3 and glycerol)<br />
* [[Team:Groningen/Literature#Fu, DX, et al.2000|Fu, DX, et al.2000]] (Structure of the GlpF channel)<br />
*[[Team:Groningen/Literature#Heller, KB, et al.1980|Heller, KB, et al.1980]] (Glycerol transport properties of GlpF)<br />
<br />
==Copper/zinc uptake via HmtA==<br />
<br />
===HmtA===<br />
====Introduction====<br />
HmtA(heavy metal transporter A) from <i>Pseudomonas aeruginosa</i> [http://www.ncbi.nlm.nih.gov/protein/81857196 Q9I147] is a P-type ATPase importer. This membrane protein mediates the uptake of copper (Cu) and zinc (Zn) and was functionally expressed in E.coli ([http://www.ncbi.nlm.nih.gov/pubmed/19264958 Lewinson 2009]). We want to use this membrane protein to accumulate copper and zinc into the cells. we believe this ATP-driven pump is capable of generating an elevated intracellular concentration of these compounds enabling the harvesting of copper and zinc from the medium.<br />
<br />
====Cloning strategy====<br />
<br />
There are several restriction sites to be modified from [https://static.igem.org/mediawiki/2009/8/85/PBAD-HmtA-ClonemanagerFile.zip Lewinson's] pBAD construct. A vector with amp resistance with L-arabinose inducible HmtA-6HIS. The restriction sites have been silently mutated maintaining the amino acid sequence.<br />
We will create these mutations via PCR than digest the old methylated template and clone the product into competent cells.<br />
<br />
====Results==== <br />
[[Image:HmtA_SDS_gel.jpg|200px|thumb|right|[Team:Groningen/Team|HmtA-6HIS on SDS-page]]<br />
So far we have cloned HmtA as a biobrick without EcoRI site in the coding region into the iGEM vector. Unfortunately a mutation occurred at base 103 from the start of the orf. By a point mutation c to t in the first nucleotide of the codon changed arginine 35 to a Cysteine. We do not know the effects but we suspect it might have a great influence due to the very reactive side chain of Cysteine, eventhough it is not in the channel itself based on [http://www.cbs.dtu.dk/services/TMHMM/ TMHMM] predictions which indicate trans membrane helices of a protein. Further cloning is expected to be unsuccessful because the iPTG induced clones grow even slightly better than the empty vector control. This is most likely cause by the missing pLAC-RBS in front of the gene. There was no positive controle with the L-arabinose inducable HmtA-6His in pBAD. We did do expression experiments with the pBAD construct to purified the membrane protein as quality controle. result shown in the figure on the right.<br />
<br />
==Heavy metal uptake coupled to citrate via ''ef''CitH ''bs''CitM==<br />
<br />
Force feeding of the heavy metals into the cell is possible when citrate is the only available carbon source. Citrate in complex with heavy metals can be translocated over the membrane into the cell via citrate transporters.<br />
This can be a very efficient strategy to accumulate vast ammounts of heavy metals.<br />
The two membrane proteins are CitM from ''Bacillus subtilis'' studied by [http://www.ncbi.nlm.nih.gov/pubmed/11053381 B.P Krom]. <i>Bs</i>CitM can transport citrate in complex with Mg<sup>2+</sup>, Ni<sup>2+</sup>, Mn<sup>2+</sup>, Co<sup>2+</sup>, and Zn<sup>2+</sup>. <br />
The other is CitH from ''Enterococcus faecalis'' described by [http://www.ncbi.nlm.nih.gov/pubmed/17042778 V.S Blancato]. <i>Ef</i>CitH catalyzes translocation of the citrate in complex with Ca<sup>2+</sup>, Sr<sup>2+</sup> Mn<sup>2+</sup> Mn<sup>2+</sup> Cd<sup>2+</sup> and Pb<sup>2+</sup>.<br />
<br />
<br />
===Additional sources===<br />
<br />
More information on this topic can be found in:<br />
<br />
Bastiaan Krom. Citrate transporters of <i>Bacillus subtilis</i> PhD thesis. [[http://dissertations.ub.rug.nl/faculties/science/2002/b.p.krom/ Dissertation Groningen]]<br />
<br />
Jessica B. Warner. Regulation and expression of the metal citrate transporter CitM PhD thesis. [[http://dissertations.ub.rug.nl/faculties/science/2002/j.b.warner/ Dissertation Groningen]]<br />
<br />
==Periplasmic accumulation of heavy metals via Mer Operon==<br />
Periplasmic accumulation of heavy metals via Mer proteins enables the harvesting of heavy metals from the medium by binding the cytosolic and periplasmic metals to metallothionein and transporting the metal-protein complex into the periplasm.<br />
The MerR family consists of different proteins for one specific metal (<i>i.e.</i><br />
PbrR (lead), CueR (copper), ZntR (zinc), MerR (mercury), ArsR (arsenic), CadR (cadmium)).<br />
<br />
As the cells die after uptake of Mg (and induction of the Mer transporter), this system is not very well usable for our project. The dead cells will not produce the gas vesicles (it may be used however by having the gas vesicles consitutively expressed), thereby bouyancy may be a problem ([[Team:Groningen/Literature#Pennella2005|Pennella 2005]], [[Team:Groningen/Literature#Kao2008|Kao 2008]]).<br />
<br />
===Missing information/To Do===<br />
*Expression assesment<br />
**Stability<br />
**Level<br />
*Functional assesment<br />
**Uptake speed<br />
**Affinity<br />
**Electrolyte potential generating force<br />
*Eliminate BioBrick restriction sites<br />
<br />
==Planning and requirements==<br />
<br />
* '''Modelling:'''<br />
** Import speed<br />
** Amount <br />
** Max<br />
* '''Lab:'''<br />
** HmtA<br />
*** Zn/Cu alone<br />
*** B-type ATPase (could be use if there is a ATP shortage?)<br />
** CitM (probably not used)<br />
*** Divalent ions<br />
*** Citrate around<br />
*** Citrate can bind metals that are already bound.<br />
** Measurements (both for the "normal" cell and the cell with overexpression of the transporter)<br />
*** Transporter, on/off mechanism, up to what concentration (in the cell) does it still have metal uptake.<br />
*** Measure concentration of metal. difference between begin and end concentrations of metal outside the cell.<br />
*** How fast does it transport metal in/out the cell.<br />
**** Set up tests with (initial) extracellular concentrations of about <sup>1</sup>/<sub>3</sub>K (25% of V<sub>max</sub>), K (50% of V<sub>max</sub>), 3K (75% of V<sub>max</sub>) and 10mM (99.7% of V<sub>max</sub>, corresponding to extremely polluted water), and a control with no arsenic. Obviously, more tests is better. In general a desired fraction of V<sub>max</sub> at the initial concentration can be attained by using an initial concentration of x/(1-x) times K.<br />
**** Determine "final" (steady-state) concentration of As(III) in the solution and in the cells. (Concentration over time is even better!)<br />
**** This means that the total volume of the cells (and the solution) has to be determined. Possibly through looking at the dry weight (without arsenic!).<br />
**** By manipulating the equation for the derivative of As(III) in equilibrium, As(III) can be expressed as a function of As(III)<sub>ex</sub> (given the V and K constants). We can try to fill in the computed V and K constants for GlpF and then use a least squares fit to estimate the V and K constants for ArsB.<br />
**** '''NOTE:''' Interestingly [[Team:Groningen/Literature#Kostal2004|Kostal 2004]] already did an experiment like this with cells that overexpressed ArsR. We're looking at analysing these results under the assumption that overexpressing ArsR only gives a constant factor more accumulation (for 1-100&microM As(III)), but it would be very nice to do this ourselves for unmodified cells to determine whether this is indeed true (and to determine the factor).<br />
<br />
==Export of arsenicum via Ars operon==<br />
<br />
GlpF is the importer of arsenicum. After arsenicum enters the cell, in response the Ars operon produces ArsR. At the same time, ArsB is also produced by Ars operon. This happens because the Ars operon contains three open reading frames: the first is ArsR, second ArsB and the last one is ArsC. ArsB is the exporter of arsenicum. The ars operon is located on the chromosomal DNA of E. coli.<br />
For more information see: [http://biocyc.org/ECOLI/NEW-IMAGE?type=GENE-IN-CHROM-BROWSER&object=EG12235 biocyc].<br />
<br />
[[Image:ArsRBC_operon.PNG|600px]]<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
{{Team:Groningen/Project/Footer}}</div>JolandaWitteveenhttp://2009.igem.org/Team:Groningen/Project/WholeSystemTeam:Groningen/Project/WholeSystem2009-10-21T16:44:40Z<p>JolandaWitteveen: </p>
<hr />
<div>{{Team:Groningen/Header|}}<br />
<br />
{|style="clear:both"<br />
|<html><style type="text/css"><br />
.intro { margin-left:0px; margin-top:10px; padding:10px; border-left:solid 5px #FFF6D5; border-right:solid 5px #FFF6D5; text-align:justify;background:#FFFFE5; }<br />
</style></html><br />
<div class="intro"><br />
<h2>Whole System</h2><br />
'''The metal scavenger with a vertical gas drive is a modular system. The system is explored for arsenic in this project. The accumulation module was made out of the aquaglyverol porin, GlpF, that was used as importer and the sequestering protein fMT. On a complementary plasmid the gas vesicle protein (GVP) cluster was cloned downstream of the arsenic sensitive promotor ars. The two plasmids were transformed in one cell and analysed using the buoyancy assay. This showed succesfull transformation and a buoyancy phenotype.'''<br />
</div><br />
<br><br><br><br />
</div><br />
|}<br />
<br />
==Introduction==<br />
<br />
The metal scavenger with a vertical gas drive is a modular system which contains five modules; the transporter, an accumulation protein, a regulated promoter, a regulator and the gas vesicle cluster. The transporter imports the metal ion of choice. An accumulation protein facilitates accumulation of metal ions and prevents the cell from dying of the metal toxicity. The imported metal ion also acts as a regulator for the metal sensitive promotor which activates the expression of the gvp-cluster. Thereby the recombinant bacterium, with this system, accumulates metal and upon accumulation starts to float which is convenient in bioremediation or mining [[Team:Groningen/Application|applications]]. But in principle this system may be used in order to accumulate any component for which these modules are available!<br />
<br><br />
The system explored in this project was mainly based on arsenic, however, aspects of a copper/zinc system were also explored. For the copper/zinc system [[Team:Groningen/Project/Transport#Copper/zinc uptake via HmtA|HmtA]], (<partinfo>BBa_K190018</partinfo>) was chosen as transporter, [[Team:Groningen/Project/Accumulation#Copper|Mymt]] and [[Team:Groningen/Project/Accumulation#Zinc|SmtA]] as accumulators and [[Team:Groningen/Project/Promoters#Copper Induced Promoters|CueO]], (<partinfo>BBa_K190017</partinfo>) and [[Team:Groningen/Project/Promoters#Zinc Induced Promoters|ZntR]], (<partinfo>BBa_K190016</partinfo>) as metal sensitive promotors.<br />
<br />
Arsenic is a very toxic metal and [[Team:Groningen/Application|causes health problems all over the world]]. The transporter used in this system is [[Team:Groningen/Project/Transport#Arsenite uptake via GlpF|GlpF]] (<partinfo>BBa_K190028</partinfo>) a glycerol transported known to import arsenic as well [[Team:Groningen/Literature#Meng, YL, et al.2004|(Meng, YL, et al.2004)]], [[Team:Groningen/Literature#Rosen, BR, et al.2009|(Rosen, BR, et al.2009)]]. Two accumulation proteins were chosen to accumulate arsenic, the fusion protein [[Team:Groningen/Project/Accumulation#Arsenic| MBP-ArsR]] (<partinfo>BBa_K190027</partinfo>) and [[Team:Groningen/Project/Accumulation#Arsenic|fMT]] (<partinfo>BBa_K190019</partinfo>). Both accumulation proteins were combined with GlpF to create new accumulation devices (<partinfo>BBa_K190073</partinfo>, <partinfo>BBa_K190038</partinfo>). As metal sensitive promotor, [[Team:Groningen/Project/Promoters|pArsR]] was chosen and together with the gvp cluster it makes up a buoyancy device (<partinfo>BBa_K190033</partinfo>). The two devices, the accumulation device and the buoyancy device were transformed together in ''E. coli'', to create the metal scavenger with a vertical gas drive.<br />
<br />
==Cloning Strategy==<br />
In order to use the buoyancy device(<partinfo>BBa_K190033</partinfo>) in combination with the accumulation device (<partinfo>BBa_K190038</partinfo>, <partinfo>BBa_K190039</partinfo>) a two vector system was used (see Figure 1). A two vector system was chosen because it is not feasible to combine the 6kb gas vesicle cluster, with a 1kb transporter and 300bp metallothionein and their promoters in one vector. This could easily increase the size of a vector to 10kb, a vector size which can hardly be transformed to ''E. coli''. <br />
This two vector system is composed of a <partinfo>pSB1A2</partinfo> vector with the accumulation device(<partinfo>BBa_K190038</partinfo>, <partinfo>BBa_K190039</partinfo>) and a <partinfo>pSB2K3</partinfo> vector with the buoyancy device (<partinfo>BBa_K190033</partinfo>). This combination was chosen because this makes it possible to tranform both vectors in one ''E.coli'' cell, since the vectors contain different origin of replications and antibiotic resistance markers. They contain respectively a pMB1 origin of replication and a P1 ori.<br />
<br />
[[Image:Whole system.PNG]] <br />
:Figure 1: Whole system, combining the bouyancy device on a pSB2K3 vector and the accumulation device on a pSB1AC3 vector<br />
<br />
==Results==<br />
<br />
Combining parts <partinfo>BBa_K190033</partinfo> and <partinfo>BBa_K190038</partinfo> was done by using a normal [[Team:Groningen/Protocols|transformation protocol]] with both ampicillin and kanamycin as antibiotics. A [[Team:Groningen/Protocols|buoyancy test]] was performed as described also using both ampicillin and kanamycin as antibiotics. Iptg was also added to the dayculture to induce the <partinfo>BBa_K190033</partinfo> part. In exponential phase 10μM NaAsO<sub>2</sub> was added to half of the samples.<br />
A [[Team:Groningen/Protocols|restriction]] was done to check the transformation. <br />
<br />
====Restriction====<br />
Figure 2 shows that the transformation of both vectors succeeded.<br />
<br />
[[Image: Groningen_GelPhotoSystemRestriction.png|400px]]<br />
<br />
:Figure 2:Gel image of restricted and not restricted plasmids.<br />
<br />
<br />
The gel shows a 1Kb marker to the far left, after that two times a restriction with ''EcoR''I and ''Pst''I can be seen. This shows clearly fragments of 6000pb and 4500pb for the <partinfo>BBa_K190033</partinfo> and fragments of 2000pb and 1300 bp for the <partinfo>BBa_K190038</partinfo>. The next two slots show non-restricted plasmids, two distinc bands can be seen clearly indicating the presence of two plasmids.<br />
<br />
====Buoyancy test====<br />
The buoyancy test elegantly shows that combining both an accumulation device and the gvp-buoyancy device allows buoyancy. <br />
<br />
[[Image:Groningen_SystemBouyancy33en38.png|300px]]<br />
<br />
:Figure 3: Result of the buoyancy test, the left shows ''E.coli'' cells without arsenic induction and to the right floating ''E.coli'' cells, induced with arsenic, are shown.<br />
<br />
<br />
On the left ''E. coli'' cells (containing the whole system) without arsenic can be seen and on the right ''E. coli'' cells (containing the whole system) with arsenic is shown. It can be seen that the right sample floats. This indicates that the GlpF is transporting the arsenic inside the cells, the fMT accumulates it (otherwise the cells would be dead) and the gvp was induced by the arsenic so the cells start to float.<br />
<br />
[[Image:Groningen_BouyancyOf33en38.png|200px]]<br />
:Figure 4: Results of the buoyancy test, left ''E.coli'' with the gvp plasmid, the right tube shows ''E.coli'' cells with the fMT-glpF device. <br />
<br />
As a control ''E.coli'' cells were also transformed with the gvp plasmid and others with the fMT-GlpF plasmid. Figure 4 shows that the ''E.coli'' cells with gvp float whereas the fMT-GlpF containing ones seem to sink.<br />
<br />
==Conclusion==<br />
From the restriction analysis it can be concluded that the transformation of ''E. coli'' DH10B with the two vectors was successfull. The strain was growing slowly, but was viable. The buoyancy test proved the functionality of the buoyancy phenotype but whether the presence of the accumulation device influences the gvp expression can not be determined with this experiment. The functionality of the accumulation device will be determined by the [[Team:Groningen/Protocols|arsenic uptake assay]] and analyzed by ICP-MS.<br />
<br />
<br />
{{Team:Groningen/Project/Footer}}</div>JolandaWitteveenhttp://2009.igem.org/Team:Groningen/Project/WholeSystemTeam:Groningen/Project/WholeSystem2009-10-21T16:41:52Z<p>JolandaWitteveen: </p>
<hr />
<div>{{Team:Groningen/Header|}}<br />
<br />
{| style="clear:both"<br />
|<html><style type="text/css"><br />
.intro { margin-left:0px; margin-top:10px; padding:10px; border-left:solid 5px #FFF6D5; border-right:solid 5px #FFF6D5; text-align:justify;background:#FFFFE5; }<br />
</style></html><br />
<div class="intro"><br />
<h2>Whole System</h2><br />
'''The metal scavenger with a vertical gas drive is a modular system. The system is explored for arsenic in this project. The accumulation module was made out of the aquaglyverol porin, GlpF, that was used as importer and the sequestering protein fMT. On a complementary plasmid the gas vesicle protein (GVP) cluster was cloned downstream of the arsenic sensitive promotor ars. The two plasmids were transformed in one cell and analysed using the buoyancy assay. This showed succesfull transformation and a buoyancy phenotype.'''<br />
<br><br><br><br />
</div><br />
|}<br />
<br />
==Introduction==<br />
<br />
The metal scavenger with a vertical gas drive is a modular system which contains five modules; the transporter, an accumulation protein, a regulated promoter, a regulator and the gas vesicle cluster. The transporter imports the metal ion of choice. An accumulation protein facilitates accumulation of metal ions and prevents the cell from dying of the metal toxicity. The imported metal ion also acts as a regulator for the metal sensitive promotor which activates the expression of the gvp-cluster. Thereby the recombinant bacterium, with this system, accumulates metal and upon accumulation starts to float which is convenient in bioremediation or mining [[Team:Groningen/Application|applications]]. But in principle this system may be used in order to accumulate any component for which these modules are available!<br />
The system explored in this project was mainly based on arsenic, however, aspects of a copper/zinc system were also explored. For the copper/zinc system [[Team:Groningen/Project/Transport#Copper/zinc uptake via HmtA|HmtA]], (<partinfo>BBa_K190018</partinfo>) was chosen as transporter, [[Team:Groningen/Project/Accumulation#Copper|Mymt]] and [[Team:Groningen/Project/Accumulation#Zinc|SmtA]] as accumulators and [[Team:Groningen/Project/Promoters#Copper Induced Promoters|CueO]], (<partinfo>BBa_K190017</partinfo>) and [[Team:Groningen/Project/Promoters#Zinc Induced Promoters|ZntR]], (<partinfo>BBa_K190016</partinfo>) as metal sensitive promotors.<br />
<br />
Arsenic is a very toxic metal and [[Team:Groningen/Application|causes health problems all over the world]]. The transporter used in this system is [[Team:Groningen/Project/Transport#Arsenite uptake via GlpF|GlpF]] (<partinfo>BBa_K190028</partinfo>) a glycerol transported known to import arsenic as well [[Team:Groningen/Literature#Meng, YL, et al.2004|(Meng, YL, et al.2004)]], [[Team:Groningen/Literature#Rosen, BR, et al.2009|(Rosen, BR, et al.2009)]]. Two accumulation proteins were chosen to accumulate arsenic, the fusion protein [[Team:Groningen/Project/Accumulation#Arsenic| MBP-ArsR]] (<partinfo>BBa_K190027</partinfo>) and [[Team:Groningen/Project/Accumulation#Arsenic|fMT]] (<partinfo>BBa_K190019</partinfo>). Both accumulation proteins were combined with GlpF to create new accumulation devices (<partinfo>BBa_K190073</partinfo>, <partinfo>BBa_K190038</partinfo>). As metal sensitive promotor, [[Team:Groningen/Project/Promoters|pArsR]] was chosen and together with the gvp cluster it makes up a buoyancy device (<partinfo>BBa_K190033</partinfo>). The two devices, the accumulation device and the buoyancy device were transformed together in ''E. coli'', to create the metal scavenger with a vertical gas drive.<br />
<br />
==Cloning Strategy==<br />
In order to use the buoyancy device(<partinfo>BBa_K190033</partinfo>) in combination with the accumulation device (<partinfo>BBa_K190038</partinfo>, <partinfo>BBa_K190039</partinfo>) a two vector system was used (see Figure 1). A two vector system was chosen because it is not feasible to combine the 6kb gas vesicle cluster, with a 1kb transporter and 300bp metallothionein and their promoters in one vector. This could easily increase the size of a vector to 10kb, a vector size which can hardly be transformed to ''E. coli''. <br />
This two vector system is composed of a <partinfo>pSB1A2</partinfo> vector with the accumulation device(<partinfo>BBa_K190038</partinfo>, <partinfo>BBa_K190039</partinfo>) and a <partinfo>pSB2K3</partinfo> vector with the buoyancy device (<partinfo>BBa_K190033</partinfo>). This combination was chosen because this makes it possible to tranform both vectors in one ''E.coli'' cell, since the vectors contain different origin of replications and antibiotic resistance markers. They contain respectively a pMB1 origin of replication and a P1 ori.<br />
<br />
[[Image:Whole system.PNG]] <br />
:Figure 1: Whole system, combining the bouyancy device on a pSB2K3 vector and the accumulation device on a pSB1AC3 vector<br />
<br />
==Results==<br />
<br />
Combining parts <partinfo>BBa_K190033</partinfo> and <partinfo>BBa_K190038</partinfo> was done by using a normal [[Team:Groningen/Protocols|transformation protocol]] with both ampicillin and kanamycin as antibiotics. A [[Team:Groningen/Protocols|buoyancy test]] was performed as described also using both ampicillin and kanamycin as antibiotics. Iptg was also added to the dayculture to induce the <partinfo>BBa_K190033</partinfo> part. In exponential phase 10μM NaAsO<sub>2</sub> was added to half of the samples.<br />
A [[Team:Groningen/Protocols|restriction]] was done to check the transformation. <br />
<br />
====Restriction====<br />
Figure 2 shows that the transformation of both vectors succeeded.<br />
<br />
[[Image: Groningen_GelPhotoSystemRestriction.png|400px]]<br />
<br />
:Figure 2:Gel image of restricted and not restricted plasmids.<br />
<br />
<br />
The gel shows a 1Kb marker to the far left, after that two times a restriction with ''EcoR''I and ''Pst''I can be seen. This shows clearly fragments of 6000pb and 4500pb for the <partinfo>BBa_K190033</partinfo> and fragments of 2000pb and 1300 bp for the <partinfo>BBa_K190038</partinfo>. The next two slots show non-restricted plasmids, two distinc bands can be seen clearly indicating the presence of two plasmids.<br />
<br />
====Buoyancy test====<br />
The buoyancy test elegantly shows that combining both an accumulation device and the gvp-buoyancy device allows buoyancy. <br />
<br />
[[Image:Groningen_SystemBouyancy33en38.png|300px]]<br />
<br />
:Figure 3: Result of the buoyancy test, the left shows ''E.coli'' cells without arsenic induction and to the right floating ''E.coli'' cells, induced with arsenic, are shown.<br />
<br />
<br />
On the left ''E. coli'' cells (containing the whole system) without arsenic can be seen and on the right ''E. coli'' cells (containing the whole system) with arsenic is shown. It can be seen that the right sample floats. This indicates that the GlpF is transporting the arsenic inside the cells, the fMT accumulates it (otherwise the cells would be dead) and the gvp was induced by the arsenic so the cells start to float.<br />
<br />
[[Image:Groningen_BouyancyOf33en38.png|200px]]<br />
:Figure 4: Results of the buoyancy test, left ''E.coli'' with the gvp plasmid, the right tube shows ''E.coli'' cells with the fMT-glpF device. <br />
<br />
As a control ''E.coli'' cells were also transformed with the gvp plasmid and others with the fMT-GlpF plasmid. Figure 4 shows that the ''E.coli'' cells with gvp float whereas the fMT-GlpF containing ones seem to sink.<br />
<br />
==Conclusion==<br />
From the restriction analysis it can be concluded that the transformation of ''E. coli'' DH10B with the two vectors was successfull. The strain was growing slowly, but was viable. The buoyancy test proved the functionality of the buoyancy phenotype but whether the presence of the accumulation device influences the gvp expression can not be determined with this experiment. The functionality of the accumulation device will be determined by the [[Team:Groningen/Protocols|arsenic uptake assay]] and analyzed by ICP-MS.<br />
<br />
<br />
{{Team:Groningen/Project/Footer}}</div>JolandaWitteveenhttp://2009.igem.org/Team:Groningen/Project/WholeSystemTeam:Groningen/Project/WholeSystem2009-10-21T16:41:10Z<p>JolandaWitteveen: </p>
<hr />
<div>{{Team:Groningen/Header|}}<br />
<br />
{| style="clear:both"<br />
|<html><style type="text/css"><br />
.intro { margin-left:0px; margin-top:10px; padding:10px; border-left:solid 5px #FFF6D5; border-right:solid 5px #FFF6D5; text-align:justify;background:#FFFFE5; }<br />
</style></html><br />
<div class="intro"><br />
<h2>Whole System</h2><br />
'''The metal scavenger with a vertical gas drive is a modular system. The system is explored for arsenic in this project. The accumulation module was made out of the aquaglyverol porin, GlpF, that was used as importer and the sequestering protein fMT. On a complementary plasmid the gas vesicle protein (GVP) cluster was cloned downstream of the arsenic sensitive promotor ars. The two plasmids were transformed in one cell and analysed using the buoyancy assay. This showed succesfull transformation and a buoyancy phenotype.'''<br />
<br><br><br><br />
</div><br />
|}<br />
<br />
<br />
==Introduction==<br />
<br />
The metal scavenger with a vertical gas drive is a modular system which contains five modules; the transporter, an accumulation protein, a regulated promoter, a regulator and the gas vesicle cluster. The transporter imports the metal ion of choice. An accumulation protein facilitates accumulation of metal ions and prevents the cell from dying of the metal toxicity. The imported metal ion also acts as a regulator for the metal sensitive promotor which activates the expression of the gvp-cluster. Thereby the recombinant bacterium, with this system, accumulates metal and upon accumulation starts to float which is convenient in bioremediation or mining [[Team:Groningen/Application|applications]]. But in principle this system may be used in order to accumulate any component for which these modules are available!<br />
