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Team:Calgary/8 July 2009
From 2009.igem.org
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| - | + | Repeat Transformation | |
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| - | + | * The transformation from yesterday resulted in NO colonies | |
| + | * Attempted transformation with Quick Ligase and then transformed into Top10 cells. | ||
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| - | + | Adjusting Model frame work | |
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| - | + | Me , Carol and Kevin adjusted our model frame work . Then the whole modelling team had a good discussion with Thane . Before that I read more journals dealing with parameter estimation and looked for rates . I looked at the parameter fit function in Simbiology and tried to use this function for our purpose. | |
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| - | + | DNA Sequencing and cloning of B0015 | |
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| - | + | *Sent J13002-LuxOD47E- C3 down for DNA Sequencing this morning to verify the presence of the J13002 promoter within the construct. We will proceed with construction to try to clone in the B0015 terminator. | |
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| + | *Performed a restriction digest with EcoRI, XbaI and SpeI restriction enzymes,followed by Antarctic Phosphatase of the vector (NEB) and ligation with QuikLigase (Invitrogen). Did two trial, trial 1 where the B0015 terminator was treated as the insert and trial 2 where the J13002-LuxOD47E construct was terated as the insert. Ligated product was then transformed into TOP10 cells and plated on C plates (25μL and 50μL) and left for overnight growth. | ||
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| + | *Today I also transformed some of my J13002-LuxOD47E construct into KT1144 cells. Because my cells are currently in the psB1AC3 vector, where there are intrinsic terminators, the B0015 terminator may not be necessary in order for my circuit to work in the KT1144 cells (where we are testing them). After transformation, cells were plated on both C and K plates and left for overnight growth. | ||
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| - | + | Marketing, Ethics and Outreach for July 8th 2009 | |
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| - | + | Today I sent out our June newsletters to media organizations that had done on a story on our project. I also read some ethics papers regarding the importance of ethics in Synthetic Biology and Quorum Sensing. For outreach, I sent my highschool biology teacher an e-mail regarding doing presentations about synthetic biology and iGEM in Bio 30 Classes. Me and my teacher agreed on doing the presentations somewhere in October. | |
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| - | <a name=" | + | <a name="Jeremy"></a> |
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| - | + | JEREMY | |
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| - | + | Colony PCR on Overnight Cultures of LuxPQ in AK plasmid | |
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| - | + | Purpose: to verify the presence of LuxPQ in the psB1AK3 plasmid after the AC to AK plasmid switch. Eight colonies were picked (4 from colony 6 and 4 from colony 7) for a pTaq colony PCR with BBK CP F/R primers. The following conditions were used: 94ºC for 3 minutes; 36X (94ºC for 30 seconds; 55ºC for 45 seconds; 72ºC for 4 minutes); 72ºC for 10 minutes; held at 4ºC. The products were then run on a 0.8% agarose gel (see below). We can see from this gel that colony 1 and 2 (from colony 6) were the only colonies that revealed the desired size of band at around 3.8kb. | |
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| - | + | [[Image:2009.07.08.LuxPQ_cPCR.png|700px]] | |
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| - | + | Sequencing results came back today, revealing several mutations in LuxPQ Colony 6, revealing that C6-1 and C6-2, although they are of the desired size, cannot be proceeded with. No mutations were seen in the sequence of LuxPQ in Colony 7; however, the BBK restriction site, PstI, was not present. Therefore, a plasmid switch was set up of LuxPQ colony 7 from AC to AK in order to restore the BBK suffix. This was performed by setting up a restriction digest using EcoRI and SpeI for the insert and the psB1AK3 vector. After this restriction digest, phosphatase and ligation treatment was performed and the plasmid was subsequently transformed into TOP10 competent cells and plated overnight on LB+Kanamycin. | |
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| - | A weekly blog is being done by all sections of our team, including wetlab and modelling which I am part of. Every | + | A weekly blog is being done by all sections of our team, including wetlab and modelling which I am part of. Every Wednesday is wetlab blog day, so one was written. The following is the link to my post: |
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| + | <a href='http://igemcalgary.blogspot.com/2009/07/kevin-loves-rainbows.html'>Kevin Loves Rainbows</a> | ||
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| - | + | Tidying up my DNA | |
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| - | + | Retrospective Notebook: This entry was not written on this day, but derived later from working notes I made that day. | |
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| + | Originally, I had planned to keep most of the functionality for my DNA parts in a single script. But, the DNA needed to know some hardcoded information about itself: what its name is, what the name of the next part in the DNA polymer is, what type of DNA element it is, and what protein it is repressed by / activated by / produces, and I didn't want to generate a new version of the script (with different hardcoded values) for every single DNA part I made. I decided to use a second script, which would have unique hardcoded information for each DNA part, this information could also be modified for purposes of building custom DNA devices later. I had thought I could make this 'identity script' nice and short, and contain most of the changes to each part in the main 'dna script'. The problem is that this introduced an extra level of communication between the main DNA script and its identity script... and because so much of the DNA's function relates to what it is, the identity script wound up being longer than the DNA script! | ||