The system explored in this project was mainly based on arsenic, however, aspects of a copper/zinc system were also explored. For the copper/zinc system [[Team:Groningen/Project/Transport#Copper/zinc uptake via HmtA|HmtA]], (<partinfo>BBa_K190018</partinfo>) was chosen as transporter, [[Team:Groningen/Project/Accumulation#Copper|Mymt]] and [[Team:Groningen/Project/Accumulation#Zinc|SmtA]] as accumulators and [[Team:Groningen/Project/Promoters#Copper Induced Promoters|CueO]], (<partinfo>BBa_K190017</partinfo>) and [[Team:Groningen/Project/Promoters#Zinc Induced Promoters|ZntR]], (<partinfo>BBa_K190016</partinfo>) as metal sensitive promotors.<br />
<br />
Arsenic is a very toxic metal and [[Team:Groningen/Application|causes health problems all over the world]]. The transporter used in this system is [[Team:Groningen/Project/Transport#Arsenite uptake via GlpF|GlpF]] (<partinfo>BBa_K190028</partinfo>) a glycerol transported known to import arsenic as well [[Team:Groningen/Literature#Meng, YL, et al.2004|(Meng, YL, et al.2004)]], [[Team:Groningen/Literature#Rosen, BR, et al.2009|(Rosen, BR, et al.2009)]]. Two accumulation proteins were chosen to accumulate arsenic, the fusion protein [[Team:Groningen/Project/Accumulation#Arsenic| MBP-ArsR]] (<partinfo>BBa_K190027</partinfo>) and [[Team:Groningen/Project/Accumulation#Arsenic|fMT]] (<partinfo>BBa_K190019</partinfo>). Both accumulation proteins were combined with GlpF to create new accumulation devices (<partinfo>BBa_K190073</partinfo>, <partinfo>BBa_K190038</partinfo>). As metal sensitive promotor, [[Team:Groningen/Project/Promoters|pArsR]] was chosen and together with the gvp cluster it makes up a buoyancy device (<partinfo>BBa_K190033</partinfo>). The two devices, the accumulation device and the buoyancy device were transformed together in ''E. coli'', to create the metal scavenger with a vertical gas drive.<br />
<br />
==Cloning Strategy==<br />
In order to use the buoyancy device(<partinfo>BBa_K190033</partinfo>) in combination with the accumulation device (<partinfo>BBa_K190038</partinfo>, <partinfo>BBa_K190039</partinfo>) a two vector system was used (see Figure 1). A two vector system was chosen because it is not feasible to combine the 6kb gas vesicle cluster, with a 1kb transporter and 300bp metallothionein and their promoters in one vector. This could easily increase the size of a vector to 10kb, a vector size which can hardly be transformed to ''E. coli''. <br />
This two vector system is composed of a <partinfo>pSB1A2</partinfo> vector with the accumulation device(<partinfo>BBa_K190038</partinfo>, <partinfo>BBa_K190039</partinfo>) and a <partinfo>pSB2K3</partinfo> vector with the buoyancy device (<partinfo>BBa_K190033</partinfo>). This combination was chosen because this makes it possible to tranform both vectors in one ''E.coli'' cell, since the vectors contain different origin of replications and antibiotic resistance markers. They contain respectively a pMB1 origin of replication and a P1 ori.<br />
<br />
[[Image:Whole system.PNG]] <br />
:Figure 1: Whole system, combining the bouyancy device on a pSB2K3 vector and the accumulation device on a pSB1AC3 vector<br />
<br />
==Results==<br />
<br />
Combining parts <partinfo>BBa_K190033</partinfo> and <partinfo>BBa_K190038</partinfo> was done by using a normal [[Team:Groningen/Protocols|transformation protocol]] with both ampicillin and kanamycin as antibiotics. A [[Team:Groningen/Protocols|buoyancy test]] was performed as described also using both ampicillin and kanamycin as antibiotics. Iptg was also added to the dayculture to induce the <partinfo>BBa_K190033</partinfo> part. In exponential phase 10μM NaAsO<sub>2</sub> was added to half of the samples.<br />
A [[Team:Groningen/Protocols|restriction]] was done to check the transformation. <br />
<br />
====Restriction====<br />
Figure 2 shows that the transformation of both vectors succeeded.<br />
<br />
[[Image: Groningen_GelPhotoSystemRestriction.png|400px]]<br />
<br />
:Figure 2:Gel image of restricted and not restricted plasmids.<br />
<br />
<br />
The gel shows a 1Kb marker to the far left, after that two times a restriction with ''EcoR''I and ''Pst''I can be seen. This shows clearly fragments of 6000pb and 4500pb for the <partinfo>BBa_K190033</partinfo> and fragments of 2000pb and 1300 bp for the <partinfo>BBa_K190038</partinfo>. The next two slots show non-restricted plasmids, two distinc bands can be seen clearly indicating the presence of two plasmids.<br />
<br />
====Buoyancy test====<br />
The buoyancy test elegantly shows that combining both an accumulation device and the gvp-buoyancy device allows buoyancy. <br />
<br />
[[Image:Groningen_SystemBouyancy33en38.png|300px]]<br />
<br />
:Figure 3: Result of the buoyancy test, the left shows ''E.coli'' cells without arsenic induction and to the right floating ''E.coli'' cells, induced with arsenic, are shown.<br />
<br />
<br />
On the left ''E. coli'' cells (containing the whole system) without arsenic can be seen and on the right ''E. coli'' cells (containing the whole system) with arsenic is shown. It can be seen that the right sample floats. This indicates that the GlpF is transporting the arsenic inside the cells, the fMT accumulates it (otherwise the cells would be dead) and the gvp was induced by the arsenic so the cells start to float.<br />
<br />
[[Image:Groningen_BouyancyOf33en38.png|200px]]<br />
:Figure 4: Results of the buoyancy test, left ''E.coli'' with the gvp plasmid, the right tube shows ''E.coli'' cells with the fMT-glpF device. <br />
<br />
As a control ''E.coli'' cells were also transformed with the gvp plasmid and others with the fMT-GlpF plasmid. Figure 4 shows that the ''E.coli'' cells with gvp float whereas the fMT-GlpF containing ones seem to sink.<br />
<br />
==Conclusion==<br />
From the restriction analysis it can be concluded that the transformation of ''E. coli'' DH10B with the two vectors was successfull. The strain was growing slowly, but was viable. The buoyancy test proved the functionality of the buoyancy phenotype but whether the presence of the accumulation device influences the gvp expression can not be determined with this experiment. The functionality of the accumulation device will be determined by the [[Team:Groningen/Protocols|arsenic uptake assay]] and analyzed by ICP-MS.<br />
<br />
<br />
{{Team:Groningen/Project/Footer}}</div>JolandaWitteveenhttp://2009.igem.org/Team:Groningen/ApplicationTeam:Groningen/Application2009-10-21T16:29:55Z<p>JolandaWitteveen: </p>
<hr />
<div>{{Team:Groningen/Header|}}<br />
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<div class="intro"><br />
<h2>Application</h2><br />
'''The metal scavenger with a vertical gas drive is a modular system which contains five modules that are interchangeble; the transporter, an accumulation protein, a regulated promoter, a regulator and the gas vesicle cluster. The transporter imports the metal ion of choice. An accumulation protein facilitates accumulation of metal ions and prevents the cell from dying of the metal toxicity. The imported metal ion also acts as a regulator for the metal sensitive promotor which activates the expression of the gvp-cluster. Thereby the recombinant bacterium with this system, accumulates metal and upon accumulation starts to float. Many applications are possible once the correct modules are selected. Water and sludge cleaning or mining of rare metals are som examples. Also the ethical concerns of these applications should be considered.'''<br />
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</div><br />
|}<br />
<br />
===Water Cleaning===<br />
Bacteria which accumulate metal ions can be used to remove toxic metals or other contaminants from ground or surface water. This microbial [https://2009.igem.org/Team:Groningen/Project/WholeSystem system] is described for scavenging of arsenite, copper and zinc. <br />
An example where removal of arsenic from subsoil water by this organism could prevent serious poisoning of millions of people. In Bangladesh and other countries over ten million water pumps where installed, to improve personal hygiene but this raised a new problem. The unforeseen consequence was that, because the groundwater level dropped dramatically and the arsenic contamination in the groundwater began oxidizing. Chronic consumption of this water will lead to arsenic toxification. Arsenic causes swears caused necrosis of cell tissue because of disruption of the ATP production ([http://en.wikipedia.org/wiki/Arsenic wikipedia]). To prevent more people from arsenic poisoning, the arsenic concentration can be decreased by bacteria (containing the system as described before) and subsequently filtering these bacteria from the water, it can be consumed by individuals.<br />
Arsenic is most prominently found in sub-soil water found as As(III) (anaerobic condition) and as As(V) in surface water (mildly aerobic condition). For humans the trivalent state was found to be most toxic. This is exactly the state which is most efficiently bound by [https://2009.igem.org/Team:Groningen/Project/Accumulation fMT] and transported by <br />
[https://2009.igem.org/Team:Groningen/Project/Transport GlpF].<br />
<br />
===Sludge Cleaning===<br />
Due to application of sewage sludge and industrial sludge into environmental soil, toxic metals accumulates in river sludge and soil. This contaminated soil is usually "diluted" with uncontaminated soil to decrease the concentration of metals below the maximum allowable concentration (10ppb (µg/L)). But by consumption of vegetables cultivated on these soil or meat from cattle that pastured on these fields, the concentration of these toxic compounds will increase in higher organisms. A bacterium that is able to absorb and encapsulate metals and that floats, would be able to separate the metals and the sediment. It would be enough to simply put the bacteria and the sediment in large container stir them together and let the cleaned sediment sink and scoop of the floating metal filled bacteria. Such a purification plant would look similar like the sewage disposal plants that we use today. Simply inject the bacteria in the sediment, the bacteria full of metals will start floating to the surface and they can readily be collected by floaters, such as the ones which are now used to collect oil pollution from the sea.<br />
<br />
===Mining===<br />
Due to the huge worldly consumption of crude oil there is a high probability that the fossil oil reserves will be exhausted within several decades, but this is not only the case for oil, also the reserves of rare metals are becoming less abundant. Therefore the mining yield should be increased to get as much of the desired metal out of the ore as possible. <br />
For mining of copper and gold a metal oxidizing bacteria species called, ''Thiobacillus ferrooxidans'' is already being used for over 10 years. This bacterium gains energy by oxidizing insoluble sulfides of metals like copper, gold, lead, zinc, nickel and even uranium to soluble sulfides. This causes an improved recovery ([[Team:Groningen/Literature#Rawlings1994|Rawlings 1994]]), but to increase the yield and specificity a genetically engineered system could be used. Therefore a robust bacterium like the thermophilic ''Thermus thermophilus'' or the radiation resistant ''Deinococcus radiodurans'' (or a species which allows easier clonig) could be used. The transporter and metallothionein for specific import and sequestering of the rare metal, should be overexpressed. For these low concentrations the kock-out of the exporter of that metal would be required. But then finally, what keeps us from mining gold from the oceans or deserted mines to collect what was first impossible to extract!?<br />
<br />
==Ethical concerns==<br />
One of the most prominant ethical issues surrounding the application of genetic modified organisms is safety.The application should not be an environmental hazard by itself nor should it be a hazard for human health. Using a confined disposal plant would for most part prevent the spread of genetically modified bacteria into the environment, however, in case of heavy rain the basins can overspill which allows the bacteria to enter the environment. So only a confined basin is not enough, a self-destruction plasmid, which induces cell death after a certain time, should be added as well. In that case, even if the bacteria accidently end up outside the confined disposal plant, they will die after a while and will be unable to spread or transfer their DNA. This death plasmid should be carefully choosen since the bacteria need time to clean the water, sludge or mine the metals. Read more on [[Team:Groningen/Safety|safety]] and [[Team:Groningen/Ethics|ethical issues]]...<br />
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<br />
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<br />
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<br />
<br />
<br />
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{{Team:Groningen/Project/Footer}}</div>JolandaWitteveenhttp://2009.igem.org/Team:Groningen/Project/WholeSystemTeam:Groningen/Project/WholeSystem2009-10-21T16:24:19Z<p>JolandaWitteveen: </p>
<hr />
<div>{{Team:Groningen/Header|}}<br />
<br />
{| style="clear:both"<br />
|<html><style type="text/css"><br />
.intro { margin-left:0px; margin-top:10px; padding:10px; border-left:solid 5px #FFF6D5; border-right:solid 5px #FFF6D5; text-align:justify;background:#FFFFE5; }<br />
</style></html><br />
<div class="intro"><br />
<h2>Whole System</h2><br />
'''The metal scavenger with a vertical gas drive is a modular system which contains five modules; the transporter, an accumulation protein, a regulated promoter, a regulator and the gas vesicle cluster. The transporter imports the metal ion of choice. An accumulation protein facilitates accumulation of metal ions and prevents the cell from dying of the metal toxicity. The imported metal ion also acts as a regulator for the metal sensitive promotor which activates the expression of the gvp-cluster. Thereby the recombinant bacterium, with this system, accumulates metal and upon accumulation starts to float which is convenient in bioremediation or mining [[Team:Groningen/Application|applications]]. But in principle this system may be used in order to accumulate any component for which these modules are available! '''<br />
<br><br><br><br />
</div><br />
|}<br />
<br />
<br />
==Introduction==<br />
The system explored in this project was mainly based on arsenic, however, aspects of a copper/zinc system were also explored. For the copper/zinc system [[Team:Groningen/Project/Transport#Copper/zinc uptake via HmtA|HmtA]], (<partinfo>BBa_K190018</partinfo>) was chosen as transporter, [[Team:Groningen/Project/Accumulation#Copper|Mymt]] and [[Team:Groningen/Project/Accumulation#Zinc|SmtA]] as accumulators and [[Team:Groningen/Project/Promoters#Copper Induced Promoters|CueO]], (<partinfo>BBa_K190017</partinfo>) and [[Team:Groningen/Project/Promoters#Zinc Induced Promoters|ZntR]], (<partinfo>BBa_K190016</partinfo>) as metal sensitive promotors.<br />
<br />
Arsenic is a very toxic metal and [[Team:Groningen/Application|causes health problems all over the world]]. The transporter used in this system is [[Team:Groningen/Project/Transport#Arsenite uptake via GlpF|GlpF]] (<partinfo>BBa_K190028</partinfo>) a glycerol transported known to import arsenic as well [[Team:Groningen/Literature#Meng, YL, et al.2004|(Meng, YL, et al.2004)]], [[Team:Groningen/Literature#Rosen, BR, et al.2009|(Rosen, BR, et al.2009)]]. Two accumulation proteins were chosen to accumulate arsenic, the fusion protein [[Team:Groningen/Project/Accumulation#Arsenic| MBP-ArsR]] (<partinfo>BBa_K190027</partinfo>) and [[Team:Groningen/Project/Accumulation#Arsenic|fMT]] (<partinfo>BBa_K190019</partinfo>). Both accumulation proteins were combined with GlpF to create new accumulation devices (<partinfo>BBa_K190073</partinfo>, <partinfo>BBa_K190038</partinfo>). As metal sensitive promotor, [[Team:Groningen/Project/Promoters|pArsR]] was chosen and together with the gvp cluster it makes up a buoyancy device (<partinfo>BBa_K190033</partinfo>). The two devices, the accumulation device and the buoyancy device were transformed together in ''E. coli'', to create the metal scavenger with a vertical gas drive.<br />
<br />
==Cloning Strategy==<br />
In order to use the buoyancy device(<partinfo>BBa_K190033</partinfo>) in combination with the accumulation device (<partinfo>BBa_K190038</partinfo>, <partinfo>BBa_K190039</partinfo>) a two vector system was used (see Figure 1). A two vector system was chosen because it is not feasible to combine the 6kb gas vesicle cluster, with a 1kb transporter and 300bp metallothionein and their promoters in one vector. This could easily increase the size of a vector to 10kb, a vector size which can hardly be transformed to ''E. coli''. <br />
This two vector system is composed of a <partinfo>pSB1A2</partinfo> vector with the accumulation device(<partinfo>BBa_K190038</partinfo>, <partinfo>BBa_K190039</partinfo>) and a <partinfo>pSB2K3</partinfo> vector with the buoyancy device (<partinfo>BBa_K190033</partinfo>). This combination was chosen because this makes it possible to tranform both vectors in one ''E.coli'' cell, since the vectors contain different origin of replications and antibiotic resistance markers. They contain respectively a pMB1 origin of replication and a P1 ori.<br />
<br />
[[Image:Whole system.PNG]] <br />
:Figure 1: Whole system, combining the bouyancy device on a pSB2K3 vector and the accumulation device on a pSB1AC3 vector<br />
<br />
==Results==<br />
<br />
Combining parts <partinfo>BBa_K190033</partinfo> and <partinfo>BBa_K190038</partinfo> was done by using a normal [[Team:Groningen/Protocols|transformation protocol]] with both ampicillin and kanamycin as antibiotics. A [[Team:Groningen/Protocols|buoyancy test]] was performed as described also using both ampicillin and kanamycin as antibiotics. Iptg was also added to the dayculture to induce the <partinfo>BBa_K190033</partinfo> part. In exponential phase 10μM NaAsO<sub>2</sub> was added to half of the samples.<br />
A [[Team:Groningen/Protocols|restriction]] was done to check the transformation. <br />
<br />
====Restriction====<br />
Figure 2 shows that the transformation of both vectors succeeded.<br />
<br />
[[Image: Groningen_GelPhotoSystemRestriction.png|400px]]<br />
<br />
:Figure 2:Gel image of restricted and not restricted plasmids.<br />
<br />
<br />
The gel shows a 1Kb marker to the far left, after that two times a restriction with ''EcoR''I and ''Pst''I can be seen. This shows clearly fragments of 6000pb and 4500pb for the <partinfo>BBa_K190033</partinfo> and fragments of 2000pb and 1300 bp for the <partinfo>BBa_K190038</partinfo>. The next two slots show non-restricted plasmids, two distinc bands can be seen clearly indicating the presence of two plasmids.<br />
<br />
====Buoyancy test====<br />
The buoyancy test elegantly shows that combining both an accumulation device and the gvp-buoyancy device allows buoyancy. <br />
<br />
[[Image:Groningen_SystemBouyancy33en38.png|300px]]<br />
<br />
:Figure 3: Result of the buoyancy test, the left shows ''E.coli'' cells without arsenic induction and to the right floating ''E.coli'' cells, induced with arsenic, are shown.<br />
<br />
<br />
On the left ''E. coli'' cells (containing the whole system) without arsenic can be seen and on the right ''E. coli'' cells (containing the whole system) with arsenic is shown. It can be seen that the right sample floats. This indicates that the GlpF is transporting the arsenic inside the cells, the fMT accumulates it (otherwise the cells would be dead) and the gvp was induced by the arsenic so the cells start to float.<br />
<br />
[[Image:Groningen_BouyancyOf33en38.png|200px]]<br />
:Figure 4: Results of the buoyancy test, left ''E.coli'' with the gvp plasmid, the right tube shows ''E.coli'' cells with the fMT-glpF device. <br />
<br />
As a control ''E.coli'' cells were also transformed with the gvp plasmid and others with the fMT-GlpF plasmid. Figure 4 shows that the ''E.coli'' cells with gvp float whereas the fMT-GlpF containing ones seem to sink.<br />
<br />
==Conclusion==<br />
From the restriction analysis it can be concluded that the transformation of ''E. coli'' DH10B with the two vectors was successfull. The strain was growing slowly, but was viable. The buoyancy test proved the functionality of the buoyancy phenotype but whether the presence of the accumulation device influences the gvp expression can not be determined with this experiment. The functionality of the accumulation device will be determined by the [[Team:Groningen/Protocols|arsenic uptake assay]] and analyzed by ICP-MS.<br />
<br />
<br />
{{Team:Groningen/Project/Footer}}</div>JolandaWitteveenhttp://2009.igem.org/Team:Groningen/Project/WholeSystemTeam:Groningen/Project/WholeSystem2009-10-21T16:22:39Z<p>JolandaWitteveen: </p>
<hr />
<div>{{Team:Groningen/Header|}}<br />
<br />
{| style="clear:both"<br />
|<html><style type="text/css"><br />
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</style></html><br />
<div class="intro"><br />
<h2>Gas vesicles</h2><br />
'''Our goal in this project is to make cells bouyant in the presence of certain concentrations of metals like copper, zinc and arsenic. Metal induced gas vesicle production can provide our cells with this bouyancy. Gas vesicles are bacterial organelles consisting entirely of proteins that envelop a gas filled space. We made, and send to the registry, parts in which the metal sensitive promoters for copper, zinc and arsenic were cloned in front of the GVP (Gas Vesicle Protein) gene cluster. For further characterization of the GVP gene cluster inducible and constitutive promoters were also cloned in front of this cluster. Buoyancy tests showed that our constructs were able to increase cell buoyancy and electron micrographs showed the presence of gas vesicles. A model was made to predict what volume fraction of a cell would have to be gas vesicle for this cell to have a density equal to that of water.'''<br />
<br><br><br><br />
</div><br />
|}<br />
<br />
<br />
==Introduction==<br />
The metal scavenger with a vertical gas drive is a modular system which contains five modules; the transporter, an accumulation protein, a regulated promoter, a regulator and the gas vesicle cluster. The transporter imports the metal ion of choice. An accumulation protein facilitates accumulation of metal ions and prevents the cell from dying of the metal toxicity. The imported metal ion also acts as a regulator for the metal sensitive promotor which activates the expression of the gvp-cluster. Thereby the recombinant bacterium, with this system, accumulates metal and upon accumulation starts to float which is convenient in bioremediation or mining [[Team:Groningen/Application|applications]]. But in principle this system may be used in order to accumulate any component for which these modules are available! <br />
<br />
The system explored in this project was mainly based on arsenic, however, aspects of a copper/zinc system were also explored. For the copper/zinc system [[Team:Groningen/Project/Transport#Copper/zinc uptake via HmtA|HmtA]], (<partinfo>BBa_K190018</partinfo>) was chosen as transporter, [[Team:Groningen/Project/Accumulation#Copper|Mymt]] and [[Team:Groningen/Project/Accumulation#Zinc|SmtA]] as accumulators and [[Team:Groningen/Project/Promoters#Copper Induced Promoters|CueO]], (<partinfo>BBa_K190017</partinfo>) and [[Team:Groningen/Project/Promoters#Zinc Induced Promoters|ZntR]], (<partinfo>BBa_K190016</partinfo>) as metal sensitive promotors.<br />
<br />
Arsenic is a very toxic metal and [[Team:Groningen/Application|causes health problems all over the world]]. The transporter used in this system is [[Team:Groningen/Project/Transport#Arsenite uptake via GlpF|GlpF]] (<partinfo>BBa_K190028</partinfo>) a glycerol transported known to import arsenic as well [[Team:Groningen/Literature#Meng, YL, et al.2004|(Meng, YL, et al.2004)]], [[Team:Groningen/Literature#Rosen, BR, et al.2009|(Rosen, BR, et al.2009)]]. Two accumulation proteins were chosen to accumulate arsenic, the fusion protein [[Team:Groningen/Project/Accumulation#Arsenic| MBP-ArsR]] (<partinfo>BBa_K190027</partinfo>) and [[Team:Groningen/Project/Accumulation#Arsenic|fMT]] (<partinfo>BBa_K190019</partinfo>). Both accumulation proteins were combined with GlpF to create new accumulation devices (<partinfo>BBa_K190073</partinfo>, <partinfo>BBa_K190038</partinfo>). As metal sensitive promotor, [[Team:Groningen/Project/Promoters|pArsR]] was chosen and together with the gvp cluster it makes up a buoyancy device (<partinfo>BBa_K190033</partinfo>). The two devices, the accumulation device and the buoyancy device were transformed together in ''E. coli'', to create the metal scavenger with a vertical gas drive.<br />
<br />
==Cloning Strategy==<br />
In order to use the buoyancy device(<partinfo>BBa_K190033</partinfo>) in combination with the accumulation device (<partinfo>BBa_K190038</partinfo>, <partinfo>BBa_K190039</partinfo>) a two vector system was used (see Figure 1). A two vector system was chosen because it is not feasible to combine the 6kb gas vesicle cluster, with a 1kb transporter and 300bp metallothionein and their promoters in one vector. This could easily increase the size of a vector to 10kb, a vector size which can hardly be transformed to ''E. coli''. <br />
This two vector system is composed of a <partinfo>pSB1A2</partinfo> vector with the accumulation device(<partinfo>BBa_K190038</partinfo>, <partinfo>BBa_K190039</partinfo>) and a <partinfo>pSB2K3</partinfo> vector with the buoyancy device (<partinfo>BBa_K190033</partinfo>). This combination was chosen because this makes it possible to tranform both vectors in one ''E.coli'' cell, since the vectors contain different origin of replications and antibiotic resistance markers. They contain respectively a pMB1 origin of replication and a P1 ori.<br />
<br />
[[Image:Whole system.PNG]] <br />
:Figure 1: Whole system, combining the bouyancy device on a pSB2K3 vector and the accumulation device on a pSB1AC3 vector<br />
<br />
==Results==<br />
<br />
Combining parts <partinfo>BBa_K190033</partinfo> and <partinfo>BBa_K190038</partinfo> was done by using a normal [[Team:Groningen/Protocols|transformation protocol]] with both ampicillin and kanamycin as antibiotics. A [[Team:Groningen/Protocols|buoyancy test]] was performed as described also using both ampicillin and kanamycin as antibiotics. Iptg was also added to the dayculture to induce the <partinfo>BBa_K190033</partinfo> part. In exponential phase 10μM NaAsO<sub>2</sub> was added to half of the samples.<br />
A [[Team:Groningen/Protocols|restriction]] was done to check the transformation. <br />
<br />
====Restriction====<br />
Figure 2 shows that the transformation of both vectors succeeded.<br />
<br />
[[Image: Groningen_GelPhotoSystemRestriction.png|400px]]<br />
<br />
:Figure 2:Gel image of restricted and not restricted plasmids.<br />
<br />
<br />
The gel shows a 1Kb marker to the far left, after that two times a restriction with ''EcoR''I and ''Pst''I can be seen. This shows clearly fragments of 6000pb and 4500pb for the <partinfo>BBa_K190033</partinfo> and fragments of 2000pb and 1300 bp for the <partinfo>BBa_K190038</partinfo>. The next two slots show non-restricted plasmids, two distinc bands can be seen clearly indicating the presence of two plasmids.<br />
<br />
====Buoyancy test====<br />
The buoyancy test elegantly shows that combining both an accumulation device and the gvp-buoyancy device allows buoyancy. <br />
<br />
[[Image:Groningen_SystemBouyancy33en38.png|300px]]<br />
<br />
:Figure 3: Result of the buoyancy test, the left shows ''E.coli'' cells without arsenic induction and to the right floating ''E.coli'' cells, induced with arsenic, are shown.<br />
<br />
<br />
On the left ''E. coli'' cells (containing the whole system) without arsenic can be seen and on the right ''E. coli'' cells (containing the whole system) with arsenic is shown. It can be seen that the right sample floats. This indicates that the GlpF is transporting the arsenic inside the cells, the fMT accumulates it (otherwise the cells would be dead) and the gvp was induced by the arsenic so the cells start to float.<br />
<br />
[[Image:Groningen_BouyancyOf33en38.png|200px]]<br />
:Figure 4: Results of the buoyancy test, left ''E.coli'' with the gvp plasmid, the right tube shows ''E.coli'' cells with the fMT-glpF device. <br />
<br />
As a control ''E.coli'' cells were also transformed with the gvp plasmid and others with the fMT-GlpF plasmid. Figure 4 shows that the ''E.coli'' cells with gvp float whereas the fMT-GlpF containing ones seem to sink.<br />
<br />
==Conclusion==<br />
From the restriction analysis it can be concluded that the transformation of ''E. coli'' DH10B with the two vectors was successfull. The strain was growing slowly, but was viable. The buoyancy test proved the functionality of the buoyancy phenotype but whether the presence of the accumulation device influences the gvp expression can not be determined with this experiment. The functionality of the accumulation device will be determined by the [[Team:Groningen/Protocols|arsenic uptake assay]] and analyzed by ICP-MS.<br />
<br />
<br />
{{Team:Groningen/Project/Footer}}</div>JolandaWitteveenhttp://2009.igem.org/Team:Groningen/Project/VesicleTeam:Groningen/Project/Vesicle2009-10-21T16:21:14Z<p>JolandaWitteveen: </p>
<hr />
<div>{{Team:Groningen/Project/Header|}}<br />
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{| style="clear:both"<br />
|<html><style type="text/css"><br />
.intro { margin-left:0px; margin-top:10px; padding:10px; border-left:solid 5px #FFF6D5; border-right:solid 5px #FFF6D5; text-align:justify;background:#FFFFE5; }<br />
</style></html><br />
<div class="intro"><br />
<h2>Gas vesicles</h2><br />
'''Our goal in this project is to make cells bouyant in the presence of certain concentrations of metals like copper, zinc and arsenic. Metal induced gas vesicle production can provide our cells with this bouyancy. Gas vesicles are bacterial organelles consisting entirely of proteins that envelop a gas filled space. We made, and send to the registry, parts in which the metal sensitive promoters for copper, zinc and arsenic were cloned in front of the GVP (Gas Vesicle Protein) gene cluster. For further characterization of the GVP gene cluster inducible and constitutive promoters were also cloned in front of this cluster. Buoyancy tests showed that our constructs were able to increase cell buoyancy and electron micrographs showed the presence of gas vesicles. A model was made to predict what volume fraction of a cell would have to be gas vesicle for this cell to have a density equal to that of water.'''<br />
<br><br><br><br />
</div><br />
|}<br />
<br />
<br />
==Background==<br />
<br />
Gas vesicles are organelles made entirely out of proteins that envelop a gas filled space. Because only gas can penetrate into the gas vesicles the total density of the cell is lowered. This lower cell density leads in turn to a buoyancy phenotype. Outside of the laboratory this buoyancy is used by microorganisms to vertically position themselves in the water column or simply to reduce their sinking rates. Organisms can regulate buoyancy by reducing gas vesicle production or by accumulation of denser compounds like carbohydrates. For certain cyanobacteria this regulation depends on light intensities. <br />
<br />
For a number of organisms it has been shown that all proteins important for the expression of gas vesicle lie in a single gene cluster. The GVP gene cluster used in this project was cloned from Bacillus megaterium into E. coli ([[Team:Groningen/Literature#Li1998|Li & Cannon 1998]]). This gene cluster now containing 11 genes was turned into a biobrick by Melbourne 2007 (link to part). Figure 1 shows the gene cluster as it was send in by Melbourne in 2007.<br />
<br />
For more info see: [[Team:Groningen/Literature#Walsby1994|Walsby 1994]].<br />
<br />
[[Image:Gas vesicle cluster bba I750016.PNG|frame|Figure 1: Gas vesicle gene cluster (<partinfo>BBa_I750016</partinfo>)<br />
]]<br />
<br />
==Goal==<br />
<br />
The goal of our project is to engineer an organism that can remove heavy metals from water. To facilitate easy separation the cells that have taken up metals should float so they can be removed from the water. The introduction of GVP gene cluster provides the cell with the required buoyancy. <br />
<br />
To make the buoyancy level of the cell responsive to the metal concentration inside the cell, metal sensitive promoters would have to be cloned in front of the GVP gene cluster. In this project these metal sensitive promoters were responsive to zinc, copper and arsenic. We also wanted to make a construct with constitutive and inducible promoters in front of the GVP cluster to show a proof of principle and as a back-up if our metal induced construct would fail. <br />
<br />
Finally we wanted to improve the Melbourne 2007 biobrick (<partinfo>BBa_I750016</partinfo>) by removing a repeat that was accidentally introduced during the removal of forbidden restriction sites.<br />
<br />
==Cloning strategy== <br />
For our msGVP (metal sensitive GVP) constructs we ordered oligo's containing the promoter region and the necessary restriction sites. When annealed these pieces of DNA have EcoRI and SpeI sticky ends. The vector containing GVP (<partinfo>BBa_I750016</partinfo>) was cut with EcoRI and XbaI and was ligated to the promoter. ([[Team:Groningen/Protocols#Annealing synthetic oligo’s|Protocol]])<br />
<br />
The metal sensitive GVP constructs are: <partinfo>BBa_K190033</partinfo>, <partinfo>BBa_K190034</partinfo> and <partinfo>BBa_K190035</partinfo>.<br />
<br />
Then because of compatibility issues when our entire system has to be assembled into one cell the whole metal sensitive promoter and GVP part were transfered to a different vector.<br />
<br />
[[Image:Cloning strategy floating device1.PNG]]<br />
:Figure 2: The floating device will be built up of an inducible promoter which can be induced by a certain intracellular concentration of metal-ions, and a gas vesicle cluster.<br />
<br />
==Bouyancy Tests==<br />
<br />
The buoyancy of GVP was tested by using the [[Team:Groningen/Protocols#Bouyancytest|buoyancy test protocol]]. The cells were grown in medium and induced and were resuspended in a salt solution (0.15mM NaCl) in a test tube and were left for a while in order to give the cells time to sink or float. <br />
<br />
''Different circumstances''<br />
<br />
Several different circumstances and small changes to the protocol were made in order to find the perfect circumstance for the buoyancy test. It appeared that with a low cell density the difference between floating and sinking could not be seen very well. The results were best visible with and cell density of OD600=1.5. Also we tried to do the buoyancy test in a longer tube since it was expected that the difference between floating and sinking would be more obvious. This, however, did not appear to be the case, unfortunately. Also doing the buoyancy test in a higher saline concentration did not have an enhanced floating effect. <br />
Another adaption we tried was the way of induction. In the standard protocol the cells were induced in the overnight culture. It was also tried if induction in the saline or at the exponential phase of growth or even induction on plate would make any difference. Unfortunately this did not make a huge difference.<br />
<br />
''Fermentor test''<br />
<br />
[[Image:Buoyancy pNL29.jpg|800px|thumb|center|Figure 3. Buoyancy test with pNL29, cells were induced in exponential phase and resuspended in NaCl in a OD600 of 1.5 A) Fermentor buoyancy test, samples taken after 1 hour, 2.5 hours, 6 hours, 8.5 hours and 22.5 hours. The cells were induced after 1 hour, at exponential phase. Photgraph taken 1 day after resuspension. B) normal, non-fermentor buoyancy test, samples taken at t=1 t/m 4. Photograph taken 1 day after resuspension. C) Same as A, photograph was taken 2 days after resuspension. D) same as C, photograph taken 2 days after resuspension.]]<br />
<br />
{|<br />
|[[Image:Buoyancy arsR GVP.jpg|500px|thumb|Figure 4. Buoyancy test with pSB1AC3 containing pArsR-GVP, cells were induced in exponential phase and resuspended in NaCl in a OD600 of 1.5 A) Fermentor buoyancy test, samples taken after 1 hour, 2 hours, 6 hours, 7.5 hours and 22 hours. The cells were induced after 1 hour, at exponential phase. Photgraph taken 1 day after resuspension. B) normal, non-fermentor buoyancy test, samples taken at t=1 t/m 4. Photograph taken 1 day after resuspension.]]<br />
|[[Image:Groningen_ODFerAndNonFer.PNG|400px|thumb|Figure 5. Graph of OD measurements at 600nm of both the fermentor and non-fermentor tests. Solid lines and points represent actual measurements, dotted lines represent the expected curve between the last two measurements]]<br />
|}<br />
<div style="clear:both"></div><br />
<br />
So the length of the tube, the saline concentration and the time of induction did work for the buoyancy test. It was also suggested that there was not enough gas in the surrounding of the cells and a better result could be achieved if this could be improve or another gas could be used. To test this we tried to grow the cells in a [[Team:Groningen/Protocols#Fermentation|fermentor]]. It was also suggested that the fermentor test could be done with helium, however, modelling showed that it would not make a difference in floating which gas is used as long as it is lighter than water (check this yourself by changing the density (ρ) of the gas [[Team:Groningen/Project/Vesicle#Modelling|here]]). Therefore the fermentor test was done by using O<sub>2</sub>. This resulted in better buoyancy results. As can be seen in figure 3A the positive control pNL29 showed better buoyancy over time. After 2 hours the cells were in exponential phase and were induced with IPTG. After 8,5 hours the buoyancy is best, after 22.5 hours the buoyancy the cell level is declining. This suggests that there is an optimum after 8,5 hours. The cells at t=22.5 are probably in stationary phase whereas the cells at t=8.5h could still be in exponential phase, this could explain the difference in buoyancy found. It suggests that in stationary phase less gas vesicles are produced. Figure 3C shows the same tubes 24 hours later. This still shows buoyancy for the t=6 and t=8 tubes and no buoyancy for the others. This suggest that the buoyancy last for at least 24 hours. Simultaneously a normal, non-fermentor, buoyancy test was also performed with the same construct. In figure 3B these results at day 1 can be seen, this shows nothing. After a day no buoyancy can be seen for t=1 and t=2 a more dense cell suspension can be seen for t=3 and t=4 (figure 3D), however still no confincing buoyancy can be seen.<br />
Figure 4 shows the results from one of our own constructs, pArsR-GvP (<partinfo>BBa_K190033</partinfo>) grown in a fermentor. This shows an increase in buoyancy in time, however, at t=22.5h no buoyant cells can be seen. A buoyancy test done at the same time without a fermentor shows the same increase in buoyancy but does show buoyancy at t=22.5h (figure 4B). This difference can be explained since the cells in the fermentor are probably already dead or dying. In a fermentor the cell density is large this causes the cells to die.<br />
<br />
==Electron Microscopy==<br />
<br />
To check whether gas vesicles really were present in the cells we did some electron microscopy.<br />
<br />
In Figure 5 a picture of gas vesicles in a protoplast can be seen. This protoplast comes from an ''E. coli'' cell that contained a plasmid with the GVP gene cluster behind an arsenic sensitive promoter (<partinfo>BBa_K190033</partinfo>).<br />
<br />
<br />
[[Image:GasvesiclesEM.jpg|thumb|500px|left|Figure 5. Gas vesicles in ''E. coli'' protoplasts (<partinfo>BBa_K190033</partinfo>). The cells were treated with Lysozyme and SDS to create the protoplasts, uranyl acetate was used for staining. Magnification: 11500x.]]<br />
<br />
<div style="clear:both"></div><br />
<br />
==Modelling==<br />
<br />
===Buoyancy===<br />
The gas vesicles are shaped roughly like a cylinder with a cone at each end, whose cross-section we model as (based mostly on [[Team:Groningen/Literature#Walsby1994|Walsby 1994]]):<br />
<br />
[[Image:Vesicle_Shape.png]]<br />
<br />
We assume the interior of the wall of the gas vesicle is similarly shaped to the exterior, just slightly smaller (the right-most part of the image above illustrates this situation for the left tip of the gas vesicle). This means the different dimensions are related through the equations below. To determine the total volume, just use them with the given width/diameter (at least for the dimensions given in [[Team:Groningen/Literature#Walsby1994|Walsby 1994]]). To determine the gas volume, use them with w<sub>gas</sub> and d<sub>gas</sub>.<br />
<br />
{|<br />
|style="vertical-align:top;"|<html><br />
<div style="background:#efe;border:1px solid #9c9;padding:1em;"><br />
<table style="border-collapse:collapse;background:none;"><tr><br />
<td style="border-right:1px solid #9c9;padding-right:1em;"><br />
w = <input type="text" id="w" value="300"/> nm (</html>[[:Image:Ars-lyzo-007.png|TEM picture]]<html>)<br/><br />
d = <input type="text" id="d" value="75"/> nm (</html>[[:Image:Ars-lyzo-007.png|TEM picture]]<html>)<br/><br />
tw = <input type="text" id="tw" value="1.8"/> nm (</html>[[Team:Groningen/Literature#Walsby1994|Walsby1994]]<html>)<br/><br />
a = <input type="text" id="a" value="77"/> &deg; (</html>[[Team:Groningen/Literature#Walsby1994|Walsby1994]]<html>)<br/><br />
&rho;<sub>gas</sub> = <input type="text" id="rhogas" value="1.2"/> kg/m<sup>3</sup> (</html>[[Team:Groningen/Literature#Walsby1994|Walsby1994]]<html>)<br/> <!-- Walsby1994, for moist air at atmospheric pressure --><br />
&rho;<sub>wall</sub> = <input type="text" id="rhowall" value="1320"/> kg/m<sup>3</sup> (</html>[[Team:Groningen/Literature#Walsby1994|Walsby1994]]<html>)<br/> <!-- Walsby1994 --><br />
<br />
<button onClick="computeVolumes()">Compute</button><br/><br />
</td><br />
<br />
<td style="padding-left:1em;"><br />
<div id="volumeError" style="color:red"></div><br />
V<sub>gas</sub> = <span id="Vgas"></span> nm<sup>3</sup><br/><br />
M<sub>gas</sub> = <span id="Mgas"></span> yg<br/><br />
V<sub>wall</sub> = <span id="Vwall"></span> nm<sup>3</sup><br/><br />
M<sub>wall</sub> = <span id="Mwall"></span> yg<br/><br />
V<sub>vesicle</sub> = <span id="Vvesicle"></span> nm<sup>3</sup><br/><br />
M<sub>vesicle</sub> = <span id="Mvesicle"></span> yg<br/><br />
<b>&rho;<sub>vesicle</sub> = <span id="rhovesicle"></span> kg/m<sup>3</sup></b><br/><br />
</td><br />
</tr></table><br />
</div><br />
<script type="text/javascript"><br />
<br />
addOnloadHook(computeVolumes);<br />
<br />
function computeVolumes() {<br />
// Input<br />
var wNode = document.getElementById("w");<br />
var twNode = document.getElementById("tw");<br />
var dNode = document.getElementById("d");<br />
var aNode = document.getElementById("a");<br />
var rhogasNode = document.getElementById("rhogas");<br />
var rhowallNode = document.getElementById("rhowall");<br />
<br />
// Intermediates (mostly useful for debugging)<br />
var volumeErrorNode = document.getElementById("volumeError");<br />
var wwtNode = document.getElementById("wwt");<br />
volumeErrorNode.innerHTML = '';<br />
<br />
// Outputs<br />
var VgasNode = document.getElementById("Vgas");<br />
var VwallNode = document.getElementById("Vwall");<br />
var MgasNode = document.getElementById("Mgas");<br />
var MwallNode = document.getElementById("Mwall");<br />
var VvesicleNode = document.getElementById("Vvesicle");<br />
var MvesicleNode = document.getElementById("Mvesicle");<br />
var rhovesicleNode = document.getElementById("rhovesicle");<br />
<br />
// Read inputs<br />
var w = Number(wNode.value);<br />
var tw = Number(twNode.value);<br />
var d = Number(dNode.value);<br />
var a = Number(aNode.value) * Math.PI / 180.0;<br />
var rhogas = Number(rhogasNode.value);<br />
var rhowall = Number(rhowallNode.value);<br />
<br />
// Compute Vgas and Vwall<br />
try {<br />
var wwt = tw/Math.sin(a/2);<br />
var Vvesicle = computeVolume(w, d, a);<br />
var Vgas = computeVolume(w-2*wwt,d-2*tw,a);<br />
var Vwall = Vvesicle - Vgas;<br />
var Mgas = rhogas*Vgas;<br />
var Mwall = rhowall*Vwall;<br />
var Mvesicle = Mgas+Mwall;<br />
var rhovesicle = Mvesicle/Vvesicle;<br />
} catch(err) {<br />
volumeErrorNode.innerHTML = err.message;<br />
}<br />
<br />
// Set intermediates if they exist<br />
if (wwtNode) setOutput(wwtNode, wwt);<br />
<br />
// Set outputs<br />
setOutput(VgasNode, Vgas);<br />
setOutput(VwallNode, Vwall);<br />
setOutput(MgasNode, Mgas);<br />
setOutput(MwallNode, Mwall);<br />
setOutput(VvesicleNode, Vvesicle);<br />
setOutput(MvesicleNode, Mvesicle);<br />
setOutput(rhovesicleNode, rhovesicle);<br />
}<br />
<br />
function computeVolume(w,d,a) {<br />
// This computes the volume of cylinder with a cone at each end as defined in the text.<br />
var wt = (1/2)*d/Math.tan(a/2);<br />
var wc = w-2*wt;<br />
var Vc = (1/4)*Math.PI*Math.pow(d,2)*wc;<br />
var Vt = (1/12)*Math.PI*Math.pow(d,2)*wt;<br />
if (wc<0) throw Error("The given diameter would imply a larger width.<br/>(Do not trust the computed volumes!)");<br />
return Vc+2*Vt;<br />
}<br />
<br />
function formatNumberToHTML(v,p) {<br />
if (p===undefined) p = 5;<br />
return v.toPrecision(p)<br />
.replace(/e\+([0-9]+)$/i,'&middot;10<sup>$1</sup>')<br />
.replace(/e\-([0-9]+)$/i,'&middot;10<sup>-$1</sup>');<br />
}<br />
<br />
function setOutput(node,v,p) {<br />
node.innerHTML = formatNumberToHTML(v);<br />
node.value = v;<br />
}<br />
</script><br />
</html><br />
|style="vertical-align:top;"|<pre><br />
w = total width<br />
tw = thickness of wall (1.8-1.95nm)<br />
d = diameter<br />
a = 77 degrees<br />
&rho;gas = density of gas in vesicle (kg/m^3 = yg/nm^3)<br />
&rho;wall = density of vesicle wall (kg/m^3)<br />
wwt = tw/sin(a/2)<br />
wt = (1/2)*d/tan(a/2)<br />
wc = w - 2*wt<br />
Vc = (1/4)*pi*d^2*wc<br />
Vt = (1/12)*pi*d^2*wt<br />
V = Vc+2*Vt<br />
M = &rho;*V<br />
<br />
wgas = w-2*wwt = width of gas space<br />
dgas = d-2*tw = diameter of gas space<br />
V = Vgas + Vwall<br />
</pre><br />
|}<br />
<br />
Now we can consider the buoyant density of <i>E. coli</i> with gas vesicles. We have chosen to approach this problem using densities and volume ratios. According to [[Team:Groningen/Literature#Baldwin1995|Baldwin 1995]], [[Team:Groningen/Literature#Bylund1991|Bylund 1991]] and [[Team:Groningen/Literature#Poole1977|Poole 1977]], the density of (wild-type) <i>E. coli</i> is 1100 kg/m<sup>3</sup> &plusmn;3% under wildly varying conditions. This makes our method easier than trying to directly compute the density of a single cell, due to the fact that the volume can differ wildly (both during the life cycle and from strain to strain) and a lack of concrete data on the number of gas vesicles produced (in <i>E. coli</i>). Note that the computations below assume that the gas vesicles simply add to the existing structures.<br />
<br />
{|<br />
|style="vertical-align:top;"|<html><br />
<div style="background:#efe;border:1px solid #9c9;padding:1em;"><br />
<table style="border-collapse:collapse;background:none;"><tr><br />
<td style="border-right:1px solid #9c9;padding-right:1em;"><br />
<nobr>&rho;<sub>medium</sub> = <input type="text" id="rhomedium" value="1000"/> kg/m<sup>3</sup></nobr><br/><br />
&rho;<sub>cell</sub> = <input type="text" id="rhocell" value="1100"/> kg/m<sup>3</sup><br/> <!-- Reasonable estimate, TODO: more precision+reference --><br />
<br />
<button onClick="computeEColiDensity()">Compute</button><br/><br />
</td><br />
<br />
<td style="padding-left:1em;"><br />
<div id="densityError" style="color:red"></div><br />
V<sub>v</sub> / V<sub>cv</sub> > <span id="relVvesicles"></span><br/><br />
</td><br />
</tr></table><br />
</div><br />
<script type="text/javascript"><br />
<br />
addOnloadHook(computeEColiDensity);<br />
<br />
function computeEColiDensity() {<br />
// Input<br />
var rhomediumNode = document.getElementById("rhomedium");<br />
var rhovesicleNode = document.getElementById("rhovesicle");<br />
var rhocellNode = document.getElementById("rhocell");<br />
<br />
// Intermediates (mostly useful for debugging)<br />
var densityErrorNode = document.getElementById("densityError");<br />
densityErrorNode.innerHTML = '';<br />
<br />
// Outputs<br />
var relVvesiclesNode = document.getElementById("relVvesicles");<br />
<br />
// Read inputs<br />
var rhomedium = Number(rhomediumNode.value);<br />
var rhovesicle = Number(rhovesicleNode.value);<br />
var rhocell = Number(rhocellNode.value);<br />
<br />
// Compute density(/-ies)<br />
try {<br />
var relVvesicles = 1.0 - (rhomedium-rhovesicle)/(rhocell-rhovesicle);<br />
if (rhovesicle>=rhocell) throw Error("Vesicle denser than cell, > should be <.");<br />
} catch(err) {<br />
densityErrorNode.innerHTML = err.message;<br />
}<br />
<br />
// Set intermediates if they exist<br />
<br />
// Set outputs<br />
setOutput(relVvesiclesNode, relVvesicles);<br />
document.getElementById('densityLimitGraph').refresh();<br />
}<br />
</script><br />
</html><br />
{{graph|Team:Groningen/Graphs/DensityLimit|id=densityLimitGraph}}<br />
|style="vertical-align:top;"|<pre><br />
Vc = volume of a cell without gas vesicles<br />
Vv = volume of gas vesicles in cell<br />
Vcv = volume of a cell with gas vesicles (assumed to be Vc+Vv)<br />
&rho;c = density of a cell without gas vesicles<br />
&rho;v = density of gas vesicles<br />
&rho;m = density of medium<br />
<br />
The following has to be true if the cell floats:<br />
Vc*&rho;c + Vv*&rho;v < Vcv*&rho;m<br />
(Vcv-Vv)*&rho;c + Vv*&rho;v < Vcv*&rho;m<br />
&rho;c + (Vv/Vcv)*(&rho;v-&rho;c) < &rho;m<br />
Assume (&rho;v - &rho;c)<0<br />
Vv/Vcv > (&rho;m - &rho;c)/(&rho;v - &rho;c)<br />
Vv/Vcv > 1 - (&rho;m - &rho;v)/(&rho;c - &rho;v)<br />
</pre><br />
'''Explanation of the graph'''<br />
<br />
Four curves are shown, corresponding to how many gas vesicles a cell needs with "our" gas vesicles (unless you changed the constants in the calculator above), the gas vesicles documented in [[Team:Groningen/Literature#Li1998|Li 1998]]{{infoBox|Using a width and diameter of 75nm and 50nm, respectively. Here we assume that their "width" should be interpreted as our diameter, as doing it the other way around would leave no room for a cylinder and they specifically mention that the vesicles appear to be shaped like cylinders with conical ends.}}, the gas vesicles from Anabaena in [[Team:Groningen/Literature#Walsby1994|Walsby 1994]]{{infoBox|Using a width and diameter of 500nm and 84nm, respectively.}} and our gas vesicles when the medium has the density of seawater.<br />
<br />
'''The X-axis''' depicts the cell density of the part of the cell not occupied by gas vesicles.<br />
<br />
'''The Y-axis''' depicts the minimum volume fraction of the cell that should consist of gas vesicles to make the cell float.<br />
|}<br />
<br />
{{GraphHeader}}<br />
<br />
==Conclusion & Discussion==<br />
<br />
We have experimented with two different constructs containing the GVP gene cluster i.e. pNL29 containing the 6 kb gene cluster from ''Bacillus megaterium'' ([[Team:Groningen/Literature#Li1998|Li & Cannon 1998]]) and [http://partsregistry.org/wiki/index.php/Part:BBa_I750016 BBa_I750016] from the [http://parts.mit.edu/igem07/index.php/Melbourne Melbourne 2007] iGEM team.<br />
We observed that it is best to have an OD600 of 1.5 when doing bouyancy tests, for withnessing differences with lower values is difficult. Furthermore buoyancy tests carried out in sea water or normal (LB) medium also give rise to difficult to interpret results. <br />
<br />
For cells cultivated in a more aerobic environment, such as the ones carried out in a fermentor, an enhanced bouyancy phenotype is observed. The extra O<sub>2</sub> added probably causes a higher concentration of intracellular oxygen, that can diffuse to the gas vesicles that are produced. The best buoyancy phenotype is withnessed at t=8.5 hours, however at t=22.5 hours no buoyancy can be seen. This suggests that there is an optimum after 8,5 hours.The cells at t=22.5 are probably in stationary phase whereas the cells at t=8.5h could still be in exponential phase, this could explain the difference in buoyancy found. It suggests that in stationary phase less gas vesicles are produced. Buoyancy is observed after 1 day, and also still after 2 days, which suggest that the buoyancy last for at least 24 hours. This is in accordance with experimental data from [[Team:Groningen/Literature#Walsby1994|Walsby, 1994]], who also observed a buoyant phenotype for over 2 days.<br />
<br />
<br />