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| + | I wanted to merge these two scripts into something better organized, but I still needed a way to record the DNA's important details. I briefly used an internal notecard (text document) to store this information, but Second Life doesn't allow objects to edit notecards, which would be a problem when we get to building custom DNA in the Biobricker. In the end I settled on storing this information in the part's name and description fields, the only text fields that are persistent across sessions and resets, but also editable. The only problem with doing this is that these fields are user accessible, it's very possible for someone to edit their object's name and break its functionality the next time it resets or is moved from inventory! Yet, it's the only way I could find to get the DNA construction system to work, and it also has a hidden feature: capable users can change the traits of their biobrick parts without needing to build entire new devices in the HUD. | ||
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| + | The new DNA script uses a much simplified system to keep track of what it is, and what it can do. There are three parameters: number one is the part's type, with each of constitutive promoter, repressable promoter, coding sequence... etc as possible types. The DNA learns its type when it is created from inventory or reset, and that type is permanent until the next reset or recreation. Type also informs the DNA of what transcription factor it should pay attention to, if any. | ||
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| + | The type of the DNA determines the second parameter: possible behaviours with respect to RNA Polymerase: | ||
| + | * Permit to bind, and pass through (like a promoter) | ||
| + | * Permit to pass through, but not bind (like regular DNA) | ||
| + | * Do not permit binding or passing through (like a terminator) | ||
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| + | The DNA's behaviour is set to one of these when its type is determined, but this behaviour can then change depending on the state of a third parameter: whether it is bound by a repressor or activator or not. The information of which type of protein is binding or unbinding, combined with the DNAs type, is enough to determine what behaviour the DNA is now responsible for. | ||
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| - | + | Marketing | |
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| - | + | I began looking at WestJet and Air Canada flights flying to Boston. I had callled a few travel agencies to try to get a quote. I also looked at Hotels at MIT. I need to determine how far it is from MIT, whether it would be economical to stay there and take a cab or get a hotel closer (but a little bit more expensive) and walk to MIT. I also made a list of hotels including their rates, discounts, services, distance from MIT, etc. | |
| - | + | I began following up with companies via phone and email. I found 16 more companies that I could contact. I began researching those companies and writing up emails to them. | |
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| - | + | The team also sat down to discuss logo designs, colors, shapes, etc. | |
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| - | + | Glycerol stock preparation of successfully-biobricked parts | |
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| - | + | Today was a day of plasmid isolation and glycerol stock preparation. I made overnight cultures and restreaks last night of the parts that I had biobricked. We need some isolated DNA for the registry, as well as glycerol stocks for long-term storage. We have samples of everything that I prepared, with concentrations on the order of 100 ng/uL. One of the sets - the LuxO D47A BBk - was not isolated well, so I made new overnights and will prepare glycerol stocks of those tomorrow. | |
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Latest revision as of 21:48, 20 October 2009
UNIVERSITY OF CALGARY
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CAROL
Repeat Transformation
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CHINMOYEE
Adjusting Model frame work
Me , Carol and Kevin adjusted our model frame work . Then the whole modelling team had a good discussion with Thane . Before that I read more journals dealing with parameter estimation and looked for rates . I looked at the parameter fit function in Simbiology and tried to use this function for our purpose.
|
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EMILY
DNA Sequencing and cloning of B0015
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FAHD
Marketing, Ethics and Outreach for July 8th 2009
Today I sent out our June newsletters to media organizations that had done on a story on our project. I also read some ethics papers regarding the importance of ethics in Synthetic Biology and Quorum Sensing. For outreach, I sent my highschool biology teacher an e-mail regarding doing presentations about synthetic biology and iGEM in Bio 30 Classes. Me and my teacher agreed on doing the presentations somewhere in October.
|
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JEREMY
Colony PCR on Overnight Cultures of LuxPQ in AK plasmid
Purpose: to verify the presence of LuxPQ in the psB1AK3 plasmid after the AC to AK plasmid switch. Eight colonies were picked (4 from colony 6 and 4 from colony 7) for a pTaq colony PCR with BBK CP F/R primers. The following conditions were used: 94ºC for 3 minutes; 36X (94ºC for 30 seconds; 55ºC for 45 seconds; 72ºC for 4 minutes); 72ºC for 10 minutes; held at 4ºC. The products were then run on a 0.8% agarose gel (see below). We can see from this gel that colony 1 and 2 (from colony 6) were the only colonies that revealed the desired size of band at around 3.8kb.