{{Team:Groningen/Project/Footer}}</div>JolandaWitteveenhttp://2009.igem.org/Team:Groningen/Project/VesicleTeam:Groningen/Project/Vesicle2009-10-21T16:19:29Z<p>JolandaWitteveen: </p>
<hr />
<div>{{Team:Groningen/Project/Header|}}<br />
<br />
{| style type="clear:both"<br />
|<html><style type="text/css"><br />
.intro { margin-left:0px; margin-top:10px; padding:10px; border-left:solid 5px #FFF6D5; border-right:solid 5px #FFF6D5; text-align:justify;background:#FFFFE5; }<br />
</style></html><br />
<div class="intro"><br />
<h2>Gas vesicles</h2><br />
'''Our goal in this project is to make cells bouyant in the presence of certain concentrations of metals like copper, zinc and arsenic. Metal induced gas vesicle production can provide our cells with this bouyancy. Gas vesicles are bacterial organelles consisting entirely of proteins that envelop a gas filled space. We made, and send to the registry, parts in which the metal sensitive promoters for copper, zinc and arsenic were cloned in front of the GVP (Gas Vesicle Protein) gene cluster. For further characterization of the GVP gene cluster inducible and constitutive promoters were also cloned in front of this cluster. Buoyancy tests showed that our constructs were able to increase cell buoyancy and electron micrographs showed the presence of gas vesicles. A model was made to predict what volume fraction of a cell would have to be gas vesicle for this cell to have a density equal to that of water.'''<br />
<br><br><br><br />
</div><br />
|}<br />
<br />
<br />
==Background==<br />
<br />
Gas vesicles are organelles made entirely out of proteins that envelop a gas filled space. Because only gas can penetrate into the gas vesicles the total density of the cell is lowered. This lower cell density leads in turn to a buoyancy phenotype. Outside of the laboratory this buoyancy is used by microorganisms to vertically position themselves in the water column or simply to reduce their sinking rates. Organisms can regulate buoyancy by reducing gas vesicle production or by accumulation of denser compounds like carbohydrates. For certain cyanobacteria this regulation depends on light intensities. <br />
<br />
For a number of organisms it has been shown that all proteins important for the expression of gas vesicle lie in a single gene cluster. The GVP gene cluster used in this project was cloned from Bacillus megaterium into E. coli ([[Team:Groningen/Literature#Li1998|Li & Cannon 1998]]). This gene cluster now containing 11 genes was turned into a biobrick by Melbourne 2007 (link to part). Figure 1 shows the gene cluster as it was send in by Melbourne in 2007.<br />
<br />
For more info see: [[Team:Groningen/Literature#Walsby1994|Walsby 1994]].<br />
<br />
[[Image:Gas vesicle cluster bba I750016.PNG|frame|Figure 1: Gas vesicle gene cluster (<partinfo>BBa_I750016</partinfo>)<br />
]]<br />
<br />
==Goal==<br />
<br />
The goal of our project is to engineer an organism that can remove heavy metals from water. To facilitate easy separation the cells that have taken up metals should float so they can be removed from the water. The introduction of GVP gene cluster provides the cell with the required buoyancy. <br />
<br />
To make the buoyancy level of the cell responsive to the metal concentration inside the cell, metal sensitive promoters would have to be cloned in front of the GVP gene cluster. In this project these metal sensitive promoters were responsive to zinc, copper and arsenic. We also wanted to make a construct with constitutive and inducible promoters in front of the GVP cluster to show a proof of principle and as a back-up if our metal induced construct would fail. <br />
<br />
Finally we wanted to improve the Melbourne 2007 biobrick (<partinfo>BBa_I750016</partinfo>) by removing a repeat that was accidentally introduced during the removal of forbidden restriction sites.<br />
<br />
==Cloning strategy== <br />
For our msGVP (metal sensitive GVP) constructs we ordered oligo's containing the promoter region and the necessary restriction sites. When annealed these pieces of DNA have EcoRI and SpeI sticky ends. The vector containing GVP (<partinfo>BBa_I750016</partinfo>) was cut with EcoRI and XbaI and was ligated to the promoter. ([[Team:Groningen/Protocols#Annealing synthetic oligo’s|Protocol]])<br />
<br />
The metal sensitive GVP constructs are: <partinfo>BBa_K190033</partinfo>, <partinfo>BBa_K190034</partinfo> and <partinfo>BBa_K190035</partinfo>.<br />
<br />
Then because of compatibility issues when our entire system has to be assembled into one cell the whole metal sensitive promoter and GVP part were transfered to a different vector.<br />
<br />
[[Image:Cloning strategy floating device1.PNG]]<br />
:Figure 2: The floating device will be built up of an inducible promoter which can be induced by a certain intracellular concentration of metal-ions, and a gas vesicle cluster.<br />
<br />
==Bouyancy Tests==<br />
<br />
The buoyancy of GVP was tested by using the [[Team:Groningen/Protocols#Bouyancytest|buoyancy test protocol]]. The cells were grown in medium and induced and were resuspended in a salt solution (0.15mM NaCl) in a test tube and were left for a while in order to give the cells time to sink or float. <br />
<br />
''Different circumstances''<br />
<br />
Several different circumstances and small changes to the protocol were made in order to find the perfect circumstance for the buoyancy test. It appeared that with a low cell density the difference between floating and sinking could not be seen very well. The results were best visible with and cell density of OD600=1.5. Also we tried to do the buoyancy test in a longer tube since it was expected that the difference between floating and sinking would be more obvious. This, however, did not appear to be the case, unfortunately. Also doing the buoyancy test in a higher saline concentration did not have an enhanced floating effect. <br />
Another adaption we tried was the way of induction. In the standard protocol the cells were induced in the overnight culture. It was also tried if induction in the saline or at the exponential phase of growth or even induction on plate would make any difference. Unfortunately this did not make a huge difference.<br />
<br />
''Fermentor test''<br />
<br />
[[Image:Buoyancy pNL29.jpg|800px|thumb|center|Figure 3. Buoyancy test with pNL29, cells were induced in exponential phase and resuspended in NaCl in a OD600 of 1.5 A) Fermentor buoyancy test, samples taken after 1 hour, 2.5 hours, 6 hours, 8.5 hours and 22.5 hours. The cells were induced after 1 hour, at exponential phase. Photgraph taken 1 day after resuspension. B) normal, non-fermentor buoyancy test, samples taken at t=1 t/m 4. Photograph taken 1 day after resuspension. C) Same as A, photograph was taken 2 days after resuspension. D) same as C, photograph taken 2 days after resuspension.]]<br />
<br />
{|<br />
|[[Image:Buoyancy arsR GVP.jpg|500px|thumb|Figure 4. Buoyancy test with pSB1AC3 containing pArsR-GVP, cells were induced in exponential phase and resuspended in NaCl in a OD600 of 1.5 A) Fermentor buoyancy test, samples taken after 1 hour, 2 hours, 6 hours, 7.5 hours and 22 hours. The cells were induced after 1 hour, at exponential phase. Photgraph taken 1 day after resuspension. B) normal, non-fermentor buoyancy test, samples taken at t=1 t/m 4. Photograph taken 1 day after resuspension.]]<br />
|[[Image:Groningen_ODFerAndNonFer.PNG|400px|thumb|Figure 5. Graph of OD measurements at 600nm of both the fermentor and non-fermentor tests. Solid lines and points represent actual measurements, dotted lines represent the expected curve between the last two measurements]]<br />
|}<br />
<div style="clear:both"></div><br />
<br />
So the length of the tube, the saline concentration and the time of induction did work for the buoyancy test. It was also suggested that there was not enough gas in the surrounding of the cells and a better result could be achieved if this could be improve or another gas could be used. To test this we tried to grow the cells in a [[Team:Groningen/Protocols#Fermentation|fermentor]]. It was also suggested that the fermentor test could be done with helium, however, modelling showed that it would not make a difference in floating which gas is used as long as it is lighter than water (check this yourself by changing the density (ρ) of the gas [[Team:Groningen/Project/Vesicle#Modelling|here]]). Therefore the fermentor test was done by using O<sub>2</sub>. This resulted in better buoyancy results. As can be seen in figure 3A the positive control pNL29 showed better buoyancy over time. After 2 hours the cells were in exponential phase and were induced with IPTG. After 8,5 hours the buoyancy is best, after 22.5 hours the buoyancy the cell level is declining. This suggests that there is an optimum after 8,5 hours. The cells at t=22.5 are probably in stationary phase whereas the cells at t=8.5h could still be in exponential phase, this could explain the difference in buoyancy found. It suggests that in stationary phase less gas vesicles are produced. Figure 3C shows the same tubes 24 hours later. This still shows buoyancy for the t=6 and t=8 tubes and no buoyancy for the others. This suggest that the buoyancy last for at least 24 hours. Simultaneously a normal, non-fermentor, buoyancy test was also performed with the same construct. In figure 3B these results at day 1 can be seen, this shows nothing. After a day no buoyancy can be seen for t=1 and t=2 a more dense cell suspension can be seen for t=3 and t=4 (figure 3D), however still no confincing buoyancy can be seen.<br />
Figure 4 shows the results from one of our own constructs, pArsR-GvP (<partinfo>BBa_K190033</partinfo>) grown in a fermentor. This shows an increase in buoyancy in time, however, at t=22.5h no buoyant cells can be seen. A buoyancy test done at the same time without a fermentor shows the same increase in buoyancy but does show buoyancy at t=22.5h (figure 4B). This difference can be explained since the cells in the fermentor are probably already dead or dying. In a fermentor the cell density is large this causes the cells to die.<br />
<br />
==Electron Microscopy==<br />
<br />
To check whether gas vesicles really were present in the cells we did some electron microscopy.<br />
<br />
In Figure 5 a picture of gas vesicles in a protoplast can be seen. This protoplast comes from an ''E. coli'' cell that contained a plasmid with the GVP gene cluster behind an arsenic sensitive promoter (<partinfo>BBa_K190033</partinfo>).<br />
<br />
<br />
[[Image:GasvesiclesEM.jpg|thumb|500px|left|Figure 5. Gas vesicles in ''E. coli'' protoplasts (<partinfo>BBa_K190033</partinfo>). The cells were treated with Lysozyme and SDS to create the protoplasts, uranyl acetate was used for staining. Magnification: 11500x.]]<br />
<br />
<div style="clear:both"></div><br />
<br />
==Modelling==<br />
<br />
===Buoyancy===<br />
The gas vesicles are shaped roughly like a cylinder with a cone at each end, whose cross-section we model as (based mostly on [[Team:Groningen/Literature#Walsby1994|Walsby 1994]]):<br />
<br />
[[Image:Vesicle_Shape.png]]<br />
<br />
We assume the interior of the wall of the gas vesicle is similarly shaped to the exterior, just slightly smaller (the right-most part of the image above illustrates this situation for the left tip of the gas vesicle). This means the different dimensions are related through the equations below. To determine the total volume, just use them with the given width/diameter (at least for the dimensions given in [[Team:Groningen/Literature#Walsby1994|Walsby 1994]]). To determine the gas volume, use them with w<sub>gas</sub> and d<sub>gas</sub>.<br />
<br />
{|<br />
|style="vertical-align:top;"|<html><br />
<div style="background:#efe;border:1px solid #9c9;padding:1em;"><br />
<table style="border-collapse:collapse;background:none;"><tr><br />
<td style="border-right:1px solid #9c9;padding-right:1em;"><br />
w = <input type="text" id="w" value="300"/> nm (</html>[[:Image:Ars-lyzo-007.png|TEM picture]]<html>)<br/><br />
d = <input type="text" id="d" value="75"/> nm (</html>[[:Image:Ars-lyzo-007.png|TEM picture]]<html>)<br/><br />
tw = <input type="text" id="tw" value="1.8"/> nm (</html>[[Team:Groningen/Literature#Walsby1994|Walsby1994]]<html>)<br/><br />
a = <input type="text" id="a" value="77"/> &deg; (</html>[[Team:Groningen/Literature#Walsby1994|Walsby1994]]<html>)<br/><br />
&rho;<sub>gas</sub> = <input type="text" id="rhogas" value="1.2"/> kg/m<sup>3</sup> (</html>[[Team:Groningen/Literature#Walsby1994|Walsby1994]]<html>)<br/> <!-- Walsby1994, for moist air at atmospheric pressure --><br />
&rho;<sub>wall</sub> = <input type="text" id="rhowall" value="1320"/> kg/m<sup>3</sup> (</html>[[Team:Groningen/Literature#Walsby1994|Walsby1994]]<html>)<br/> <!-- Walsby1994 --><br />
<br />
<button onClick="computeVolumes()">Compute</button><br/><br />
</td><br />
<br />
<td style="padding-left:1em;"><br />
<div id="volumeError" style="color:red"></div><br />
V<sub>gas</sub> = <span id="Vgas"></span> nm<sup>3</sup><br/><br />
M<sub>gas</sub> = <span id="Mgas"></span> yg<br/><br />
V<sub>wall</sub> = <span id="Vwall"></span> nm<sup>3</sup><br/><br />
M<sub>wall</sub> = <span id="Mwall"></span> yg<br/><br />
V<sub>vesicle</sub> = <span id="Vvesicle"></span> nm<sup>3</sup><br/><br />
M<sub>vesicle</sub> = <span id="Mvesicle"></span> yg<br/><br />
<b>&rho;<sub>vesicle</sub> = <span id="rhovesicle"></span> kg/m<sup>3</sup></b><br/><br />
</td><br />
</tr></table><br />
</div><br />
<script type="text/javascript"><br />
<br />
addOnloadHook(computeVolumes);<br />
<br />
function computeVolumes() {<br />
// Input<br />
var wNode = document.getElementById("w");<br />
var twNode = document.getElementById("tw");<br />
var dNode = document.getElementById("d");<br />
var aNode = document.getElementById("a");<br />
var rhogasNode = document.getElementById("rhogas");<br />
var rhowallNode = document.getElementById("rhowall");<br />
<br />
// Intermediates (mostly useful for debugging)<br />
var volumeErrorNode = document.getElementById("volumeError");<br />
var wwtNode = document.getElementById("wwt");<br />
volumeErrorNode.innerHTML = '';<br />
<br />
// Outputs<br />
var VgasNode = document.getElementById("Vgas");<br />
var VwallNode = document.getElementById("Vwall");<br />
var MgasNode = document.getElementById("Mgas");<br />
var MwallNode = document.getElementById("Mwall");<br />
var VvesicleNode = document.getElementById("Vvesicle");<br />
var MvesicleNode = document.getElementById("Mvesicle");<br />
var rhovesicleNode = document.getElementById("rhovesicle");<br />
<br />
// Read inputs<br />
var w = Number(wNode.value);<br />
var tw = Number(twNode.value);<br />
var d = Number(dNode.value);<br />
var a = Number(aNode.value) * Math.PI / 180.0;<br />
var rhogas = Number(rhogasNode.value);<br />
var rhowall = Number(rhowallNode.value);<br />
<br />
// Compute Vgas and Vwall<br />
try {<br />
var wwt = tw/Math.sin(a/2);<br />
var Vvesicle = computeVolume(w, d, a);<br />
var Vgas = computeVolume(w-2*wwt,d-2*tw,a);<br />
var Vwall = Vvesicle - Vgas;<br />
var Mgas = rhogas*Vgas;<br />
var Mwall = rhowall*Vwall;<br />
var Mvesicle = Mgas+Mwall;<br />
var rhovesicle = Mvesicle/Vvesicle;<br />
} catch(err) {<br />
volumeErrorNode.innerHTML = err.message;<br />
}<br />
<br />
// Set intermediates if they exist<br />
if (wwtNode) setOutput(wwtNode, wwt);<br />
<br />
// Set outputs<br />
setOutput(VgasNode, Vgas);<br />
setOutput(VwallNode, Vwall);<br />
setOutput(MgasNode, Mgas);<br />
setOutput(MwallNode, Mwall);<br />
setOutput(VvesicleNode, Vvesicle);<br />
setOutput(MvesicleNode, Mvesicle);<br />
setOutput(rhovesicleNode, rhovesicle);<br />
}<br />
<br />
function computeVolume(w,d,a) {<br />
// This computes the volume of cylinder with a cone at each end as defined in the text.<br />
var wt = (1/2)*d/Math.tan(a/2);<br />
var wc = w-2*wt;<br />
var Vc = (1/4)*Math.PI*Math.pow(d,2)*wc;<br />
var Vt = (1/12)*Math.PI*Math.pow(d,2)*wt;<br />
if (wc<0) throw Error("The given diameter would imply a larger width.<br/>(Do not trust the computed volumes!)");<br />
return Vc+2*Vt;<br />
}<br />
<br />
function formatNumberToHTML(v,p) {<br />
if (p===undefined) p = 5;<br />
return v.toPrecision(p)<br />
.replace(/e\+([0-9]+)$/i,'&middot;10<sup>$1</sup>')<br />
.replace(/e\-([0-9]+)$/i,'&middot;10<sup>-$1</sup>');<br />
}<br />
<br />
function setOutput(node,v,p) {<br />
node.innerHTML = formatNumberToHTML(v);<br />
node.value = v;<br />
}<br />
</script><br />
</html><br />
|style="vertical-align:top;"|<pre><br />
w = total width<br />
tw = thickness of wall (1.8-1.95nm)<br />
d = diameter<br />
a = 77 degrees<br />
&rho;gas = density of gas in vesicle (kg/m^3 = yg/nm^3)<br />
&rho;wall = density of vesicle wall (kg/m^3)<br />
wwt = tw/sin(a/2)<br />
wt = (1/2)*d/tan(a/2)<br />
wc = w - 2*wt<br />
Vc = (1/4)*pi*d^2*wc<br />
Vt = (1/12)*pi*d^2*wt<br />
V = Vc+2*Vt<br />
M = &rho;*V<br />
<br />
wgas = w-2*wwt = width of gas space<br />
dgas = d-2*tw = diameter of gas space<br />
V = Vgas + Vwall<br />
</pre><br />
|}<br />
<br />
Now we can consider the buoyant density of <i>E. coli</i> with gas vesicles. We have chosen to approach this problem using densities and volume ratios. According to [[Team:Groningen/Literature#Baldwin1995|Baldwin 1995]], [[Team:Groningen/Literature#Bylund1991|Bylund 1991]] and [[Team:Groningen/Literature#Poole1977|Poole 1977]], the density of (wild-type) <i>E. coli</i> is 1100 kg/m<sup>3</sup> &plusmn;3% under wildly varying conditions. This makes our method easier than trying to directly compute the density of a single cell, due to the fact that the volume can differ wildly (both during the life cycle and from strain to strain) and a lack of concrete data on the number of gas vesicles produced (in <i>E. coli</i>). Note that the computations below assume that the gas vesicles simply add to the existing structures.<br />
<br />
{|<br />
|style="vertical-align:top;"|<html><br />
<div style="background:#efe;border:1px solid #9c9;padding:1em;"><br />
<table style="border-collapse:collapse;background:none;"><tr><br />
<td style="border-right:1px solid #9c9;padding-right:1em;"><br />
<nobr>&rho;<sub>medium</sub> = <input type="text" id="rhomedium" value="1000"/> kg/m<sup>3</sup></nobr><br/><br />
&rho;<sub>cell</sub> = <input type="text" id="rhocell" value="1100"/> kg/m<sup>3</sup><br/> <!-- Reasonable estimate, TODO: more precision+reference --><br />
<br />
<button onClick="computeEColiDensity()">Compute</button><br/><br />
</td><br />
<br />
<td style="padding-left:1em;"><br />
<div id="densityError" style="color:red"></div><br />
V<sub>v</sub> / V<sub>cv</sub> > <span id="relVvesicles"></span><br/><br />
</td><br />
</tr></table><br />
</div><br />
<script type="text/javascript"><br />
<br />
addOnloadHook(computeEColiDensity);<br />
<br />
function computeEColiDensity() {<br />
// Input<br />
var rhomediumNode = document.getElementById("rhomedium");<br />
var rhovesicleNode = document.getElementById("rhovesicle");<br />
var rhocellNode = document.getElementById("rhocell");<br />
<br />
// Intermediates (mostly useful for debugging)<br />
var densityErrorNode = document.getElementById("densityError");<br />
densityErrorNode.innerHTML = '';<br />
<br />
// Outputs<br />
var relVvesiclesNode = document.getElementById("relVvesicles");<br />
<br />
// Read inputs<br />
var rhomedium = Number(rhomediumNode.value);<br />
var rhovesicle = Number(rhovesicleNode.value);<br />
var rhocell = Number(rhocellNode.value);<br />
<br />
// Compute density(/-ies)<br />
try {<br />
var relVvesicles = 1.0 - (rhomedium-rhovesicle)/(rhocell-rhovesicle);<br />
if (rhovesicle>=rhocell) throw Error("Vesicle denser than cell, > should be <.");<br />
} catch(err) {<br />
densityErrorNode.innerHTML = err.message;<br />
}<br />
<br />
// Set intermediates if they exist<br />
<br />
// Set outputs<br />
setOutput(relVvesiclesNode, relVvesicles);<br />
document.getElementById('densityLimitGraph').refresh();<br />
}<br />
</script><br />
</html><br />
{{graph|Team:Groningen/Graphs/DensityLimit|id=densityLimitGraph}}<br />
|style="vertical-align:top;"|<pre><br />
Vc = volume of a cell without gas vesicles<br />
Vv = volume of gas vesicles in cell<br />
Vcv = volume of a cell with gas vesicles (assumed to be Vc+Vv)<br />
&rho;c = density of a cell without gas vesicles<br />
&rho;v = density of gas vesicles<br />
&rho;m = density of medium<br />
<br />
The following has to be true if the cell floats:<br />
Vc*&rho;c + Vv*&rho;v < Vcv*&rho;m<br />
(Vcv-Vv)*&rho;c + Vv*&rho;v < Vcv*&rho;m<br />
&rho;c + (Vv/Vcv)*(&rho;v-&rho;c) < &rho;m<br />
Assume (&rho;v - &rho;c)<0<br />
Vv/Vcv > (&rho;m - &rho;c)/(&rho;v - &rho;c)<br />
Vv/Vcv > 1 - (&rho;m - &rho;v)/(&rho;c - &rho;v)<br />
</pre><br />
'''Explanation of the graph'''<br />
<br />
Four curves are shown, corresponding to how many gas vesicles a cell needs with "our" gas vesicles (unless you changed the constants in the calculator above), the gas vesicles documented in [[Team:Groningen/Literature#Li1998|Li 1998]]{{infoBox|Using a width and diameter of 75nm and 50nm, respectively. Here we assume that their "width" should be interpreted as our diameter, as doing it the other way around would leave no room for a cylinder and they specifically mention that the vesicles appear to be shaped like cylinders with conical ends.}}, the gas vesicles from Anabaena in [[Team:Groningen/Literature#Walsby1994|Walsby 1994]]{{infoBox|Using a width and diameter of 500nm and 84nm, respectively.}} and our gas vesicles when the medium has the density of seawater.<br />
<br />
'''The X-axis''' depicts the cell density of the part of the cell not occupied by gas vesicles.<br />
<br />
'''The Y-axis''' depicts the minimum volume fraction of the cell that should consist of gas vesicles to make the cell float.<br />
|}<br />
<br />
{{GraphHeader}}<br />
<br />
==Conclusion & Discussion==<br />
<br />
We have experimented with two different constructs containing the GVP gene cluster i.e. pNL29 containing the 6 kb gene cluster from ''Bacillus megaterium'' ([[Team:Groningen/Literature#Li1998|Li & Cannon 1998]]) and [http://partsregistry.org/wiki/index.php/Part:BBa_I750016 BBa_I750016] from the [http://parts.mit.edu/igem07/index.php/Melbourne Melbourne 2007] iGEM team.<br />
We observed that it is best to have an OD600 of 1.5 when doing bouyancy tests, for withnessing differences with lower values is difficult. Furthermore buoyancy tests carried out in sea water or normal (LB) medium also give rise to difficult to interpret results. <br />
<br />
For cells cultivated in a more aerobic environment, such as the ones carried out in a fermentor, an enhanced bouyancy phenotype is observed. The extra O<sub>2</sub> added probably causes a higher concentration of intracellular oxygen, that can diffuse to the gas vesicles that are produced. The best buoyancy phenotype is withnessed at t=8.5 hours, however at t=22.5 hours no buoyancy can be seen. This suggests that there is an optimum after 8,5 hours.The cells at t=22.5 are probably in stationary phase whereas the cells at t=8.5h could still be in exponential phase, this could explain the difference in buoyancy found. It suggests that in stationary phase less gas vesicles are produced. Buoyancy is observed after 1 day, and also still after 2 days, which suggest that the buoyancy last for at least 24 hours. This is in accordance with experimental data from [[Team:Groningen/Literature#Walsby1994|Walsby, 1994]], who also observed a buoyant phenotype for over 2 days.<br />
<br />
<br />
{{Team:Groningen/Project/Footer}}</div>JolandaWitteveenhttp://2009.igem.org/File:Groningen_ODFerAndNonFer.PNGFile:Groningen ODFerAndNonFer.PNG2009-10-21T16:01:14Z<p>JolandaWitteveen: </p>
<hr />
<div></div>JolandaWitteveenhttp://2009.igem.org/Team:Groningen/Project/VesicleTeam:Groningen/Project/Vesicle2009-10-21T14:15:38Z<p>JolandaWitteveen: /* Conclusion & Discussion */</p>
<hr />
<div>{{Team:Groningen/Project/Header|}}<br />
<br />
=Gas vesicles=<br />
'''Our goal in this project is to make cells bouyant in the presence of certain concentrations of metals like copper, zinc and arsenic. Metal induced gas vesicle production can provide our cells with this bouyancy. Gas vesicles are bacterial organelles consisting entirely of proteins that envelop a gas filled space. We made, and send to the registry, parts in which the metal sensitive promoters for copper, zinc and arsenic were cloned in front of the GVP (Gas Vesicle Protein) gene cluster. For further characterization of the GVP gene cluster inducible and constitutive promoters were also cloned in front of this cluster. Buoyancy tests showed that our constructs were able to increase cell buoyancy and electron micrographs showed the presence of gas vesicles. A model was made to predict what volume fraction of a cell would have to be gas vesicle for this cell to have a density equal to that of water.'''<br />
<br />
==Background==<br />
<br />
Gas vesicles are organelles made entirely out of proteins that envelop a gas filled space. Because only gas can penetrate into the gas vesicles the total density of the cell is lowered. This lower cell density leads in turn to a buoyancy phenotype. Outside of the laboratory this buoyancy is used by microorganisms to vertically position themselves in the water column or simply to reduce their sinking rates. Organisms can regulate buoyancy by reducing gas vesicle production or by accumulation of denser compounds like carbohydrates. For certain cyanobacteria this regulation depends on light intensities. <br />
<br />
For a number of organisms it has been shown that all proteins important for the expression of gas vesicle lie in a single gene cluster. The GVP gene cluster used in this project was cloned from Bacillus megaterium into E. coli ([[Team:Groningen/Literature#Li1998|Li & Cannon 1998]]). This gene cluster now containing 11 genes was turned into a biobrick by Melbourne 2007 (link to part). Figure 1 shows the gene cluster as it was send in by Melbourne in 2007.<br />
<br />
For more info see: [[Team:Groningen/Literature#Walsby1994|Walsby 1994]].<br />
<br />
[[Image:Gas vesicle cluster bba I750016.PNG|frame|Figure 1: Gas vesicle gene cluster (<partinfo>BBa_I750016</partinfo>)<br />
]]<br />
<br />
==Goal==<br />
<br />
The goal of our project is to engineer an organism that can remove heavy metals from water. To facilitate easy separation the cells that have taken up metals should float so they can be removed from the water. The introduction of GVP gene cluster provides the cell with the required buoyancy. <br />
<br />
To make the buoyancy level of the cell responsive to the metal concentration inside the cell, metal sensitive promoters would have to be cloned in front of the GVP gene cluster. In this project these metal sensitive promoters were responsive to zinc, copper and arsenic. We also wanted to make a construct with constitutive and inducible promoters in front of the GVP cluster to show a proof of principle and as a back-up if our metal induced construct would fail. <br />
<br />
Finally we wanted to improve the Melbourne 2007 biobrick (<partinfo>BBa_I750016</partinfo>) by removing a repeat that was accidentally introduced during the removal of forbidden restriction sites.<br />
<br />
==Cloning strategy== <br />
For our msGVP (metal sensitive GVP) constructs we ordered oligo's containing the promoter region and the necessary restriction sites. When annealed these pieces of DNA have EcoRI and SpeI sticky ends. The vector containing GVP (<partinfo>BBa_I750016</partinfo>) was cut with EcoRI and XbaI and was ligated to the promoter. ([[Team:Groningen/Protocols#Annealing synthetic oligo’s|Protocol]])<br />
<br />
The metal sensitive GVP constructs are: <partinfo>BBa_K190033</partinfo>, <partinfo>BBa_K190034</partinfo> and <partinfo>BBa_K190035</partinfo>.<br />
<br />
Then because of compatibility issues when our entire system has to be assembled into one cell the whole metal sensitive promoter and GVP part were transfered to a different vector.<br />
<br />
[[Image:Cloning strategy floating device1.PNG]]<br />
:Figure 2: The floating device will be built up of an inducible promoter which can be induced by a certain intracellular concentration of metal-ions, and a gas vesicle cluster.<br />
<br />
==Bouyancy Tests==<br />
<br />
The buoyancy of GVP was tested by using the [[Team:Groningen/Protocols#Bouyancytest|buoyancy test protocol]]. The cells were grown in medium and induced and were resuspended in a salt solution (0.15mM NaCl) in a test tube and were left for a while in order to give the cells time to sink or float. <br />
<br />
''Different circumstances''<br />
<br />
Several different circumstances and small changes to the protocol were made in order to find the perfect circumstance for the buoyancy test. It appeared that with a low cell density the difference between floating and sinking could not be seen very well. The results were best visible with and cell density of OD600=1.5. Also we tried to do the buoyancy test in a longer tube since it was expected that the difference between floating and sinking would be more obvious. This, however, did not appear to be the case, unfortunately. Also doing the buoyancy test in a higher saline concentration did not have an enhanced floating effect. <br />
Another adaption we tried was the way of induction. In the standard protocol the cells were induced in the overnight culture. It was also tried if induction in the saline or at the exponential phase of growth or even induction on plate would make any difference. Unfortunately this did not make a huge difference.<br />
<br />
''Fermentor test''<br />
<br />
<br />
<br />
[[Image:Buoyancy pNL29.jpg|800px|thumb|center|Figure 3. Buoyancy test with pNL29, cells were induced in exponential phase and resuspended in NaCl in a OD600 of 1.5 A) Fermentor buoyancy test, samples taken after 1 hour, 2.5 hours, 6 hours, 8.5 hours and 22.5 hours. The cells were induced after 1 hour, at exponential phase. Photgraph taken 1 day after resuspension. B) normal, non-fermentor buoyancy test, samples taken at t=1 t/m 4. Photograph taken 1 day after resuspension. C) Same as A, photograph was taken 2 days after resuspension. D) same as C, photograph taken 2 days after resuspension.]]<br />
<br />
<br />
<br />
[[Image:Buoyancy arsR GVP.jpg|800px|thumb|center|Figure 4. Buoyancy test with pSB1AC3 containing pArsR-GVP, cells were induced in exponential phase and resuspended in NaCl in a OD600 of 1.5 A) Fermentor buoyancy test, samples taken after 1 hour, 2 hours, 6 hours, 7.5 hours and 22 hours. The cells were induced after 1 hour, at exponential phase. Photgraph taken 1 day after resuspension. B) normal, non-fermentor buoyancy test, samples taken at t=1 t/m 4. Photograph taken 1 day after resuspension.]] <br />
<br />
<div style="clear:both"></div><br />
<br />
So the length of the tube, the saline concentration and the time of induction did work for the buoyancy test. It was also suggested that there was not enough gas in the surrounding of the cells and a better result could be achieved if this could be improve or another gas could be used. To test this we tried to grow the cells in a [[Team:Groningen/Protocols#Fermentation|fermentor]]. It was also suggested that the fermentor test could be done with helium, however, modelling showed that it would not make a difference in floating which gas is used as long as it is lighter than water (check this yourself by changing the density (ρ) of the gas [[Team:Groningen/Project/Vesicle#Modelling|here]]). Therefore the fermentor test was done by using O<sub>2</sub>. This resulted in better buoyancy results. As can be seen in figure 3A the positive control pNL29 showed better buoyancy over time. After 2 hours the cells were in exponential phase and were induced with IPTG. After 8,5 hours the buoyancy is best, after 22.5 hours the buoyancy the cell level is declining. This suggests that there is an optimum after 8,5 hours. The cells at t=22.5 are probably in stationary phase whereas the cells at t=8.5h could still be in exponential phase, this could explain the difference in buoyancy found. It suggests that in stationary phase less gas vesicles are produced. Figure 3C shows the same tubes 24 hours later. This still shows buoyancy for the t=6 and t=8 tubes and no buoyancy for the others. This suggest that the buoyancy last for at least 24 hours. Simultaneously a normal, non-fermentor, buoyancy test was also performed with the same construct. In figure 3B these results at day 1 can be seen, this shows nothing. After a day no buoyancy can be seen for t=1 and t=2 a more dense cell suspension can be seen for t=3 and t=4 (figure 3D), however still no confincing buoyancy can be seen.<br />
Figure 4 shows the results from one of our own constructs, pArsR-GvP (<partinfo>BBa_K190033</partinfo>) grown in a fermentor. This shows an increase in buoyancy in time, however, at t=22.5h no buoyant cells can be seen. A buoyancy test done at the same time without a fermentor shows the same increase in buoyancy but does show buoyancy at t=22.5h (figure 4B). This difference can be explained since the cells in the fermentor are probably already dead or dying. In a fermentor the cell density is large this causes the cells to die.<br />
<br />
==Electron Microscopy==<br />
<br />
To check whether gas vesicles really were present in the cells we did some electron microscopy.<br />
<br />
In Figure 5 a picture of gas vesicles in a protoplast can be seen. This protoplast comes from an ''E. coli'' cell that contained a plasmid with the GVP gene cluster behind an arsenic sensitive promoter (<partinfo>BBa_K190033</partinfo>).<br />
<br />
<br />
[[Image:GasvesiclesEM.jpg|thumb|500px|left|Figure 5. Gas vesicles in ''E. coli'' protoplasts (<partinfo>BBa_K190033</partinfo>). The cells were treated with Lysozyme and SDS to create the protoplasts, uranyl acetate was used for staining. Magnification: 11500x.]]<br />
<br />
<div style="clear:both"></div><br />
<br />
==Modelling==<br />
<br />
===Buoyancy===<br />
The gas vesicles are shaped roughly like a cylinder with a cone at each end, whose cross-section we model as (based mostly on [[Team:Groningen/Literature#Walsby1994|Walsby 1994]]):<br />
<br />
[[Image:Vesicle_Shape.png]]<br />
<br />
We assume the interior of the wall of the gas vesicle is similarly shaped to the exterior, just slightly smaller (the right-most part of the image above illustrates this situation for the left tip of the gas vesicle). This means the different dimensions are related through the equations below. To determine the total volume, just use them with the given width/diameter (at least for the dimensions given in [[Team:Groningen/Literature#Walsby1994|Walsby 1994]]). To determine the gas volume, use them with w<sub>gas</sub> and d<sub>gas</sub>.<br />
<br />
{|<br />
|style="vertical-align:top;"|<html><br />
<div style="background:#efe;border:1px solid #9c9;padding:1em;"><br />
<table style="border-collapse:collapse;background:none;"><tr><br />
<td style="border-right:1px solid #9c9;padding-right:1em;"><br />
w = <input type="text" id="w" value="300"/> nm (</html>[[:Image:Ars-lyzo-007.png|TEM picture]]<html>)<br/><br />
d = <input type="text" id="d" value="75"/> nm (</html>[[:Image:Ars-lyzo-007.png|TEM picture]]<html>)<br/><br />
tw = <input type="text" id="tw" value="1.8"/> nm (</html>[[Team:Groningen/Literature#Walsby1994|Walsby1994]]<html>)<br/><br />
a = <input type="text" id="a" value="77"/> &deg; (</html>[[Team:Groningen/Literature#Walsby1994|Walsby1994]]<html>)<br/><br />
&rho;<sub>gas</sub> = <input type="text" id="rhogas" value="1.2"/> kg/m<sup>3</sup> (</html>[[Team:Groningen/Literature#Walsby1994|Walsby1994]]<html>)<br/> <!-- Walsby1994, for moist air at atmospheric pressure --><br />
&rho;<sub>wall</sub> = <input type="text" id="rhowall" value="1320"/> kg/m<sup>3</sup> (</html>[[Team:Groningen/Literature#Walsby1994|Walsby1994]]<html>)<br/> <!-- Walsby1994 --><br />
<br />
<button onClick="computeVolumes()">Compute</button><br/><br />
</td><br />
<br />
<td style="padding-left:1em;"><br />
<div id="volumeError" style="color:red"></div><br />
V<sub>gas</sub> = <span id="Vgas"></span> nm<sup>3</sup><br/><br />
M<sub>gas</sub> = <span id="Mgas"></span> yg<br/><br />
V<sub>wall</sub> = <span id="Vwall"></span> nm<sup>3</sup><br/><br />
M<sub>wall</sub> = <span id="Mwall"></span> yg<br/><br />
V<sub>vesicle</sub> = <span id="Vvesicle"></span> nm<sup>3</sup><br/><br />
M<sub>vesicle</sub> = <span id="Mvesicle"></span> yg<br/><br />
<b>&rho;<sub>vesicle</sub> = <span id="rhovesicle"></span> kg/m<sup>3</sup></b><br/><br />
</td><br />
</tr></table><br />
</div><br />
<script type="text/javascript"><br />
<br />
addOnloadHook(computeVolumes);<br />
<br />
function computeVolumes() {<br />
// Input<br />
var wNode = document.getElementById("w");<br />
var twNode = document.getElementById("tw");<br />
var dNode = document.getElementById("d");<br />
var aNode = document.getElementById("a");<br />
var rhogasNode = document.getElementById("rhogas");<br />
var rhowallNode = document.getElementById("rhowall");<br />
<br />
// Intermediates (mostly useful for debugging)<br />
var volumeErrorNode = document.getElementById("volumeError");<br />
var wwtNode = document.getElementById("wwt");<br />
volumeErrorNode.innerHTML = '';<br />
<br />
// Outputs<br />
var VgasNode = document.getElementById("Vgas");<br />
var VwallNode = document.getElementById("Vwall");<br />
var MgasNode = document.getElementById("Mgas");<br />
var MwallNode = document.getElementById("Mwall");<br />
var VvesicleNode = document.getElementById("Vvesicle");<br />
var MvesicleNode = document.getElementById("Mvesicle");<br />
var rhovesicleNode = document.getElementById("rhovesicle");<br />
<br />
// Read inputs<br />
var w = Number(wNode.value);<br />
var tw = Number(twNode.value);<br />
var d = Number(dNode.value);<br />
var a = Number(aNode.value) * Math.PI / 180.0;<br />
var rhogas = Number(rhogasNode.value);<br />
var rhowall = Number(rhowallNode.value);<br />
<br />
// Compute Vgas and Vwall<br />
try {<br />
var wwt = tw/Math.sin(a/2);<br />
var Vvesicle = computeVolume(w, d, a);<br />
var Vgas = computeVolume(w-2*wwt,d-2*tw,a);<br />
var Vwall = Vvesicle - Vgas;<br />
var Mgas = rhogas*Vgas;<br />
var Mwall = rhowall*Vwall;<br />
var Mvesicle = Mgas+Mwall;<br />
var rhovesicle = Mvesicle/Vvesicle;<br />
} catch(err) {<br />
volumeErrorNode.innerHTML = err.message;<br />
}<br />
<br />
// Set intermediates if they exist<br />
if (wwtNode) setOutput(wwtNode, wwt);<br />
<br />
// Set outputs<br />
setOutput(VgasNode, Vgas);<br />
setOutput(VwallNode, Vwall);<br />
setOutput(MgasNode, Mgas);<br />
setOutput(MwallNode, Mwall);<br />
setOutput(VvesicleNode, Vvesicle);<br />
setOutput(MvesicleNode, Mvesicle);<br />
setOutput(rhovesicleNode, rhovesicle);<br />
}<br />
<br />
function computeVolume(w,d,a) {<br />
// This computes the volume of cylinder with a cone at each end as defined in the text.<br />
var wt = (1/2)*d/Math.tan(a/2);<br />
var wc = w-2*wt;<br />
var Vc = (1/4)*Math.PI*Math.pow(d,2)*wc;<br />
var Vt = (1/12)*Math.PI*Math.pow(d,2)*wt;<br />
if (wc<0) throw Error("The given diameter would imply a larger width.<br/>(Do not trust the computed volumes!)");<br />
return Vc+2*Vt;<br />
}<br />
<br />
function formatNumberToHTML(v,p) {<br />
if (p===undefined) p = 5;<br />
return v.toPrecision(p)<br />
.replace(/e\+([0-9]+)$/i,'&middot;10<sup>$1</sup>')<br />
.replace(/e\-([0-9]+)$/i,'&middot;10<sup>-$1</sup>');<br />
}<br />
<br />
function setOutput(node,v,p) {<br />
node.innerHTML = formatNumberToHTML(v);<br />
node.value = v;<br />
}<br />
</script><br />
</html><br />
|style="vertical-align:top;"|<pre><br />
w = total width<br />
tw = thickness of wall (1.8-1.95nm)<br />
d = diameter<br />
a = 77 degrees<br />
&rho;gas = density of gas in vesicle (kg/m^3 = yg/nm^3)<br />
&rho;wall = density of vesicle wall (kg/m^3)<br />
wwt = tw/sin(a/2)<br />
wt = (1/2)*d/tan(a/2)<br />
wc = w - 2*wt<br />
Vc = (1/4)*pi*d^2*wc<br />
Vt = (1/12)*pi*d^2*wt<br />
V = Vc+2*Vt<br />
M = &rho;*V<br />
<br />
wgas = w-2*wwt = width of gas space<br />
dgas = d-2*tw = diameter of gas space<br />
V = Vgas + Vwall<br />
</pre><br />
|}<br />
<br />
Now we can consider the buoyant density of <i>E. coli</i> with gas vesicles. We have chosen to approach this problem using densities and volume ratios. According to [[Team:Groningen/Literature#Baldwin1995|Baldwin 1995]], [[Team:Groningen/Literature#Bylund1991|Bylund 1991]] and [[Team:Groningen/Literature#Poole1977|Poole 1977]], the density of (wild-type) <i>E. coli</i> is 1100 kg/m<sup>3</sup> &plusmn;3% under wildly varying conditions. This makes our method easier than trying to directly compute the density of a single cell, due to the fact that the volume can differ wildly (both during the life cycle and from strain to strain) and a lack of concrete data on the number of gas vesicles produced (in <i>E. coli</i>). Note that the computations below assume that the gas vesicles simply add to the existing structures.<br />
<br />
{|<br />
|style="vertical-align:top;"|<html><br />
<div style="background:#efe;border:1px solid #9c9;padding:1em;"><br />
<table style="border-collapse:collapse;background:none;"><tr><br />
<td style="border-right:1px solid #9c9;padding-right:1em;"><br />
<nobr>&rho;<sub>medium</sub> = <input type="text" id="rhomedium" value="1000"/> kg/m<sup>3</sup></nobr><br/><br />
&rho;<sub>cell</sub> = <input type="text" id="rhocell" value="1100"/> kg/m<sup>3</sup><br/> <!-- Reasonable estimate, TODO: more precision+reference --><br />
<br />
<button onClick="computeEColiDensity()">Compute</button><br/><br />
</td><br />
<br />
<td style="padding-left:1em;"><br />
<div id="densityError" style="color:red"></div><br />
V<sub>v</sub> / V<sub>cv</sub> > <span id="relVvesicles"></span><br/><br />
</td><br />
</tr></table><br />
</div><br />
<script type="text/javascript"><br />
<br />
addOnloadHook(computeEColiDensity);<br />
<br />
function computeEColiDensity() {<br />
// Input<br />
var rhomediumNode = document.getElementById("rhomedium");<br />
var rhovesicleNode = document.getElementById("rhovesicle");<br />
var rhocellNode = document.getElementById("rhocell");<br />
<br />
// Intermediates (mostly useful for debugging)<br />
var densityErrorNode = document.getElementById("densityError");<br />
densityErrorNode.innerHTML = '';<br />
<br />
// Outputs<br />
var relVvesiclesNode = document.getElementById("relVvesicles");<br />
<br />
// Read inputs<br />
var rhomedium = Number(rhomediumNode.value);<br />
var rhovesicle = Number(rhovesicleNode.value);<br />
var rhocell = Number(rhocellNode.value);<br />
<br />
// Compute density(/-ies)<br />
try {<br />
var relVvesicles = 1.0 - (rhomedium-rhovesicle)/(rhocell-rhovesicle);<br />
if (rhovesicle>=rhocell) throw Error("Vesicle denser than cell, > should be <.");<br />
} catch(err) {<br />
densityErrorNode.innerHTML = err.message;<br />
}<br />
<br />
// Set intermediates if they exist<br />
<br />
// Set outputs<br />
setOutput(relVvesiclesNode, relVvesicles);<br />
document.getElementById('densityLimitGraph').refresh();<br />
}<br />
</script><br />
</html><br />
{{graph|Team:Groningen/Graphs/DensityLimit|id=densityLimitGraph}}<br />
|style="vertical-align:top;"|<pre><br />
Vc = volume of a cell without gas vesicles<br />
Vv = volume of gas vesicles in cell<br />
Vcv = volume of a cell with gas vesicles (assumed to be Vc+Vv)<br />
&rho;c = density of a cell without gas vesicles<br />
&rho;v = density of gas vesicles<br />
&rho;m = density of medium<br />
<br />
The following has to be true if the cell floats:<br />
Vc*&rho;c + Vv*&rho;v < Vcv*&rho;m<br />
(Vcv-Vv)*&rho;c + Vv*&rho;v < Vcv*&rho;m<br />
&rho;c + (Vv/Vcv)*(&rho;v-&rho;c) < &rho;m<br />
Assume (&rho;v - &rho;c)<0<br />
Vv/Vcv > (&rho;m - &rho;c)/(&rho;v - &rho;c)<br />
Vv/Vcv > 1 - (&rho;m - &rho;v)/(&rho;c - &rho;v)<br />
</pre><br />
'''Explanation of the graph'''<br />
<br />
Four curves are shown, corresponding to how many gas vesicles a cell needs with "our" gas vesicles (unless you changed the constants in the calculator above), the gas vesicles documented in [[Team:Groningen/Literature#Li1998|Li 1998]]{{infoBox|Using a width and diameter of 75nm and 50nm, respectively. Here we assume that their "width" should be interpreted as our diameter, as doing it the other way around would leave no room for a cylinder and they specifically mention that the vesicles appear to be shaped like cylinders with conical ends.}}, the gas vesicles from Anabaena in [[Team:Groningen/Literature#Walsby1994|Walsby 1994]]{{infoBox|Using a width and diameter of 500nm and 84nm, respectively.}} and our gas vesicles when the medium has the density of seawater.<br />
<br />
'''The X-axis''' depicts the cell density of the part of the cell not occupied by gas vesicles.<br />
<br />
'''The Y-axis''' depicts the minimum volume fraction of the cell that should consist of gas vesicles to make the cell float.<br />
|}<br />
<br />
{{GraphHeader}}<br />
<br />
==Conclusion & Discussion==<br />
<br />
We have experimented with two different constructs containing the GVP gene cluster i.e. pNL29 containing the 6 kb gene cluster from ''Bacillus megaterium'' ([[Team:Groningen/Literature#Li1998|Li & Cannon 1998]]) and [http://partsregistry.org/wiki/index.php/Part:BBa_I750016 BBa_I750016] from the [http://parts.mit.edu/igem07/index.php/Melbourne Melbourne 2007] iGEM team.<br />
We observed that it is best to have an OD600 of 1.5 when doing bouyancy tests, for withnessing differences with lower values is difficult. Furthermore buoyancy tests carried out in sea water or normal (LB) medium also give rise to difficult to interpret results. <br />
<br />
For cells cultivated in a more aerobic environment, such as the ones carried out in a fermentor, an enhanced bouyancy phenotype is observed. The extra O<sub>2</sub> added probably causes a higher concentration of intracellular oxygen, that can diffuse to the gas vesicles that are produced. The best buoyancy phenotype is withnessed at t=8.5 hours, however at t=22.5 hours no buoyancy can be seen. This suggests that there is an optimum after 8,5 hours.The cells at t=22.5 are probably in stationary phase whereas the cells at t=8.5h could still be in exponential phase, this could explain the difference in buoyancy found. It suggests that in stationary phase less gas vesicles are produced. Buoyancy is observed after 1 day, and also still after 2 days, which suggest that the buoyancy last for at least 24 hours. This is in occordance with Walsby.<br />
<br />
<br />
{{Team:Groningen/Project/Footer}}</div>JolandaWitteveenhttp://2009.igem.org/Team:Groningen/Project/VesicleTeam:Groningen/Project/Vesicle2009-10-21T14:01:20Z<p>JolandaWitteveen: /* Bouyancy Tests */</p>
<hr />
<div>{{Team:Groningen/Project/Header|}}<br />
<br />
=Gas vesicles=<br />
'''Our goal in this project is to make cells bouyant in the presence of certain concentrations of metals like copper, zinc and arsenic. Metal induced gas vesicle production can provide our cells with this bouyancy. Gas vesicles are bacterial organelles consisting entirely of proteins that envelop a gas filled space. We made, and send to the registry, parts in which the metal sensitive promoters for copper, zinc and arsenic were cloned in front of the GVP (Gas Vesicle Protein) gene cluster. For further characterization of the GVP gene cluster inducible and constitutive promoters were also cloned in front of this cluster. Buoyancy tests showed that our constructs were able to increase cell buoyancy and electron micrographs showed the presence of gas vesicles. A model was made to predict what volume fraction of a cell would have to be gas vesicle for this cell to have a density equal to that of water.'''<br />
<br />
==Background==<br />
<br />
Gas vesicles are organelles made entirely out of proteins that envelop a gas filled space. Because only gas can penetrate into the gas vesicles the total density of the cell is lowered. This lower cell density leads in turn to a buoyancy phenotype. Outside of the laboratory this buoyancy is used by microorganisms to vertically position themselves in the water column or simply to reduce their sinking rates. Organisms can regulate buoyancy by reducing gas vesicle production or by accumulation of denser compounds like carbohydrates. For certain cyanobacteria this regulation depends on light intensities. <br />
<br />
For a number of organisms it has been shown that all proteins important for the expression of gas vesicle lie in a single gene cluster. The GVP gene cluster used in this project was cloned from Bacillus megaterium into E. coli ([[Team:Groningen/Literature#Li1998|Li & Cannon 1998]]). This gene cluster now containing 11 genes was turned into a biobrick by Melbourne 2007 (link to part). Figure 1 shows the gene cluster as it was send in by Melbourne in 2007.<br />
<br />
For more info see: [[Team:Groningen/Literature#Walsby1994|Walsby 1994]].<br />
<br />
[[Image:Gas vesicle cluster bba I750016.PNG|frame|Figure 1: Gas vesicle gene cluster (<partinfo>BBa_I750016</partinfo>)<br />
]]<br />
<br />
==Goal==<br />
<br />
The goal of our project is to engineer an organism that can remove heavy metals from water. To facilitate easy separation the cells that have taken up metals should float so they can be removed from the water. The introduction of GVP gene cluster provides the cell with the required buoyancy. <br />
<br />
To make the buoyancy level of the cell responsive to the metal concentration inside the cell, metal sensitive promoters would have to be cloned in front of the GVP gene cluster. In this project these metal sensitive promoters were responsive to zinc, copper and arsenic. We also wanted to make a construct with constitutive and inducible promoters in front of the GVP cluster to show a proof of principle and as a back-up if our metal induced construct would fail. <br />
<br />
Finally we wanted to improve the Melbourne 2007 biobrick (<partinfo>BBa_I750016</partinfo>) by removing a repeat that was accidentally introduced during the removal of forbidden restriction sites.<br />
<br />
==Cloning strategy== <br />
For our msGVP (metal sensitive GVP) constructs we ordered oligo's containing the promoter region and the necessary restriction sites. When annealed these pieces of DNA have EcoRI and SpeI sticky ends. The vector containing GVP (<partinfo>BBa_I750016</partinfo>) was cut with EcoRI and XbaI and was ligated to the promoter. ([[Team:Groningen/Protocols#Annealing synthetic oligo’s|Protocol]])<br />
<br />
The metal sensitive GVP constructs are: <partinfo>BBa_K190033</partinfo>, <partinfo>BBa_K190034</partinfo> and <partinfo>BBa_K190035</partinfo>.<br />
<br />
Then because of compatibility issues when our entire system has to be assembled into one cell the whole metal sensitive promoter and GVP part were transfered to a different vector.<br />
<br />
[[Image:Cloning strategy floating device1.PNG]]<br />
:Figure 2: The floating device will be built up of an inducible promoter which can be induced by a certain intracellular concentration of metal-ions, and a gas vesicle cluster.<br />
<br />
==Bouyancy Tests==<br />
<br />
The buoyancy of GVP was tested by using the [[Team:Groningen/Protocols#Bouyancytest|buoyancy test protocol]]. The cells were grown in medium and induced and were resuspended in a salt solution (0.15mM NaCl) in a test tube and were left for a while in order to give the cells time to sink or float. <br />
<br />
''Different circumstances''<br />
<br />
Several different circumstances and small changes to the protocol were made in order to find the perfect circumstance for the buoyancy test. It appeared that with a low cell density the difference between floating and sinking could not be seen very well. The results were best visible with and cell density of OD600=1.5. Also we tried to do the buoyancy test in a longer tube since it was expected that the difference between floating and sinking would be more obvious. This, however, did not appear to be the case, unfortunately. Also doing the buoyancy test in a higher saline concentration did not have an enhanced floating effect. <br />
Another adaption we tried was the way of induction. In the standard protocol the cells were induced in the overnight culture. It was also tried if induction in the saline or at the exponential phase of growth or even induction on plate would make any difference. Unfortunately this did not make a huge difference.<br />
<br />
''Fermentor test''<br />
<br />
<br />
<br />
[[Image:Buoyancy pNL29.jpg|800px|thumb|center|Figure 3. Buoyancy test with pNL29, cells were induced in exponential phase and resuspended in NaCl in a OD600 of 1.5 A) Fermentor buoyancy test, samples taken after 1 hour, 2.5 hours, 6 hours, 8.5 hours and 22.5 hours. The cells were induced after 1 hour, at exponential phase. Photgraph taken 1 day after resuspension. B) normal, non-fermentor buoyancy test, samples taken at t=1 t/m 4. Photograph taken 1 day after resuspension. C) Same as A, photograph was taken 2 days after resuspension. D) same as C, photograph taken 2 days after resuspension.]]<br />
<br />
<br />
<br />