Sequencing results came back today, revealing several mutations in LuxPQ Colony 6, revealing that C6-1 and C6-2, although they are of the desired size, cannot be proceeded with. No mutations were seen in the sequence of LuxPQ in Colony 7; however, the BBK restriction site, PstI, was not present. Therefore, a plasmid switch was set up of LuxPQ colony 7 from AC to AK in order to restore the BBK suffix. This was performed by setting up a restriction digest using EcoRI and SpeI for the insert and the psB1AK3 vector. After this restriction digest, phosphatase and ligation treatment was performed and the plasmid was subsequently transformed into TOP10 competent cells and plated overnight on LB+Kanamycin.
|
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KATIE
Creation and Modification of Scripts and Notecards
I have completed creating instructional and information note cards for the various activities within the virtual lab on our island, including:
Note cards that are almost finished and will be completed by tomorrow include:
I have also been creating and modifying the scripts for PCR, Gel Electrophoresis, Restriction Digest and have just started Bacterial Transformation. They have been modified to use a more user friendly prompt system (using llDialog function in second life scripting language) so asking avatars for information and having them answer looks cleaner and is more intuitive.
I am hoping to have restriction digest functioning by Friday and will be working on finishing notecards, Restriction Digest and Bacterial Transformation in the upcoming days. I will also be organizing a sequencing station, which may be used for a section of the lab activities.
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KEVIN
Restreak of the glowing cells
A brightest colony from yesterday's transformation were restreaked. These cells have to grow overnight, once again.
Blog update
A weekly blog is being done by all sections of our team, including wetlab and modelling which I am part of. Every Wednesday is wetlab blog day, so one was written. The following is the link to my post:
Kevin Loves Rainbows
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PATRICK
Tidying up my DNA
Retrospective Notebook: This entry was not written on this day, but derived later from working notes I made that day.
Originally, I had planned to keep most of the functionality for my DNA parts in a single script. But, the DNA needed to know some hardcoded information about itself: what its name is, what the name of the next part in the DNA polymer is, what type of DNA element it is, and what protein it is repressed by / activated by / produces, and I didn't want to generate a new version of the script (with different hardcoded values) for every single DNA part I made. I decided to use a second script, which would have unique hardcoded information for each DNA part, this information could also be modified for purposes of building custom DNA devices later. I had thought I could make this 'identity script' nice and short, and contain most of the changes to each part in the main 'dna script'. The problem is that this introduced an extra level of communication between the main DNA script and its identity script... and because so much of the DNA's function relates to what it is, the identity script wound up being longer than the DNA script! I wanted to merge these two scripts into something better organized, but I still needed a way to record the DNA's important details. I briefly used an internal notecard (text document) to store this information, but Second Life doesn't allow objects to edit notecards, which would be a problem when we get to building custom DNA in the Biobricker. In the end I settled on storing this information in the part's name and description fields, the only text fields that are persistent across sessions and resets, but also editable. The only problem with doing this is that these fields are user accessible, it's very possible for someone to edit their object's name and break its functionality the next time it resets or is moved from inventory! Yet, it's the only way I could find to get the DNA construction system to work, and it also has a hidden feature: capable users can change the traits of their biobrick parts without needing to build entire new devices in the HUD. The new DNA script uses a much simplified system to keep track of what it is, and what it can do. There are three parameters: number one is the part's type, with each of constitutive promoter, repressable promoter, coding sequence... etc as possible types. The DNA learns its type when it is created from inventory or reset, and that type is permanent until the next reset or recreation. Type also informs the DNA of what transcription factor it should pay attention to, if any. The type of the DNA determines the second parameter: possible behaviours with respect to RNA Polymerase:
The DNA's behaviour is set to one of these when its type is determined, but this behaviour can then change depending on the state of a third parameter: whether it is bound by a repressor or activator or not. The information of which type of protein is binding or unbinding, combined with the DNAs type, is enough to determine what behaviour the DNA is now responsible for.
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PRIMA
Marketing
I began looking at WestJet and Air Canada flights flying to Boston. I had callled a few travel agencies to try to get a quote. I also looked at Hotels at MIT. I need to determine how far it is from MIT, whether it would be economical to stay there and take a cab or get a hotel closer (but a little bit more expensive) and walk to MIT. I also made a list of hotels including their rates, discounts, services, distance from MIT, etc.
I began following up with companies via phone and email. I found 16 more companies that I could contact. I began researching those companies and writing up emails to them. The team also sat down to discuss logo designs, colors, shapes, etc.
|
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VICKI
Glycerol stock preparation of successfully-biobricked parts
Today was a day of plasmid isolation and glycerol stock preparation. I made overnight cultures and restreaks last night of the parts that I had biobricked. We need some isolated DNA for the registry, as well as glycerol stocks for long-term storage. We have samples of everything that I prepared, with concentrations on the order of 100 ng/uL. One of the sets - the LuxO D47A BBk - was not isolated well, so I made new overnights and will prepare glycerol stocks of those tomorrow.
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