[[Image:Buoyancy arsR GVP.jpg|800px|thumb|center|Figure 4. Buoyancy test with pSB1AC3 containing pArsR-GVP, cells were induced in exponential phase and resuspended in NaCl in a OD600 of 1.5 A) Fermentor buoyancy test, samples taken after 1 hour, 2 hours, 6 hours, 7.5 hours and 22 hours. The cells were induced after 1 hour, at exponential phase. Photgraph taken 1 day after resuspension. B) normal, non-fermentor buoyancy test, samples taken at t=1 t/m 4. Photograph taken 1 day after resuspension.]] <br />
<br />
<div style="clear:both"></div><br />
<br />
So the length of the tube, the saline concentration and the time of induction did work for the buoyancy test. It was also suggested that there was not enough gas in the surrounding of the cells and a better result could be achieved if this could be improve or another gas could be used. To test this we tried to grow the cells in a [[Team:Groningen/Protocols#Fermentation|fermentor]]. It was also suggested that the fermentor test could be done with helium, however, modelling showed that it would not make a difference in floating which gas is used as long as it is lighter than water (check this yourself by changing the density (ρ) of the gas [[Team:Groningen/Project/Vesicle#Modelling|here]]). Therefore the fermentor test was done by using O<sub>2</sub>. This resulted in better buoyancy results. As can be seen in figure 3A the positive control pNL29 showed better buoyancy over time. After 2 hours the cells were in exponential phase and were induced with IPTG. After 8,5 hours the buoyancy is best, after 22.5 hours the buoyancy the cell level is declining. This suggests that there is an optimum after 8,5 hours. The cells at t=22.5 are probably in stationary phase whereas the cells at t=8.5h could still be in exponential phase, this could explain the difference in buoyancy found. It suggests that in stationary phase less gas vesicles are produced. Figure 3C shows the same tubes 24 hours later. This still shows buoyancy for the t=6 and t=8 tubes and no buoyancy for the others. This suggest that the buoyancy last for at least 24 hours. Simultaneously a normal, non-fermentor, buoyancy test was also performed with the same construct. In figure 3B these results at day 1 can be seen, this shows nothing. After a day no buoyancy can be seen for t=1 and t=2 a more dense cell suspension can be seen for t=3 and t=4 (figure 3D), however still no confincing buoyancy can be seen.<br />
Figure 4 shows the results from one of our own constructs, pArsR-GvP (<partinfo>BBa_K190033</partinfo>) grown in a fermentor. This shows an increase in buoyancy in time, however, at t=22.5h no buoyant cells can be seen. A buoyancy test done at the same time without a fermentor shows the same increase in buoyancy but does show buoyancy at t=22.5h (figure 4B). This difference can be explained since the cells in the fermentor are probably already dead or dying. In a fermentor the cell density is large this causes the cells to die.<br />
<br />
==Electron Microscopy==<br />
<br />
To check whether gas vesicles really were present in the cells we did some electron microscopy.<br />
<br />
In Figure 5 a picture of gas vesicles in a protoplast can be seen. This protoplast comes from an ''E. coli'' cell that contained a plasmid with the GVP gene cluster behind an arsenic sensitive promoter (<partinfo>BBa_K190033</partinfo>).<br />
<br />
<br />
[[Image:GasvesiclesEM.jpg|thumb|500px|left|Figure 5. Gas vesicles in ''E. coli'' protoplasts (<partinfo>BBa_K190033</partinfo>). The cells were treated with Lysozyme and SDS to create the protoplasts, uranyl acetate was used for staining. Magnification: 11500x.]]<br />
<br />
<div style="clear:both"></div><br />
<br />
==Modelling==<br />
<br />
===Buoyancy===<br />
The gas vesicles are shaped roughly like a cylinder with a cone at each end, whose cross-section we model as (based mostly on [[Team:Groningen/Literature#Walsby1994|Walsby 1994]]):<br />
<br />
[[Image:Vesicle_Shape.png]]<br />
<br />
We assume the interior of the wall of the gas vesicle is similarly shaped to the exterior, just slightly smaller (the right-most part of the image above illustrates this situation for the left tip of the gas vesicle). This means the different dimensions are related through the equations below. To determine the total volume, just use them with the given width/diameter (at least for the dimensions given in [[Team:Groningen/Literature#Walsby1994|Walsby 1994]]). To determine the gas volume, use them with w<sub>gas</sub> and d<sub>gas</sub>.<br />
<br />
{|<br />
|style="vertical-align:top;"|<html><br />
<div style="background:#efe;border:1px solid #9c9;padding:1em;"><br />
<table style="border-collapse:collapse;background:none;"><tr><br />
<td style="border-right:1px solid #9c9;padding-right:1em;"><br />
w = <input type="text" id="w" value="300"/> nm (</html>[[:Image:Ars-lyzo-007.png|TEM picture]]<html>)<br/><br />
d = <input type="text" id="d" value="75"/> nm (</html>[[:Image:Ars-lyzo-007.png|TEM picture]]<html>)<br/><br />
tw = <input type="text" id="tw" value="1.8"/> nm (</html>[[Team:Groningen/Literature#Walsby1994|Walsby1994]]<html>)<br/><br />
a = <input type="text" id="a" value="77"/> &deg; (</html>[[Team:Groningen/Literature#Walsby1994|Walsby1994]]<html>)<br/><br />
&rho;<sub>gas</sub> = <input type="text" id="rhogas" value="1.2"/> kg/m<sup>3</sup> (</html>[[Team:Groningen/Literature#Walsby1994|Walsby1994]]<html>)<br/> <!-- Walsby1994, for moist air at atmospheric pressure --><br />
&rho;<sub>wall</sub> = <input type="text" id="rhowall" value="1320"/> kg/m<sup>3</sup> (</html>[[Team:Groningen/Literature#Walsby1994|Walsby1994]]<html>)<br/> <!-- Walsby1994 --><br />
<br />
<button onClick="computeVolumes()">Compute</button><br/><br />
</td><br />
<br />
<td style="padding-left:1em;"><br />
<div id="volumeError" style="color:red"></div><br />
V<sub>gas</sub> = <span id="Vgas"></span> nm<sup>3</sup><br/><br />
M<sub>gas</sub> = <span id="Mgas"></span> yg<br/><br />
V<sub>wall</sub> = <span id="Vwall"></span> nm<sup>3</sup><br/><br />
M<sub>wall</sub> = <span id="Mwall"></span> yg<br/><br />
V<sub>vesicle</sub> = <span id="Vvesicle"></span> nm<sup>3</sup><br/><br />
M<sub>vesicle</sub> = <span id="Mvesicle"></span> yg<br/><br />
<b>&rho;<sub>vesicle</sub> = <span id="rhovesicle"></span> kg/m<sup>3</sup></b><br/><br />
</td><br />
</tr></table><br />
</div><br />
<script type="text/javascript"><br />
<br />
addOnloadHook(computeVolumes);<br />
<br />
function computeVolumes() {<br />
// Input<br />
var wNode = document.getElementById("w");<br />
var twNode = document.getElementById("tw");<br />
var dNode = document.getElementById("d");<br />
var aNode = document.getElementById("a");<br />
var rhogasNode = document.getElementById("rhogas");<br />
var rhowallNode = document.getElementById("rhowall");<br />
<br />
// Intermediates (mostly useful for debugging)<br />
var volumeErrorNode = document.getElementById("volumeError");<br />
var wwtNode = document.getElementById("wwt");<br />
volumeErrorNode.innerHTML = '';<br />
<br />
// Outputs<br />
var VgasNode = document.getElementById("Vgas");<br />
var VwallNode = document.getElementById("Vwall");<br />
var MgasNode = document.getElementById("Mgas");<br />
var MwallNode = document.getElementById("Mwall");<br />
var VvesicleNode = document.getElementById("Vvesicle");<br />
var MvesicleNode = document.getElementById("Mvesicle");<br />
var rhovesicleNode = document.getElementById("rhovesicle");<br />
<br />
// Read inputs<br />
var w = Number(wNode.value);<br />
var tw = Number(twNode.value);<br />
var d = Number(dNode.value);<br />
var a = Number(aNode.value) * Math.PI / 180.0;<br />
var rhogas = Number(rhogasNode.value);<br />
var rhowall = Number(rhowallNode.value);<br />
<br />
// Compute Vgas and Vwall<br />
try {<br />
var wwt = tw/Math.sin(a/2);<br />
var Vvesicle = computeVolume(w, d, a);<br />
var Vgas = computeVolume(w-2*wwt,d-2*tw,a);<br />
var Vwall = Vvesicle - Vgas;<br />
var Mgas = rhogas*Vgas;<br />
var Mwall = rhowall*Vwall;<br />
var Mvesicle = Mgas+Mwall;<br />
var rhovesicle = Mvesicle/Vvesicle;<br />
} catch(err) {<br />
volumeErrorNode.innerHTML = err.message;<br />
}<br />
<br />
// Set intermediates if they exist<br />
if (wwtNode) setOutput(wwtNode, wwt);<br />
<br />
// Set outputs<br />
setOutput(VgasNode, Vgas);<br />
setOutput(VwallNode, Vwall);<br />
setOutput(MgasNode, Mgas);<br />
setOutput(MwallNode, Mwall);<br />
setOutput(VvesicleNode, Vvesicle);<br />
setOutput(MvesicleNode, Mvesicle);<br />
setOutput(rhovesicleNode, rhovesicle);<br />
}<br />
<br />
function computeVolume(w,d,a) {<br />
// This computes the volume of cylinder with a cone at each end as defined in the text.<br />
var wt = (1/2)*d/Math.tan(a/2);<br />
var wc = w-2*wt;<br />
var Vc = (1/4)*Math.PI*Math.pow(d,2)*wc;<br />
var Vt = (1/12)*Math.PI*Math.pow(d,2)*wt;<br />
if (wc<0) throw Error("The given diameter would imply a larger width.<br/>(Do not trust the computed volumes!)");<br />
return Vc+2*Vt;<br />
}<br />
<br />
function formatNumberToHTML(v,p) {<br />
if (p===undefined) p = 5;<br />
return v.toPrecision(p)<br />
.replace(/e\+([0-9]+)$/i,'&middot;10<sup>$1</sup>')<br />
.replace(/e\-([0-9]+)$/i,'&middot;10<sup>-$1</sup>');<br />
}<br />
<br />
function setOutput(node,v,p) {<br />
node.innerHTML = formatNumberToHTML(v);<br />
node.value = v;<br />
}<br />
</script><br />
</html><br />
|style="vertical-align:top;"|<pre><br />
w = total width<br />
tw = thickness of wall (1.8-1.95nm)<br />
d = diameter<br />
a = 77 degrees<br />
&rho;gas = density of gas in vesicle (kg/m^3 = yg/nm^3)<br />
&rho;wall = density of vesicle wall (kg/m^3)<br />
wwt = tw/sin(a/2)<br />
wt = (1/2)*d/tan(a/2)<br />
wc = w - 2*wt<br />
Vc = (1/4)*pi*d^2*wc<br />
Vt = (1/12)*pi*d^2*wt<br />
V = Vc+2*Vt<br />
M = &rho;*V<br />
<br />
wgas = w-2*wwt = width of gas space<br />
dgas = d-2*tw = diameter of gas space<br />
V = Vgas + Vwall<br />
</pre><br />
|}<br />
<br />
Now we can consider the buoyant density of <i>E. coli</i> with gas vesicles. We have chosen to approach this problem using densities and volume ratios. According to [[Team:Groningen/Literature#Baldwin1995|Baldwin 1995]], [[Team:Groningen/Literature#Bylund1991|Bylund 1991]] and [[Team:Groningen/Literature#Poole1977|Poole 1977]], the density of (wild-type) <i>E. coli</i> is 1100 kg/m<sup>3</sup> &plusmn;3% under wildly varying conditions. This makes our method easier than trying to directly compute the density of a single cell, due to the fact that the volume can differ wildly (both during the life cycle and from strain to strain) and a lack of concrete data on the number of gas vesicles produced (in <i>E. coli</i>). Note that the computations below assume that the gas vesicles simply add to the existing structures.<br />
<br />
{|<br />
|style="vertical-align:top;"|<html><br />
<div style="background:#efe;border:1px solid #9c9;padding:1em;"><br />
<table style="border-collapse:collapse;background:none;"><tr><br />
<td style="border-right:1px solid #9c9;padding-right:1em;"><br />
<nobr>&rho;<sub>medium</sub> = <input type="text" id="rhomedium" value="1000"/> kg/m<sup>3</sup></nobr><br/><br />
&rho;<sub>cell</sub> = <input type="text" id="rhocell" value="1100"/> kg/m<sup>3</sup><br/> <!-- Reasonable estimate, TODO: more precision+reference --><br />
<br />
<button onClick="computeEColiDensity()">Compute</button><br/><br />
</td><br />
<br />
<td style="padding-left:1em;"><br />
<div id="densityError" style="color:red"></div><br />
V<sub>v</sub> / V<sub>cv</sub> > <span id="relVvesicles"></span><br/><br />
</td><br />
</tr></table><br />
</div><br />
<script type="text/javascript"><br />
<br />
addOnloadHook(computeEColiDensity);<br />
<br />
function computeEColiDensity() {<br />
// Input<br />
var rhomediumNode = document.getElementById("rhomedium");<br />
var rhovesicleNode = document.getElementById("rhovesicle");<br />
var rhocellNode = document.getElementById("rhocell");<br />
<br />
// Intermediates (mostly useful for debugging)<br />
var densityErrorNode = document.getElementById("densityError");<br />
densityErrorNode.innerHTML = '';<br />
<br />
// Outputs<br />
var relVvesiclesNode = document.getElementById("relVvesicles");<br />
<br />
// Read inputs<br />
var rhomedium = Number(rhomediumNode.value);<br />
var rhovesicle = Number(rhovesicleNode.value);<br />
var rhocell = Number(rhocellNode.value);<br />
<br />
// Compute density(/-ies)<br />
try {<br />
var relVvesicles = 1.0 - (rhomedium-rhovesicle)/(rhocell-rhovesicle);<br />
if (rhovesicle>=rhocell) throw Error("Vesicle denser than cell, > should be <.");<br />
} catch(err) {<br />
densityErrorNode.innerHTML = err.message;<br />
}<br />
<br />
// Set intermediates if they exist<br />
<br />
// Set outputs<br />
setOutput(relVvesiclesNode, relVvesicles);<br />
document.getElementById('densityLimitGraph').refresh();<br />
}<br />
</script><br />
</html><br />
{{graph|Team:Groningen/Graphs/DensityLimit|id=densityLimitGraph}}<br />
|style="vertical-align:top;"|<pre><br />
Vc = volume of a cell without gas vesicles<br />
Vv = volume of gas vesicles in cell<br />
Vcv = volume of a cell with gas vesicles (assumed to be Vc+Vv)<br />
&rho;c = density of a cell without gas vesicles<br />
&rho;v = density of gas vesicles<br />
&rho;m = density of medium<br />
<br />
The following has to be true if the cell floats:<br />
Vc*&rho;c + Vv*&rho;v < Vcv*&rho;m<br />
(Vcv-Vv)*&rho;c + Vv*&rho;v < Vcv*&rho;m<br />
&rho;c + (Vv/Vcv)*(&rho;v-&rho;c) < &rho;m<br />
Assume (&rho;v - &rho;c)<0<br />
Vv/Vcv > (&rho;m - &rho;c)/(&rho;v - &rho;c)<br />
Vv/Vcv > 1 - (&rho;m - &rho;v)/(&rho;c - &rho;v)<br />
</pre><br />
'''Explanation of the graph'''<br />
<br />
Four curves are shown, corresponding to how many gas vesicles a cell needs with "our" gas vesicles (unless you changed the constants in the calculator above), the gas vesicles documented in [[Team:Groningen/Literature#Li1998|Li 1998]]{{infoBox|Using a width and diameter of 75nm and 50nm, respectively. Here we assume that their "width" should be interpreted as our diameter, as doing it the other way around would leave no room for a cylinder and they specifically mention that the vesicles appear to be shaped like cylinders with conical ends.}}, the gas vesicles from Anabaena in [[Team:Groningen/Literature#Walsby1994|Walsby 1994]]{{infoBox|Using a width and diameter of 500nm and 84nm, respectively.}} and our gas vesicles when the medium has the density of seawater.<br />
<br />
'''The X-axis''' depicts the cell density of the part of the cell not occupied by gas vesicles.<br />
<br />
'''The Y-axis''' depicts the minimum volume fraction of the cell that should consist of gas vesicles to make the cell float.<br />
|}<br />
<br />
{{GraphHeader}}<br />
<br />
==Conclusion & Discussion==<br />
<br />
We have experimented with two different constructs containing the GVP gene cluster i.e. pNL29 containing the 6 kb gene cluster from ''Bacillus megaterium'' ([[Team:Groningen/Literature#Li1998|Li & Cannon 1998]]) and [http://partsregistry.org/wiki/index.php/Part:BBa_I750016 BBa_I750016] from the [http://parts.mit.edu/igem07/index.php/Melbourne Melbourne 2007] iGEM team.<br />
We observed that it is best to have an OD600 of 1.5 when doing bouyancy tests, for withnessing differences with lower values is difficult. Furthermore buoyancy tests carried out in sea water or normal (LB) medium also give rise to difficult to interpret results. <br />
<br />
In cultures to which extra O<sub>2</sub> is added in the inducing stage??, such as the ones carried out in a fermentor, a better bouyancy phenotype is withnessed. The extra O<sub>2</sub> added probably causes a higher concentration of intracellular oxygen, that can diffuse to the gas vesicles that are produced.<br />
<br />
{{Team:Groningen/Project/Footer}}</div>JolandaWitteveenhttp://2009.igem.org/Team:Groningen/Project/PromotersTeam:Groningen/Project/Promoters2009-10-21T13:50:14Z<p>JolandaWitteveen: </p>
<hr />
<div>{{Team:Groningen/Project/Header|}}<br />
<br />
<br />
<br />
{|<br />
|<html><style type="text/css"><br />
.intro { margin-left:0px; margin-top:10px; padding:10px; border-left:solid 5px #FFF6D5; border-right:solid 5px #FFF6D5; text-align:justify;background:#FFFFE5; }<br />
</style></html><br />
<div class="intro"><br />
<h2>Promotors</h2><br />
'''A promoter is a part of DNA involved in the regulation of gene transcription by RNA polymerase. In general RNA polymerase tends to bind weakly to a strand of DNA until a suitable promoter is encountered and the binding becomes strong. Promoters are used to express genes of interest in cells in either a constitutive or induced manner. The constitutive promoters are used when a constant expression of enzymes is desired, and the amount of activity can be regulated by choosing from a range of promoters varying from low to high expression. If, however, expression is desired at certain points in time, or growth stage, inducible promoters are the best choice for regulating gene expression. In our system, we want to induce GVP production when the concentration of desired metal in the cells reaches a certain level. By choosing metal sensitive promoters already present in ''E. coli'' cells, the cells contain the necessary components for controlling the promoters, and the promoter sequence has only to be placed in front of the genes of interest.'''<br />
We take into consideration the following promoters:<br />
{| cellpadding="30"<br />
|align="center"|[[#Arsenic Induced Promoters|<big>As</big><br>Arsenic Induced Promoters]]<br />
|align="center"|[[#Copper Induced Promoters|<big>Cu</big><br>Copper Induced Promoters]]<br />
|align="center"|[[#Zinc Induced Promoters|<big>Zn</big><br>Zinc Induced Promoters]]<br />
|align="center"|[[#Mercury Induced Promoters|<big>Hg</big><br>Mercury Induced Promoters]]<br />
|}<br />
<br><br><br><br />
</div><br />
|}<br />
<br />
<br />
==Background==<br />
<br />
Metal sensitive promoters are widely used by bacteria in defence stategies against high concentrations of metals, which would have a destructive result on the cell. The promoters activate transcription of metal binding proteins to encapsule the ions, or transporters to pump the metals outside of the cell. In order to find different promoters to induce genes in the presence of different heavy metals we used the following list of databases and sites:<br />
{|<br />
|<br />
# [http://www.genome.jp/kegg/kegg2.html KEGG]<br />
# [http://www.ncbi.nlm.nih.gov NCBI]<br />
# [http://regtransbase.lbl.gov Regtransbase]<br />
|}<br />
<br />
<br />
==Arsenic Induced Promoters==<br />
<br />
Because of the similarity to phosphate, sometimes arsenate is mistaken for phosphate, which is how it is introduced into living organisms, including <i>E. coli</i>, by the phosphate uptake system. Other molecules such as As(III) can also be introduced into the cells by various membrane transporters. (needs a ref.)<br />
<br />
====<i>E. coli</i>====<br />
<br />
Promoter arsRp is associated with the dimer of ArsR for the arsenic induced transcription of genes involved in arsenic efflux (arsR, arsB and arsC, which is present on the genome of <i>Escherichia coli</i> str. K-12 substrain MG1655). The sequence shows the typical -10 and -35 region of the promoter and can be found through the following [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU00239 link]. A second region, located at -41.5 from the transcription start site, is thought to bind dimeric ArsR. Upon binding of arsenic, the dimer dissociates and allows the RNA polymerase space to attach itself, and can also be found in the same [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU00239 link].<br />
<br />
*ArsR belongs to the ArsR/SmtB family of transcriptional regulators that respond to a variety of metals. ArsR has a helix-turn-helix motif for DNA binding, a metal-binding site, and a dimerization domain. In ArsR the inducer-binding site contains three cysteine residues that bind arsenite and antimonite specifically and with high affinity. Dimerization of ArsR is required for DNA binding and its ability to act as a transcriptional repressor. The dimer recognizes and binds to a 12-2-12 inverted repeat, but the binding of arsenic or antimonite to ArsR causes a conformational change in it, leading to dissociation from DNA and hence derepression (KEGG).<br />
<br />
*ArsR negatively controls the expression of the genes involved in arsenical and antimonite metals resistance, whose expression is induced in the presence of these metals. The protein is autoregulated, because arsR is the first gene in the arsRBC operon that it regulates. Overexpression of ArsR in <i>Escherichia coli</i> has been used for removal of arsenite from contaminated water (KEGG).<br />
<br />
(ArsR)<sub>2</sub>-DNA &rarr; ArsR-Ar + ArsR-Ar + DNA &rarr; Activation of transription<br />
<br />
The presence of all genes and promoters on the chromosome of <i>E. coli</i> makes the use of the arsRp for induction of the GVP cluster relatively straith forward. The promoter sequence of arsRp, with the upstream binding box for ArsR dimer, can either be synthesized completely with the required restriction sites, or acquired using PCR and carefully designed primers. It might even be an option to alter the -10/-35 promoter region for higher or lower transcription of the genes.<br />
<br />
====cloning strategy====<br />
<br />
====Results====<br />
The functionality of pArsR was tested by using a test construct, composed of pArsR and RFP (Figure 1).<br />
<br />
[[Image:Promoter measurement device.png|200px]]<br />
:Figure 1: The promoter testing device in J61002, where RFP expression is under control of the promoter which is placed in front of it. <br />
<br />
The fluorescence (and OD600) was measured as described in [https://2009.igem.org/Team:Groningen/Protocols| protocols]. Upon induction of the ArsR promoter the expression of RFP increased with a relative promoter unit of 2.3 (calculated according to formula 9 as described by [[Team:Groningen/Literature#Kelly2009|Kelly 2009]]). This induction of promoter activity was also found for other metal sensitive promoter (used in expression of MTs) (personal communication, Dr. D. Wilcox). The increase in fluorescence over time is shown in figure 2 and the fluorescence change due to a change in the internal as(III) concentration in figure 3. <br />
<br />
[[Image:Fluorescence over time.PNG]]<br />
:Figure 2: Increase of fluorescence (RFP = 590nm) upon induction of the pArsR promoter with 100uM As(III). The data was a bit noisy therefore a trendline was calculated and used to calculate the relative promoter units(RPU) with. <br />
<br />
[[Image:RFP over As conc2.PNG]]<br />
:Figure 3: The increase of RFP over an increased intracellular As(III) concentration. The internal arsenic concentration upon induction of cells with 100uM As(III), was calculated by extrapolating the the As(III) uptake curve (incubated 10uM As(III) over 1hr) of ''E. coli'' with pArsR-RFP (in J61002). The polynominal trendline was used to calculate the internal As concentration at the time point used for the fluorescence measurement. <br />
<br />
The raw data can be found at [https://2009.igem.org/Team:Groningen/Modelling/Downloads| downloads].<br />
<br />
===Modelling===<br />
{{GraphHeader}}<br />
<html><br />
<script type="text/javascript" src="/Team:Groningen/Modelling/Model.js?action=raw"></script><br />
<script type="text/javascript" src="/Team:Groningen/Modelling/Arsenic.js?action=raw"></script><br />
</html><br />
<br />
The three graphs below illustrate the promoter response after induction with arsenic (directly in the cell, with the equivalent of 1&micro;M in the solution) with and without constitutive expression of ArsR (the first two graphs) and with slower production and degradation of ArsR (the two left graphs). Also, each graph has a line showing the formation of a product behind the ars promoter that does not degrade (and has production rate 1), subtracting the production that would have occurred without induction to show the effect of adding arsenic. Some conclusions:<br />
<br />
* Constitutive expression of ArsR greatly reduces (and slows) the promoter response.<br />
* On the other hand, if we divide the production and degradation rates of ArsR by ten the promoter response is ten times slower, producing ten times as much product.<br />
* In the bottom-right graph the induction is done gradually (the amount of arsenic increases linearly during the first five minutes), showing the high-pass behaviour of the promoter and that this can negatively impact product formation.<br />
<br />
<html><br />
<script type="text/javascript"><br />
addOnloadHook(computePromoterActivation);<br />
<br />
function computePromoterActivation() {<br />
// Set up constants<br />
var maxt = 600;<br />
var c = arsenicModelConstants();<br />
var cNP = {}, cS = {}, cG = {};<br />
c.v5 = 0;<br />
c.k8 = 0;<br />
c.pro = 0;<br />
c.ars2T = 0;<br />
for(var a in c) {<br />
cNP[a] = c[a];<br />
cS[a] = c[a];<br />
cG[a] = c[a];<br />
}<br />
<br />
var Vcell = 1 * 1e-15; // micrometer^3/cell -> liter/cell<br />
var avogadro = 6.02214179e23; // 1/mol<br />
c.pro = 2/(avogadro*Vcell); // 1/cell -> mol/L<br />
cS.tauR *= 10;<br />
cS.beta1 /= 10;<br />
cS.beta3 /= 10;<br />
cG.ars2T = 100*cG.ars1T;<br />
<br />
// Initialize<br />
var x0 = arsenicModelInitialization(c,0);<br />
var xNP0 = arsenicModelInitialization(cNP,0);<br />
var xS0 = arsenicModelInitialization(cS,0);<br />
var x20 = arsenicModelInitialization(c,0);<br />
var xG0 = arsenicModelInitialization(cG,0);<br />
var AsT = 1e-6*c.Vs;<br />
x0.AsinT = AsT/c.Vc;<br />
xNP0.AsinT = AsT/c.Vc;<br />
xS0.AsinT = AsT/c.Vc;<br />
x20.AsinT = 0;<br />
xG0.AsinT = AsT/c.Vc;<br />
<br />
// Simulate<br />
var x = simulate(x0,maxt,function(t,d){return arsenicModelGradient(c,d);});<br />
var xNP = simulate(xNP0,maxt,function(t,d){return arsenicModelGradient(cNP,d);});<br />
var xS = simulate(xS0,maxt*10,function(t,d){return arsenicModelGradient(cS,d);});<br />
var xG = simulate(xG0,maxt,function(t,d){return arsenicModelGradient(cG,d);});<br />
var x2 = simulate(x0,maxt,function(t,d){<br />
var Dx = arsenicModelGradient(c,d);<br />
if (t<maxt/2) Dx.AsinT += (AsT/c.Vc)*2/maxt;<br />
return Dx;<br />
});<br />
<br />
// Output<br />
function convertToSeries(c,x0,x) {<br />
var bAsin, cAsin, ArsR, ars, arsP, arsE;<br />
var arsInt = 0;<br />
var series = [[],[]];<br />
var preTime = -x.time[x._arsF.length-1]/(60*20);<br />
arsE = x0._arsF;<br />
series[0].push({x:preTime,y:100*arsE});<br />
series[0].push({x:0,y:100*arsE});<br />
series[1].push({x:preTime,y:0});<br />
for(var i=0; i<x._arsF.length; i++) {<br />
ars = x._arsF[i];<br />
if (i>0) arsInt += (x.time[i]-x.time[i-1])*(ars+arsP)/2;<br />
series[0].push({x:x.time[i]/60,y:100*ars});<br />
series[1].push({x:x.time[i]/60,y:(arsInt-x.time[i]*arsE)});<br />
arsP = ars;<br />
}<br />
return series;<br />
}<br />
document.getElementById("promoterActivationData").data = {<br />
ars:convertToSeries(c,x0,x),<br />
arsNP:convertToSeries(cNP,xNP0,xNP),<br />
arsS:convertToSeries(cS,xS0,xS),<br />
arsG:convertToSeries(cG,xG0,xG),<br />
ars2:convertToSeries(c,x20,x2)};<br />
var graphNodes = [document.getElementById("promoterActivationGraph"),<br />
document.getElementById("promoterActivationGraphNP"),<br />
document.getElementById("promoterActivationGraphS"),<br />
document.getElementById("promoterActivationGraphG"),<br />
document.getElementById("promoterActivationGraph2")];<br />
for(var i in graphNodes) if (graphNodes[i]) graphNodes[i].refresh();<br />
}<br />
</script><br />
</html><br />
<span id="promoterActivationData"></span><br />
{|<br />
!Wild-type<br />
!+ ArsR overexpression<br />
!+ extra ars promoters<br />
|-<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationNP|promoterActivitationGraphNP}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivation|promoterActivitationGraph}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationG|promoterActivitationGraphG}}<br />
|-<br />
!Slower response<br />
!Gradual induction<br />
|-<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationSlow|promoterActivitationGraphS}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivation2|promoterActivitationGraph2}}<br />
|}<br />
<br />
===Other organisms===<br />
''Bacillus subtilis''<br />
<br />
In <i>B. subtilis</i>, an ArsR family repressor (ArsR<sub>BS</sub>) responds to As(III) and Sb(III) and regulates the ars operon encoding itself (ArsR), and arsenate reductase (ArsC), an arsenite efflux pump (ArsB) and a protein of unknown function (YqcK). The order in which ArsR<sub>BS</sub> recognises metals is as follows: As(III)>As(V)>Cd(II)~Ag(I).<br />
<br />
A second protein, AseR, negatively regulates itself and AseA, an As(III) efflux pump which contributes to arsenite resistance in cells lacking a functional ars operon. The order in which AseR recognises metals is as follows: As(III)>As(V).<br />
<br />
==Copper Induced Promoters==<br />
<br />
Copper is an essential element that becomes highly cytotoxic when concentrations exceed the capacity of cells to sequester the ion. The toxicity of copper is largely due to its tendency to alternate between its cuprous, Cu(I), and cupric, Cu(II), oxidation states, differentiating copper from other trace metals, such as zinc or nickel. Under aerobic conditions, this redox cycling leads to the generation of highly reactive hydroxyl radicals that readily and efficiently damage biomolecules, such as DNA, proteins, and lipids.(needs a ref.). Most organisms have specialized mechanisms to deal with dangerous levels of heavy metals, like the production of efflux pumps. These genes are regulated by promoters, which are inducible by the respective metals.<br />
<br />
====<i>E. coli </i>====<br />
<br />
"The intracellular level of copper in ''E. coli'' is controlled by the export of excess copper, but the entire systems of copper uptake and intracellular copper delivery are not fully understood. Two regulatory systems, the<br />
CueR and CusR systems, have been identified to be involved in transcription regulation of the genes for copper<br />
homeostasis (Rensing et al., 2000; Rensing and Grass, 2003). CueR, a MerR-family transcription factor, stimulates<br />
copper-induced transcription of both copA encoding Cu(I)-translocating P-type ATPase pump (exporter), that is the central component for maintenance of the copper homeostasis, and cueO encoding a periplasmic multicopper<br />
oxidase for detoxification (Outten et al., 2000; Petersen and Moller, 2000)." (from Yamamoto K., 2005)<br />
<br />
Promoter cusCp is associated with the two component system CusR and CusS for the copper induced transcription of genes involved in copper efflux (cusC, cusF, cusB and cusA, which is present on the genome of <i>Escherichia coli </i> str. K-12 substrain MG1655). The sequence shows the typical -10 and -35 region of the promoter and can be found through the following [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU0-1821 link]. A second region, located at -53.5 from the transcription start site, is thought to bind CusR. Upon binding of CusR, the RNA polymerase is able to recognize the site and attach itself, and can also be found in the same [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU0-1821 link].<br />
<br />
*CusS, a sensory histidine kinase in a two-component regulatory system with CusR, is able to recognize copper ions, phosphorilate, and form a complex with CusR. It's a 480 amino acid long protein of which the sequence (aa and nt) can be found [http://www.genome.jp/dbget-bin/www_bget?eco+b0570 here] along with other information.<br />
<br />
*CusR, "Cu-sensing regulator", regulates genes related to the copper and silver efflux systems under '''anaerobic growth''' and under '''extreme copper stress''' in aerobic growth . It's a 227 amino acid long protein of which the sequence (aa and nt) can be found [http://www.genome.jp/dbget-bin/www_bget?eco+b0571 here] along with other information. <br />
<br />
Cu &rarr; CusS &rarr; +P &rarr; CusR &rarr; Activation of transription<br />
<br />
The problem so far is the site of detection of copper. The CusS protein senses the external copper concentrations and not the internal. For our project it would be nice to have an internal sensor for the induction of the floatation genes, so it will float after uptake. In addition to CusR, three other systems involved in copper resistence are present (CueR, CpxR and YedW). Both CpxR and YedW have the same problem of sensing external copper instead of internal copper, CueR is thought to respond to intracellular concentrations of copper. The choice for CusR over CueR would be based on the frequency of binding sites of both on the genome of <i>E. coli</i> (1 vs. 197 times), which gives CusR more chance of binding to our promoter. However, the idea behind our project is to induce GVP transtriction at a high intracellular concentration, and results in the CueR related promoter.<br />
<br />
===Parts Registry===<br />
<br />
Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>CusR/CusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before).<br />
<br />
'''Abs''': This nucleotide sequence is believed to be able to bind with phosphorylated CusR transcription factor in <i>E. coli</i>. CusR protein is phosphorylated by CusS transmembrane protein in a case of high extracellular concentration of copper ions. After phosphorylation CusR interacts with described DNA sequence and activates the transcription of <i>cusA</i>, Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>cusR/cusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before).<i>CusB</i>, <i>cusC</i> and Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>cusR/cusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before). <i>CusF</i> genes coding the proteins of copper metabolic system were used by Saint-Petersburg Team of 2007 for constructing a copper biosensor system.<br />
*{{part|BBa_I760005}}<br />
*Cu-sensitive promoter <br />
*Part-only sequence (16 bp):<br />
::atgacaaaattgtcat<br />
<br />
====Other organisms====<br />
<br />
''Mycobacterium tuberculosis'' <br><br />
'''Abs.''': Cu(I) binding to the CsoR–DNA complex induces a conformational change in the dimer that decreases its affinity for the DNA [[Team:Groningen/Literature#Liu2006|Liu 2006]].<br />
<br />
''Pseudomonas syringae'' <br><br />
'''Abs.''': The copper resistance (cop) operon promoter (Pcop) of <i>Pseudomonas syringae</i> is copper-inducible, and requires the regulatory genes <i>copR</i> and <i>copS</i>. Primer extension analysis identified the transcriptional initiation site of Pcop 59 bp 5' to the translational start site of <i>copA</i> [[Team:Groningen/Literature#Mills1994|Mills 1994]].<br />
<br />
''Sulfolobus solfataricus'' <br><br />
'''Abs.''': That CopT binds to the copMA promoter at multiple sites, both upstream and downstream of the predicted TATA-BRE site. Copper was found to specifically modulate the affinity of DNA binding by CopT. This study describes a copper-responsive operon in archaea, a new family of archaeal DNA-binding proteins, and supports the idea that this domain plays a prominent role in the archaeal copper response. A model is proposed for copper-responsive transcriptional regulation of the <i>copMA</i> gene cluster [[Team:Groningen/Literature#Ettema2006|Ettema 2006]].<br />
<br />
''Lactococcus lactis'' <br><br />
'''Abs.''': Two regulatory genes (<i>lcoR</i> and <i>lcoS</i>) were identified from a plasmid-borne lactococcal copper resistance determinant and characterized by transcriptional fusion to the promoterless chloramphenicol acetyltransferase gene (<i>cat</i>). The transcription start site involved in copper induction was mapped by primer extension [[Team:Groningen/Literature#Khunajakr1999|Khunajakr 1999]].<br />
<br />
==Zinc Induced Promoters==<br />
<br />
====Other organisms====<br />
''Bacillus subtilis''<br />
<br />
'''Abs.''': The ''Bacillus subtilis'' cation efflux pump czcD, which mediates resistance against Zn<sup>2+</sup>, Co<sup>2+</sup>, Ni<sup>2+</sup> and Cu<sup>2+</sup>, is regulated by an ArsR-type repressor (CzrABS) as well [[Team:Groningen/Literature#Moore2005|Moore 2005]].<br />
<br />
''Streptococcus pneumoniae''<br />
<br />
'''Abs.''': Activation of the czcD promoter by SczA is shown to proceed by Zn<sup>2+</sup>-dependent binding of SczA to a conserved DNA motif. In the absence of Zn<sup>2+</sup>, SczA binds to a second site in the czcD promoter, thereby fully blocking czcD expression. A metalloregulatory protein belonging to the TetR family<br />
Kloosterman T.G., et al. (O.P. Kuipers), The novel transcriptional regulator SczA mediates protection against Zn<sup>2+</sup> stress by activation of the Zn<sup>2+</sup>-resistance gene czcD in ''Streptococcus pneumoniae'', Molecular Microbiology, 2007, 65(4), 1049–1063. Retrieved from "https://2009.igem.org/Team:Groningen/Project/Promoters" <br />
<br />
<br />
''Staphylococcus aureus''<br />
<br />
'''Abs.''': In ''Staphylococcus aureus'' CzrA, a member of the ArsR/SmtB family of DNA binding proteins, functions as a repressor of the czr operon, that consists of czrA and the gene encoding the CzcD homologue CzrB (Xiong and Jayaswal, 1998; Kuroda et al., 1999; Singh et al., 1999). CzrA-mediated repression is alleviated in the presence of Zn<sup>2+</sup> and Co<sup>2+</sup> (Xiong and Jayaswal, 1998; Kuroda et al., 1999; Singh et al., 1999).<br />
<br />
==Mercury Induced Promoters==<br />
<br />
===MerR===<br />
<br />
<div title="Arsie Says UP TO GAS VESICLES" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Project/Vesicle|}}</div><br />
{{Team:Groningen/Project/Footer}}</div>JolandaWitteveenhttp://2009.igem.org/Team:Groningen/Project/PromotersTeam:Groningen/Project/Promoters2009-10-21T13:47:12Z<p>JolandaWitteveen: </p>
<hr />
<div>{{Team:Groningen/Project/Header|}}<br />
<br />
<br />
<br />
{|<br />
|<html><style type="text/css"><br />
.intro { margin-left:0px; margin-top:10px; padding:10px; border-left:solid 5px #FFF6D5; border-right:solid 5px #FFF6D5; text-align:justify;background:#FFFFE5; }<br />
</style></html><br />
<div class="intro"><br />
<h2>Promotors</h2><br />
'''A promoter is a part of DNA involved in the regulation of gene transcription by RNA polymerase. In general RNA polymerase tends to bind weakly to a strand of DNA until a suitable promoter is encountered and the binding becomes strong. Promoters are used to express genes of interest in cells in either a constitutive or induced manner. The constitutive promoters are used when a constant expression of enzymes is desired, and the amount of activity can be regulated by choosing from a range of promoters varying from low to high expression. If, however, expression is desired at certain points in time, or growth stage, inducible promoters are the best choice for regulating gene expression. In our system, we want to induce GVP production when the concentration of desired metal in the cells reaches a certain level. By choosing metal sensitive promoters already present in ''E. coli'' cells, the cells contain the necessary components for controlling the promoters, and the promoter sequence has only to be placed in front of the genes of interest.'''<br />
We take into consideration the following promoters:<br />
{| cellpadding="30"<br />
|align="center"|[[#Arsenic Induced Promoters|<big>As</big><br>Arsenic Induced Promoters]]<br />
|align="center"|[[#Copper Induced Promoters|<big>Cu</big><br>Copper Induced Promoters]]<br />
|align="center"|[[#Zinc Induced Promoters|<big>Zn</big><br>Zinc Induced Promoters]]<br />
|align="center"|[[#Mercury Induced Promoters|<big>Hg</big><br>Mercury Induced Promoters]]<br />
|}<br />
<br />
</div><br />
|}<br />
<br />
<br />
==Background==<br />
<br />
Metal sensitive promoters are widely used by bacteria in defence stategies against high concentrations of metals, which would have a destructive result on the cell. The promoters activate transcription of metal binding proteins to encapsule the ions, or transporters to pump the metals outside of the cell. In order to find different promoters to induce genes in the presence of different heavy metals we used the following list of databases and sites:<br />
{|<br />
|<br />
# [http://www.genome.jp/kegg/kegg2.html KEGG]<br />
# [http://www.ncbi.nlm.nih.gov NCBI]<br />
# [http://regtransbase.lbl.gov Regtransbase]<br />
|}<br />
<br />
<br />
==Arsenic Induced Promoters==<br />
<br />
Because of the similarity to phosphate, sometimes arsenate is mistaken for phosphate, which is how it is introduced into living organisms, including <i>E. coli</i>, by the phosphate uptake system. Other molecules such as As(III) can also be introduced into the cells by various membrane transporters. (needs a ref.)<br />
<br />
====<i>E. coli</i>====<br />
<br />
Promoter arsRp is associated with the dimer of ArsR for the arsenic induced transcription of genes involved in arsenic efflux (arsR, arsB and arsC, which is present on the genome of <i>Escherichia coli</i> str. K-12 substrain MG1655). The sequence shows the typical -10 and -35 region of the promoter and can be found through the following [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU00239 link]. A second region, located at -41.5 from the transcription start site, is thought to bind dimeric ArsR. Upon binding of arsenic, the dimer dissociates and allows the RNA polymerase space to attach itself, and can also be found in the same [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU00239 link].<br />
<br />
*ArsR belongs to the ArsR/SmtB family of transcriptional regulators that respond to a variety of metals. ArsR has a helix-turn-helix motif for DNA binding, a metal-binding site, and a dimerization domain. In ArsR the inducer-binding site contains three cysteine residues that bind arsenite and antimonite specifically and with high affinity. Dimerization of ArsR is required for DNA binding and its ability to act as a transcriptional repressor. The dimer recognizes and binds to a 12-2-12 inverted repeat, but the binding of arsenic or antimonite to ArsR causes a conformational change in it, leading to dissociation from DNA and hence derepression (KEGG).<br />
<br />
*ArsR negatively controls the expression of the genes involved in arsenical and antimonite metals resistance, whose expression is induced in the presence of these metals. The protein is autoregulated, because arsR is the first gene in the arsRBC operon that it regulates. Overexpression of ArsR in <i>Escherichia coli</i> has been used for removal of arsenite from contaminated water (KEGG).<br />
<br />
(ArsR)<sub>2</sub>-DNA &rarr; ArsR-Ar + ArsR-Ar + DNA &rarr; Activation of transription<br />
<br />
The presence of all genes and promoters on the chromosome of <i>E. coli</i> makes the use of the arsRp for induction of the GVP cluster relatively straith forward. The promoter sequence of arsRp, with the upstream binding box for ArsR dimer, can either be synthesized completely with the required restriction sites, or acquired using PCR and carefully designed primers. It might even be an option to alter the -10/-35 promoter region for higher or lower transcription of the genes.<br />
<br />
====cloning strategy====<br />
<br />
====Results====<br />
The functionality of pArsR was tested by using a test construct, composed of pArsR and RFP (Figure 1).<br />
<br />
[[Image:Promoter measurement device.png|200px]]<br />
:Figure 1: The promoter testing device in J61002, where RFP expression is under control of the promoter which is placed in front of it. <br />
<br />
The fluorescence (and OD600) was measured as described in [https://2009.igem.org/Team:Groningen/Protocols| protocols]. Upon induction of the ArsR promoter the expression of RFP increased with a relative promoter unit of 2.3 (calculated according to formula 9 as described by [[Team:Groningen/Literature#Kelly2009|Kelly 2009]]). This induction of promoter activity was also found for other metal sensitive promoter (used in expression of MTs) (personal communication, Dr. D. Wilcox). The increase in fluorescence over time is shown in figure 2 and the fluorescence change due to a change in the internal as(III) concentration in figure 3. <br />
<br />
[[Image:Fluorescence over time.PNG]]<br />
:Figure 2: Increase of fluorescence (RFP = 590nm) upon induction of the pArsR promoter with 100uM As(III). The data was a bit noisy therefore a trendline was calculated and used to calculate the relative promoter units(RPU) with. <br />
<br />
[[Image:RFP over As conc2.PNG]]<br />
:Figure 3: The increase of RFP over an increased intracellular As(III) concentration. The internal arsenic concentration upon induction of cells with 100uM As(III), was calculated by extrapolating the the As(III) uptake curve (incubated 10uM As(III) over 1hr) of ''E. coli'' with pArsR-RFP (in J61002). The polynominal trendline was used to calculate the internal As concentration at the time point used for the fluorescence measurement. <br />
<br />
The raw data can be found at [https://2009.igem.org/Team:Groningen/Modelling/Downloads| downloads].<br />
<br />
===Modelling===<br />
{{GraphHeader}}<br />
<html><br />
<script type="text/javascript" src="/Team:Groningen/Modelling/Model.js?action=raw"></script><br />
<script type="text/javascript" src="/Team:Groningen/Modelling/Arsenic.js?action=raw"></script><br />
</html><br />
<br />
The three graphs below illustrate the promoter response after induction with arsenic (directly in the cell, with the equivalent of 1&micro;M in the solution) with and without constitutive expression of ArsR (the first two graphs) and with slower production and degradation of ArsR (the two left graphs). Also, each graph has a line showing the formation of a product behind the ars promoter that does not degrade (and has production rate 1), subtracting the production that would have occurred without induction to show the effect of adding arsenic. Some conclusions:<br />
<br />
* Constitutive expression of ArsR greatly reduces (and slows) the promoter response.<br />
* On the other hand, if we divide the production and degradation rates of ArsR by ten the promoter response is ten times slower, producing ten times as much product.<br />
* In the bottom-right graph the induction is done gradually (the amount of arsenic increases linearly during the first five minutes), showing the high-pass behaviour of the promoter and that this can negatively impact product formation.<br />
<br />
<html><br />
<script type="text/javascript"><br />
addOnloadHook(computePromoterActivation);<br />
<br />
function computePromoterActivation() {<br />
// Set up constants<br />
var maxt = 600;<br />
var c = arsenicModelConstants();<br />
var cNP = {}, cS = {}, cG = {};<br />
c.v5 = 0;<br />
c.k8 = 0;<br />
c.pro = 0;<br />
c.ars2T = 0;<br />
for(var a in c) {<br />
cNP[a] = c[a];<br />
cS[a] = c[a];<br />
cG[a] = c[a];<br />
}<br />
<br />
var Vcell = 1 * 1e-15; // micrometer^3/cell -> liter/cell<br />
var avogadro = 6.02214179e23; // 1/mol<br />
c.pro = 2/(avogadro*Vcell); // 1/cell -> mol/L<br />
cS.tauR *= 10;<br />
cS.beta1 /= 10;<br />
cS.beta3 /= 10;<br />
cG.ars2T = 100*cG.ars1T;<br />
<br />
// Initialize<br />
var x0 = arsenicModelInitialization(c,0);<br />
var xNP0 = arsenicModelInitialization(cNP,0);<br />
var xS0 = arsenicModelInitialization(cS,0);<br />
var x20 = arsenicModelInitialization(c,0);<br />
var xG0 = arsenicModelInitialization(cG,0);<br />
var AsT = 1e-6*c.Vs;<br />
x0.AsinT = AsT/c.Vc;<br />
xNP0.AsinT = AsT/c.Vc;<br />
xS0.AsinT = AsT/c.Vc;<br />
x20.AsinT = 0;<br />
xG0.AsinT = AsT/c.Vc;<br />
<br />
// Simulate<br />
var x = simulate(x0,maxt,function(t,d){return arsenicModelGradient(c,d);});<br />
var xNP = simulate(xNP0,maxt,function(t,d){return arsenicModelGradient(cNP,d);});<br />
var xS = simulate(xS0,maxt*10,function(t,d){return arsenicModelGradient(cS,d);});<br />
var xG = simulate(xG0,maxt,function(t,d){return arsenicModelGradient(cG,d);});<br />
var x2 = simulate(x0,maxt,function(t,d){<br />
var Dx = arsenicModelGradient(c,d);<br />
if (t<maxt/2) Dx.AsinT += (AsT/c.Vc)*2/maxt;<br />
return Dx;<br />
});<br />
<br />
// Output<br />
function convertToSeries(c,x0,x) {<br />
var bAsin, cAsin, ArsR, ars, arsP, arsE;<br />
var arsInt = 0;<br />
var series = [[],[]];<br />
var preTime = -x.time[x._arsF.length-1]/(60*20);<br />
arsE = x0._arsF;<br />
series[0].push({x:preTime,y:100*arsE});<br />
series[0].push({x:0,y:100*arsE});<br />
series[1].push({x:preTime,y:0});<br />
for(var i=0; i<x._arsF.length; i++) {<br />
ars = x._arsF[i];<br />
if (i>0) arsInt += (x.time[i]-x.time[i-1])*(ars+arsP)/2;<br />
series[0].push({x:x.time[i]/60,y:100*ars});<br />
series[1].push({x:x.time[i]/60,y:(arsInt-x.time[i]*arsE)});<br />
arsP = ars;<br />
}<br />
return series;<br />
}<br />
document.getElementById("promoterActivationData").data = {<br />
ars:convertToSeries(c,x0,x),<br />
arsNP:convertToSeries(cNP,xNP0,xNP),<br />
arsS:convertToSeries(cS,xS0,xS),<br />
arsG:convertToSeries(cG,xG0,xG),<br />
ars2:convertToSeries(c,x20,x2)};<br />
var graphNodes = [document.getElementById("promoterActivationGraph"),<br />
document.getElementById("promoterActivationGraphNP"),<br />
document.getElementById("promoterActivationGraphS"),<br />
document.getElementById("promoterActivationGraphG"),<br />
document.getElementById("promoterActivationGraph2")];<br />
for(var i in graphNodes) if (graphNodes[i]) graphNodes[i].refresh();<br />
}<br />
</script><br />
</html><br />
<span id="promoterActivationData"></span><br />
{|<br />
!Wild-type<br />
!+ ArsR overexpression<br />
!+ extra ars promoters<br />
|-<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationNP|promoterActivitationGraphNP}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivation|promoterActivitationGraph}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationG|promoterActivitationGraphG}}<br />
|-<br />
!Slower response<br />
!Gradual induction<br />
|-<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationSlow|promoterActivitationGraphS}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivation2|promoterActivitationGraph2}}<br />
|}<br />
<br />
===Other organisms===<br />
''Bacillus subtilis''<br />
<br />
In <i>B. subtilis</i>, an ArsR family repressor (ArsR<sub>BS</sub>) responds to As(III) and Sb(III) and regulates the ars operon encoding itself (ArsR), and arsenate reductase (ArsC), an arsenite efflux pump (ArsB) and a protein of unknown function (YqcK). The order in which ArsR<sub>BS</sub> recognises metals is as follows: As(III)>As(V)>Cd(II)~Ag(I).<br />
<br />
A second protein, AseR, negatively regulates itself and AseA, an As(III) efflux pump which contributes to arsenite resistance in cells lacking a functional ars operon. The order in which AseR recognises metals is as follows: As(III)>As(V).<br />
<br />
==Copper Induced Promoters==<br />
<br />
Copper is an essential element that becomes highly cytotoxic when concentrations exceed the capacity of cells to sequester the ion. The toxicity of copper is largely due to its tendency to alternate between its cuprous, Cu(I), and cupric, Cu(II), oxidation states, differentiating copper from other trace metals, such as zinc or nickel. Under aerobic conditions, this redox cycling leads to the generation of highly reactive hydroxyl radicals that readily and efficiently damage biomolecules, such as DNA, proteins, and lipids.(needs a ref.). Most organisms have specialized mechanisms to deal with dangerous levels of heavy metals, like the production of efflux pumps. These genes are regulated by promoters, which are inducible by the respective metals.<br />
<br />
====<i>E. coli </i>====<br />
<br />
"The intracellular level of copper in ''E. coli'' is controlled by the export of excess copper, but the entire systems of copper uptake and intracellular copper delivery are not fully understood. Two regulatory systems, the<br />
CueR and CusR systems, have been identified to be involved in transcription regulation of the genes for copper<br />
homeostasis (Rensing et al., 2000; Rensing and Grass, 2003). CueR, a MerR-family transcription factor, stimulates<br />
copper-induced transcription of both copA encoding Cu(I)-translocating P-type ATPase pump (exporter), that is the central component for maintenance of the copper homeostasis, and cueO encoding a periplasmic multicopper<br />
oxidase for detoxification (Outten et al., 2000; Petersen and Moller, 2000)." (from Yamamoto K., 2005)<br />
<br />
Promoter cusCp is associated with the two component system CusR and CusS for the copper induced transcription of genes involved in copper efflux (cusC, cusF, cusB and cusA, which is present on the genome of <i>Escherichia coli </i> str. K-12 substrain MG1655). The sequence shows the typical -10 and -35 region of the promoter and can be found through the following [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU0-1821 link]. A second region, located at -53.5 from the transcription start site, is thought to bind CusR. Upon binding of CusR, the RNA polymerase is able to recognize the site and attach itself, and can also be found in the same [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU0-1821 link].<br />
<br />
*CusS, a sensory histidine kinase in a two-component regulatory system with CusR, is able to recognize copper ions, phosphorilate, and form a complex with CusR. It's a 480 amino acid long protein of which the sequence (aa and nt) can be found [http://www.genome.jp/dbget-bin/www_bget?eco+b0570 here] along with other information.<br />
<br />
*CusR, "Cu-sensing regulator", regulates genes related to the copper and silver efflux systems under '''anaerobic growth''' and under '''extreme copper stress''' in aerobic growth . It's a 227 amino acid long protein of which the sequence (aa and nt) can be found [http://www.genome.jp/dbget-bin/www_bget?eco+b0571 here] along with other information. <br />
<br />
Cu &rarr; CusS &rarr; +P &rarr; CusR &rarr; Activation of transription<br />
<br />
The problem so far is the site of detection of copper. The CusS protein senses the external copper concentrations and not the internal. For our project it would be nice to have an internal sensor for the induction of the floatation genes, so it will float after uptake. In addition to CusR, three other systems involved in copper resistence are present (CueR, CpxR and YedW). Both CpxR and YedW have the same problem of sensing external copper instead of internal copper, CueR is thought to respond to intracellular concentrations of copper. The choice for CusR over CueR would be based on the frequency of binding sites of both on the genome of <i>E. coli</i> (1 vs. 197 times), which gives CusR more chance of binding to our promoter. However, the idea behind our project is to induce GVP transtriction at a high intracellular concentration, and results in the CueR related promoter.<br />
<br />
===Parts Registry===<br />
<br />
Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>CusR/CusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before).<br />
<br />
'''Abs''': This nucleotide sequence is believed to be able to bind with phosphorylated CusR transcription factor in <i>E. coli</i>. CusR protein is phosphorylated by CusS transmembrane protein in a case of high extracellular concentration of copper ions. After phosphorylation CusR interacts with described DNA sequence and activates the transcription of <i>cusA</i>, Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>cusR/cusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before).<i>CusB</i>, <i>cusC</i> and Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>cusR/cusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before). <i>CusF</i> genes coding the proteins of copper metabolic system were used by Saint-Petersburg Team of 2007 for constructing a copper biosensor system.<br />
*{{part|BBa_I760005}}<br />
*Cu-sensitive promoter <br />
*Part-only sequence (16 bp):<br />
::atgacaaaattgtcat<br />
<br />
====Other organisms====<br />
<br />
''Mycobacterium tuberculosis'' <br><br />
'''Abs.''': Cu(I) binding to the CsoR–DNA complex induces a conformational change in the dimer that decreases its affinity for the DNA [[Team:Groningen/Literature#Liu2006|Liu 2006]].<br />
<br />
''Pseudomonas syringae'' <br><br />
'''Abs.''': The copper resistance (cop) operon promoter (Pcop) of <i>Pseudomonas syringae</i> is copper-inducible, and requires the regulatory genes <i>copR</i> and <i>copS</i>. Primer extension analysis identified the transcriptional initiation site of Pcop 59 bp 5' to the translational start site of <i>copA</i> [[Team:Groningen/Literature#Mills1994|Mills 1994]].<br />
<br />
''Sulfolobus solfataricus'' <br><br />
'''Abs.''': That CopT binds to the copMA promoter at multiple sites, both upstream and downstream of the predicted TATA-BRE site. Copper was found to specifically modulate the affinity of DNA binding by CopT. This study describes a copper-responsive operon in archaea, a new family of archaeal DNA-binding proteins, and supports the idea that this domain plays a prominent role in the archaeal copper response. A model is proposed for copper-responsive transcriptional regulation of the <i>copMA</i> gene cluster [[Team:Groningen/Literature#Ettema2006|Ettema 2006]].<br />
<br />
''Lactococcus lactis'' <br><br />
'''Abs.''': Two regulatory genes (<i>lcoR</i> and <i>lcoS</i>) were identified from a plasmid-borne lactococcal copper resistance determinant and characterized by transcriptional fusion to the promoterless chloramphenicol acetyltransferase gene (<i>cat</i>). The transcription start site involved in copper induction was mapped by primer extension [[Team:Groningen/Literature#Khunajakr1999|Khunajakr 1999]].<br />
<br />
==Zinc Induced Promoters==<br />
<br />
====Other organisms====<br />
''Bacillus subtilis''<br />
<br />
'''Abs.''': The ''Bacillus subtilis'' cation efflux pump czcD, which mediates resistance against Zn<sup>2+</sup>, Co<sup>2+</sup>, Ni<sup>2+</sup> and Cu<sup>2+</sup>, is regulated by an ArsR-type repressor (CzrABS) as well [[Team:Groningen/Literature#Moore2005|Moore 2005]].<br />
<br />
''Streptococcus pneumoniae''<br />
<br />
'''Abs.''': Activation of the czcD promoter by SczA is shown to proceed by Zn<sup>2+</sup>-dependent binding of SczA to a conserved DNA motif. In the absence of Zn<sup>2+</sup>, SczA binds to a second site in the czcD promoter, thereby fully blocking czcD expression. A metalloregulatory protein belonging to the TetR family<br />
Kloosterman T.G., et al. (O.P. Kuipers), The novel transcriptional regulator SczA mediates protection against Zn<sup>2+</sup> stress by activation of the Zn<sup>2+</sup>-resistance gene czcD in ''Streptococcus pneumoniae'', Molecular Microbiology, 2007, 65(4), 1049–1063. Retrieved from "https://2009.igem.org/Team:Groningen/Project/Promoters" <br />
<br />
<br />
''Staphylococcus aureus''<br />
<br />
'''Abs.''': In ''Staphylococcus aureus'' CzrA, a member of the ArsR/SmtB family of DNA binding proteins, functions as a repressor of the czr operon, that consists of czrA and the gene encoding the CzcD homologue CzrB (Xiong and Jayaswal, 1998; Kuroda et al., 1999; Singh et al., 1999). CzrA-mediated repression is alleviated in the presence of Zn<sup>2+</sup> and Co<sup>2+</sup> (Xiong and Jayaswal, 1998; Kuroda et al., 1999; Singh et al., 1999).<br />
<br />
==Mercury Induced Promoters==<br />
<br />
===MerR===<br />
<br />
<div title="Arsie Says UP TO GAS VESICLES" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Project/Vesicle|}}</div><br />
{{Team:Groningen/Project/Footer}}</div>JolandaWitteveenhttp://2009.igem.org/Team:Groningen/Project/TransportTeam:Groningen/Project/Transport2009-10-21T13:41:54Z<p>JolandaWitteveen: </p>
<hr />
<div>{{Team:Groningen/Project/Header|}}<br />
<div title="Arsie Says UP TO ACCUMULATION" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Project/Accumulation}}</div><br />
<br />
{|<br />
|<html><style type="text/css"><br />
.intro { margin-left:0px; margin-top:10px; padding:10px; border-left:solid 5px #FFF6D5; border-right:solid 5px #FFF6D5; text-align:justify;background:#FFFFE5; }<br />
</style></html><br />
<div class="intro"><br />
<h2>Transport</h2><br />
'''To isolate heavy metals from the environment we require uptake systems. So far we found several different mechanisms to create such a system. We investigated three kinds of metal uptake:'''<br />
*Metal transporters, Membrane proteins that transport the metal from the environment (<i>i.e.</i> wastewater) to the cytoplasm<br />
**Uncoupled<br />
**Coupled with 'helper' compounds<br />
*Metal binding proteins in the periplasm<br />
<br />
'''We have investigated several systems to determine which are suitable for the final design. The following systems are under consideration:'''<br />
<br />
*Arsenite uptake via GlpF<br />
*Copper/zinc uptake via HmtA<br />
*Heavy metal uptake coupled to citrate via ''ef''CitH ''bs''CitM <br />
*Periplasmic accumulation of heavy metals via Mer Operon.<br />
<br />
'''We chose to focus on GlpF and HmtA, the final device was made with GlpF for arsenate purification. '''<br />
<br><br><br><br />
</div><br />
|}<br />
<br />
<br />
<br />
==Arsenite uptake via GlpF==<br />
<!--[[Image:GlpF.jpeg|200px|thumb|right|73As(III) and 125Sb(III) uptake into cells of E. coli is facilitated by the aquaglyceroporin channel GlpF.]]--><br />
<br />
===GlpF===<br />
<br />
====Introduction====<br />
GlpF is an aquaglycerol porin of E.coli which facilitates not only glycerol import, but also arsenic (As) and antimone (Sb) import [[Team:Groningen/Literature#Fu, DX, et al.2000|(Fu, DX, et al.2000]]), [[Team:Groningen/Literature#Meng, YL, et al.2004|(Meng, YL, et al.2004]]), [[Team:Groningen/Literature#Porquet, A, et al.2007|(Porquet, A, et al.2007]]), [[Team:Groningen/Literature#Rosen, BR, et al.2009|(Rosen, BR, et al.2009)]] . It has homologues in other organisms; Fps1p has shown to facilitate arsenic import in yeast and AQP9 is the mammalian homologue [[Team:Groningen/Literature#Porquet, A, et al.2007|(Porquet, A, et al.2007]]), [[Team:Groningen/Literature#Rosen, BR, et al.2009|(Rosen, BR, et al.2009)]] .<br />
The GlpF aquaglycerol porin is a membrane protein with a symmetric arrangement of four independent GlpF channels. One monomer of this tetramer GlpF porin consists of six transmembrane and two half membrane-spanning α-helices that form a right-handed helical bundle around the channel. The channel has a diameter of ~15Å at the periplasmid end, which constricts towards a diameter of ~3.8Å at the beginning of a 28 Å long selective channel that ends at the cytoplasmic end [[Team:Groningen/Literature#Fu, DX, et al.2000|(Fu, DX, et al.2000)]].<br />
The GlpF is a stereospecific channel that is thought to be more selective on molecular size than on chemical structure [[Team:Groningen/Literature#Fu, DX, et al.2000|(Fu, DX, et al.2000]], [[Team:Groningen/Literature#Heller, KB, et al.1980|(Heller, KB, et al.1980)]] . It does allow transport of a variance of non-charged compounds ranging from polyhydric alcohols, glycerol being one of them, arsenic to antimone [[Team:Groningen/Literature#Fu, DX, et al.2000|(Fu, DX, et al.2000]]), [[Team:Groningen/Literature#Meng, YL, et al.2004|(Meng, YL, et al.2004]]), [[Team:Groningen/Literature#Porquet, A, et al.2007|(Porquet, A, et al.2007)]], [[Team:Groningen/Literature#Rosen, BR, et al.2009|(Rosen, BR, et al.2009]]), [[Team:Groningen/Literature#Heller, KB, et al.1980|(Heller, KB, et al.1980)]]. Carbon sugars and ions are shown to be unable to be transported by GlpF [[Team:Groningen/Literature#Heller, KB, et al.1980|(Heller, KB, et al.1980)]]. At physiological pH arsenic and antimone are not present in their ionic state but rather as As(OH)3 and Sb(OH)3 [[Team:Groningen/Literature#Rosen, BR, et al.2009|(Rosen, BR, et al.2009)]]. These elements show a charge distribution similar to glycerol and a smaller but comparable volume. The structural similarities are thought to be the reason for the possibility of these elements to enter the cell by GlpF [[Team:Groningen/Literature#Porquet, A, et al.2007|(Porquet, A, et al.2007)]], GlpF facilitates transport of these compounds down there gradient (inside or outside the cell).<br />
If GlpF behaves as a nonsaturable transporter, a transport rate of 1umol of glycerol is transported per minute per mgr of cell protein [[Team:Groningen/Literature#Heller, KB, et al.1980|(Heller, KB, et al.1980)]]. The transport rate of GlpF for arsenic is estimated to be……<br />
<br />
====Cloning strategy====<br />
This part has been obtained from the genome of ''E.coli'' 356 in two steps with PCR. First the whole gene was obtained from the genome by using PCR and in the second step an ''EcoR''1 restiction site was removed.<br />
The GlpF PCR product was restricted with ''Xba''I and ''Pst''I and a psB1AC3 vector with a pMed promotor was restricted with ''Spe''I and ''Pst''I. The restriction products were ligated. This resulted in a psB1AC3 vector with a promotor and GlpF.<br />
[[Image:RestictioLigationGlpF.JPG]]<br />
<br />
====Results====<br />
The ability of GlpF (overexpressed under lactose induction) to transport As(III) was tested by an arsenite uptake [https://2009.igem.org/Team:Groningen/Protocols assay]. Also the full accumulation device (<partinfo>BBa_K190038</partinfo>) was tested using this assay. '''Data and analysis can be found [https://2009.igem.org/Team:Groningen/Project/Accumulation here]. <br />
'''<br />
The expression of GlpF was not tested by SDS-PAGE or protein purification.<br />
<br />
===Modelling uptake GlpF===<br />
<html><br />
<script type="text/javascript" src="/Team:Groningen/Modelling/Model.js?action=raw"></script><br />
<script type="text/javascript" src="/Team:Groningen/Modelling/Arsenic.js?action=raw"></script><br />
</html><br />
<html><style type="text/css"></html><br />
{{InfoBox/Style.css}}<br />
.infoIcon { display: inline; }<br />
<html></style></html><br />
The import of As(III) via GlpF is modelled as a simple import reaction with [[Team:Groningen/Glossary#MichaelisMenten|Michaelis-Menten kinetics]], in part because this makes it easy to specify, but also because we only have very high level data. The following allows a comparison with the data acquired from figure 1B from [[Team:Groningen/Literature#Meng2004|Meng 2004]].<br />
<html><br />
<div style="background:#efe;border:1px solid #9c9;padding:1em;"><br />
<table style="border-collapse:collapse;background:none;"><tr><br />
<td style="border-right:1px solid #9c9;padding-right:1em;"><br />
<dl><br />
<dt>Initial values</dt><br />
<dd><br />
As(III)<sub>ex</sub> = <input type="text" id="As3exInitial" value="9.15164271986822"/> &micro;M<br/><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(10&micro;M &middot; 1mL / 1.092mL)<br />
</dd><br />
<dt>Volumes</dt><br />
<dd><br />
V<sub>total</sub> = <input type="text" id="Vtotal" value="1.1"/> mL<br/><br />
V<sub>cells</sub> = <input type="text" id="Vcells" value="0.0073"/> mL<br/><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(0.1ml &middot; 80mg/mL / 1100mg/mL) </html>{{infoBox|E. coli has a density of approximately 1100mg/mL, see [[Team:Groningen/Project/Vesicle|our gas vesicle page]] for more information.}}<html><br />
</dd><br />
<dt>Kinetic Constants</dt><br />
<dd><br />
<nobr>v5 = <input type="text" id="v5" value="3.1862846729357852"/> &micro;mol/(s&middot;L)</nobr><br/><br />
K5 = <input type="text" id="K5" value="27.71808199428998"/> &micro;M<br/><br />
</dd><br />
</dl><br />
<br />
<button onClick="computeGlpFTransport()">Compute</button><br/><br />
</td><br />
<br />
<td style="padding-left:1em;"><br />
<div id="glpFTransportError" style="color:red"></div><br />
</html>{{graph|Team:Groningen/Graphs/GlpFTransport|id=glpFTransportGraph}}<html><br />
</td><br />
</tr></table><br />
</div><br />
<script type="text/javascript"><br />
<br />
//The graph already initializes itself (and we don't do any other computations).<br />
//addOnloadHook(computeGlpFTransport);<br />
<br />
function computeGlpFTransport() {<br />
document.getElementById('glpFTransportGraph').refresh();<br />
}<br />
</script><br />
</html><br />
<br />
To determine the constants v5 and K5 we performed the following steps:<br />
<br />
# '''Read the wild-type line in figure 1B''' of [[Team:Groningen/Literature#Meng2004|Meng 2004]] by pasting it in a drawing program and aligning/scaling the axes and then manually determining the coordinates of each data point.<br />
# '''Converted to units of concentration''' using the data in Meng 2004 and [http://gchelpdesk.ualberta.ca/CCDB/cgi-bin/STAT_NEW.cgi the CCDB] (assuming that the cells are resting/non-growing), see our [http://spreadsheets.google.com/pub?key=t4gilzCbEaCFAvpEVWUE_zQ Google Docs spreadsheet]. Here we disregarded the fact that the measurements were made by taking out 0.1mL samples, as this does not change the concentrations. Specifically (note that uptake is in nmol/mg):<br />
#* uptake<sub>total</sub> (nmol) = uptake &middot; 8mg &middot; 0.3 {{infoBox|The ratio between dry and wet weight is 0.3 (see the [http://gchelpdesk.ualberta.ca/CCDB/cgi-bin/STAT_NEW.cgi CCDB]).}}<br />
#* As(III)<sub>ex</sub> (&micro;M=nmol/mL) = (10nmol/mL &middot; 1mL - uptake<sub>total</sub>) / (1.1-0.0073)mL {{infoBox|1=The experiment started with 1mL of a 10&micro;M=10nmol/mL solution of As(III). After adding the cells the total volume of the solution was 1.1mL, and 0.0073mL is an estimate of the total volume of cells in the solution, see below.}}<br />
# '''Fit the Michaelis-Menten equation''' to find the constants v5 and K5 in Mathematica (see [http://igemgroningen.googlecode.com/svn/trunk/buoyant/Models/Meng2004%20Figure%201B.nb the Mathematica notebook in SVN]) using the method from [[Team:Groningen/Literature#Goudar1999|Goudar 1999]] (a least squares fit of a closed-form solution of the differential equation).<br />
<br />
{{GraphHeader}}<br />
<br />
<br><br />
<br />
===Missing information/To Do===<br />
*Expression assesment<br />
**Stability<br />
**Level<br />
*Functional assesment<br />
**Uptake speed<br />
**Affinity<br />
**Electrolyte potential generating force<br />
*<del>Q:Eliminate BioBrick restriction sites</del><br />
*<del>Q: What does the ars operon of our <i>E. coli</i> look like? Do we have both ArsA and ArsB? (And what about ArsR and ArsD?)</del> A: We only have ArsRBC, see [[Team:Groningen/BLAST|our BLAST results]].<br />
<br />
<br><br />
<br />
===Additional sources===<br />
<br><br />
* [[Team:Groningen/Literature#Meng2004|Meng 2004]] (As(III) and Sb(III) Uptake by GlpF and Efflux by ArsB in Escherichia coli)<br />
* [[Team:Groningen/Literature#Rosen2009|Rosen 2009]] (Transport pathways for arsenic and selenium: A minireview)<br />
*[[Team:Groningen/Literature#Porquet, A, et al.2007|Porquet, A, et al.2007]] (structural similarity between As(OH)3 and glycerol)<br />
* [[Team:Groningen/Literature#Fu, DX, et al.2000|Fu, DX, et al.2000]] (Structure of the GlpF channel)<br />
*[[Team:Groningen/Literature#Heller, KB, et al.1980|Heller, KB, et al.1980]] (Glycerol transport properties of GlpF)<br />
<br />
==Copper/zinc uptake via HmtA==<br />
<br />
===HmtA===<br />
====Introduction====<br />
HmtA(heavy metal transporter A) from <i>Pseudomonas aeruginosa</i> [http://www.ncbi.nlm.nih.gov/protein/81857196 Q9I147] is a P-type ATPase importer. This membrane protein mediates the uptake of copper (Cu) and zinc (Zn) and was functionally expressed in E.coli ([http://www.ncbi.nlm.nih.gov/pubmed/19264958 Lewinson 2009]). We want to use this membrane protein to accumulate copper and zinc into the cells. we believe this ATP-driven pump is capable of generating an elevated intracellular concentration of these compounds enabling the harvesting of copper and zinc from the medium.<br />
<br />
====Cloning strategy====<br />
<br />
There are several restriction sites to be modified from [https://static.igem.org/mediawiki/2009/8/85/PBAD-HmtA-ClonemanagerFile.zip Lewinson's] pBAD construct. A vector with amp resistance with L-arabinose inducible HmtA-6HIS. The restriction sites can be modified without changing the amino acid sequence of the protein.<br />
We will create these mutations via PCR than digest the old methylated template and clone the product into competent cells. Create ON cultures for plasmid isolation for restriction check and repeat the process.<br />
<br />
First we clone the gene into an iGEM vector pSB1AC3 and we lose the HIS tag and introducing one mutation to eliminate one EcoRI site. Than via PCR eliminate the PstI restriction site with proper primers and destroy the template with DnpI and transform into cells, repeat for the other PstI site.<br />
<br />
{| border="1"<br />
!Enzyme<br />
!Number of Sites<br />
|-<br />
|EcoRI<br />
|1<br />
|-<br />
|PstI<br />
|2<br />
|}<br />
<br />
<br />
<br />
====Results==== <br />
[[Image:HmtA_SDS_gel.jpg|200px|thumb|right|[Team:Groningen/Team|HmtA-6HIS on SDS-page]]<br />
So far we have cloned HmtA as a biobrick without EcoRI site in the coding region into the iGEM vector. Unfortunately a mutation occurred at base 103 from the start of the orf. By a point mutation c to t in the first nucleotide of the codon changed arginine 35 to a Cysteine. We do not know the effects but we suspect it might have a great influence due to the very reactive sidechain, eventhough it is not in the channel itself based on [http://www.cbs.dtu.dk/services/TMHMM/ TMHMM] predictions witch indicate trans membrane helices of a protein. Further cloning is expected to unsuccessful because the iPTG induced clones grow even better than Controle most likely because of missing pLAC-RBS. There was no positive controle with the L-arabinose inducable HmtA-6His in pBAD. We did do expression experiments with the pBAD construct to purified the membrane protein as quality controle.<br />
<br />
====Missing information/To Do====<br />
*Expression assesment<br />
**Stability<br />
**Level<br />
*Functional assesment<br />
**Uptake speed<br />
**Affinity<br />
**Electrolyte potential generating force<br />
*Eliminate BioBrick restriction sites<br />
<br />
==Heavy metal uptake coupled to citrate via ''ef''CitH ''bs''CitM==<br />
<br />
Citrate uptake coupled to heavy metals enables forcefeeding of the toxic compounds into the cell when citrate is the only carbon source available. This could be a very efficient strategy to accumulate vast ammounts of heavy metals.<br />
The current candidates are CitM from ''Bacilus subtilis'' and CitH form ''Enterococcus faecalis''. <br />
<br />
'''Missing information/To Do'''<br />
*Expression assesment<br />
**Stability<br />
**Level<br />
*Functional assesment<br />
**Uptake speed<br />
**Affinity<br />
**Electrolyte potential generating force<br />
*Eliminate BioBrick restriction sites<br />
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===Additional sources===<br />
<br />
More information on this topic can be found in:<br />
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Bastiaan Krom. Citrate transporters of <i>Bacilus subtilis</i> PhD thesis. [[http://dissertations.ub.rug.nl/faculties/science/2002/b.p.krom/ Dissertation Groningen]]<br />
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Jessica B. Warner. Regulation and expression of the metal citrate transporter CitM PhD thesis. [[http://dissertations.ub.rug.nl/faculties/science/2002/j.b.warner/ Dissertation Groningen]]<br />
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==Periplasmic accumulation of heavy metals via Mer Operon==<br />
Periplasmic accumulation of heavy metals via Mer proteins enables the harvesting of heavy metals from the medium by binding the cytosolic and periplasmic metals to metallothionein and transporting the metal-protein complex into the periplasm.<br />
The MerR family consists of different proteins for one specific metal (<i>i.e.</i><br />
PbrR (lead), CueR (copper), ZntR (zinc), MerR (mercury), ArsR (arsenic), CadR (cadmium)).<br />
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As the cells die after uptake of Mg (and induction of the Mer transporter), this system is not very well usable for our project. The dead cells will not produce the gas vesicles (it may be used however by having the gas vesicles consitutively expressed), thereby bouyancy may be a problem ([[Team:Groningen/Literature#Pennella2005|Pennella 2005]], [[Team:Groningen/Literature#Kao2008|Kao 2008]]).<br />
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===Missing information/To Do===<br />
*Expression assesment<br />
**Stability<br />
**Level<br />
*Functional assesment<br />
**Uptake speed<br />
**Affinity<br />
**Electrolyte potential generating force<br />
*Eliminate BioBrick restriction sites<br />
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==Planning and requirements==<br />
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* '''Modelling:'''<br />
** Import speed<br />
** Amount <br />
** Max<br />
* '''Lab:'''<br />
** HmtA<br />
*** Zn/Cu alone<br />
*** B-type ATPase (could be use if there is a ATP shortage?)<br />
** CitM (probably not used)<br />
*** Divalent ions<br />
*** Citrate around<br />
*** Citrate can bind metals that are already bound.<br />
** Measurements (both for the "normal" cell and the cell with overexpression of the transporter)<br />
*** Transporter, on/off mechanism, up to what concentration (in the cell) does it still have metal uptake.<br />
*** Measure concentration of metal. difference between begin and end concentrations of metal outside the cell.<br />
*** How fast does it transport metal in/out the cell.<br />
**** Set up tests with (initial) extracellular concentrations of about <sup>1</sup>/<sub>3</sub>K (25% of V<sub>max</sub>), K (50% of V<sub>max</sub>), 3K (75% of V<sub>max</sub>) and 10mM (99.7% of V<sub>max</sub>, corresponding to extremely polluted water), and a control with no arsenic. Obviously, more tests is better. In general a desired fraction of V<sub>max</sub> at the initial concentration can be attained by using an initial concentration of x/(1-x) times K.<br />
**** Determine "final" (steady-state) concentration of As(III) in the solution and in the cells. (Concentration over time is even better!)<br />
**** This means that the total volume of the cells (and the solution) has to be determined. Possibly through looking at the dry weight (without arsenic!).<br />
**** By manipulating the equation for the derivative of As(III) in equilibrium, As(III) can be expressed as a function of As(III)<sub>ex</sub> (given the V and K constants). We can try to fill in the computed V and K constants for GlpF and then use a least squares fit to estimate the V and K constants for ArsB.<br />
**** '''NOTE:''' Interestingly [[Team:Groningen/Literature#Kostal2004|Kostal 2004]] already did an experiment like this with cells that overexpressed ArsR. We're looking at analysing these results under the assumption that overexpressing ArsR only gives a constant factor more accumulation (for 1-100&microM As(III)), but it would be very nice to do this ourselves for unmodified cells to determine whether this is indeed true (and to determine the factor).<br />
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==Export of arsenicum via Ars operon==<br />
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GlpF is the importer of arsenicum. After arsenicum enters the cell, in response the Ars operon produces ArsR. At the same time, ArsB is also produced by Ars operon. This happens because the Ars operon contains three open reading frames: the first is ArsR, second ArsB and the last one is ArsC. ArsB is the exporter of arsenicum. The ars operon is located on the chromosomal DNA of E. coli.<br />
For more information see: [http://biocyc.org/ECOLI/NEW-IMAGE?type=GENE-IN-CHROM-BROWSER&object=EG12235 biocyc].<br />
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[[Image:ArsRBC_operon.PNG|600px]]<br />
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