Team:Newcastle

From 2009.igem.org

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[[Image:Newcastle BacMan.png|center|512px]]
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=<center>GOLD Medal winners 2009</center>=
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[[Image:NewcastleBac-Man bacs.png|center|350px]]
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;<center>In 2009 the Newcastle team are tackling environmental issues using ''Bacillus subtilis''. We are a team of eight with wide ranging backgrounds in the fields of Bioinformatics, Computing Science, Chemical Engineering, Genetics and Medical Sciences. This is Newcastle's second year in the iGEM competition; last year our team designed [https://2008.igem.org/Team:Newcastle_University BugBuster], which achieved a Gold Medal.</center>
;<center>In 2009 the Newcastle team are tackling environmental issues using ''Bacillus subtilis''. We are a team of eight with wide ranging backgrounds in the fields of Bioinformatics, Computing Science, Chemical Engineering, Genetics and Medical Sciences. This is Newcastle's second year in the iGEM competition; last year our team designed [https://2008.igem.org/Team:Newcastle_University BugBuster], which achieved a Gold Medal.</center>
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;<center>In 2009 we matched our Gold medal from the previous year, and hope to be back in 2010 to participate in iGEM again.</center>
=<center>Project Description</center>=
=<center>Project Description</center>=
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We engineered ''B. subtilis'' to sense and sequester cadmium from the environment into '''metallothionein containing spores''', rendering it '''bio-unavailable'''. Germination of the spores can be disabled, making retrieval of the cadmium unnecessary since they can persist intact for thousands of years.
We engineered ''B. subtilis'' to sense and sequester cadmium from the environment into '''metallothionein containing spores''', rendering it '''bio-unavailable'''. Germination of the spores can be disabled, making retrieval of the cadmium unnecessary since they can persist intact for thousands of years.
We '''computationally simulated the life cycles of individual cells''' and entire cell populations, to estimate the parameter values necessary to '''maintain sustainable populations''' of sporulating, germinating and vegetative cells. Our design required us to engineer  stochastic differentiation processes at a single cell level. A '''sporulation rate tuner''' was developed and  we also engineered a tuneable stochastic invertase switch to '''stochastically control cell differention and fate'''.  
We '''computationally simulated the life cycles of individual cells''' and entire cell populations, to estimate the parameter values necessary to '''maintain sustainable populations''' of sporulating, germinating and vegetative cells. Our design required us to engineer  stochastic differentiation processes at a single cell level. A '''sporulation rate tuner''' was developed and  we also engineered a tuneable stochastic invertase switch to '''stochastically control cell differention and fate'''.  
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</div>
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'''The following diagram gives an overview of our design, click on different areas of the diagram to go to the appropriate section of our project:'''
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<!-- full size versions [[Image:Team NewcastleOverview pic 1 2.png|590px]]
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[[Image:Team NewcastleOverview pic 2 2.png|550px]]-->
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<img src="https://static.igem.org/mediawiki/2009/9/99/Team_NewcastleOverview_pic_1_2_small.png" width="590" height="442" border="0" usemap="#Map1" />
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== Cadmium Sensing ==
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The following diagram gives an overview of what our design hopes to achieve...
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Our design allows bacteria to intake cadmium through the manganese channel MntH, as cadmium also leaks through these channels in addition to other metals [1]. The metal sensing proteins we intend to use are CzrA and ArsR which both bind cadmium as well as zinc cobalt and nickel, and arsenic, silver and copper respectively [2]. These proteins are repressor proteins which also bind DNA preventing transcription of downstream CDS. The repressor proteins however release the DNA to preferentially bind cadmium [2] allowing transcription to occur allowing selective sensing of cadmium in the form of a logic AND gate.
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[[Image:NewcastleOverview pic 1.png|550px]]
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[[Image:NewcastleOverview pic 2.png|550px]]
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== Heavy Metal Sensing ==
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Our design is for the bacteria to intake cadmium through the manganese channel MntH, as it has been found that due to the similar size and charge of the ions cadmium leaks through these channels [1].Once inside the cell the cadmium must be sensed in order for the cell to regulate some of the decisions involved in becoming a metal container. The metal sensing proteins we intend to use are ''yozA'' (''czrA'') and ''arsR'' which both bind cadmium as well as zinc cobalt and nickel, and arsenic, silver and copper respectively [2].These proteins are repressor proteins which also bind DNA preventing transcription of downstream CDS. The repressor proteins however release the DNA to preferentially bind cadmium [2] allowing transcription to occur. These proteins therefore allow selective sensing of cadmium in the form of an AND gate, a model for which can be seen in our modelling section.
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== Population Dynamics ==
== Population Dynamics ==
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As well as sensing cadmium we will attempt to engineer our bacteria’s normal population dynamics, by nudging the natural stochastic sporulation decision in favour of greater sporulation, to account for the spores that will be lost as metal containers.
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As well as sensing cadmium we plan to engineer our bacteria’s normal population dynamics, by nudging the natural stochastic sporulation decision in favour of higher sporulation rates, to account for the spores that will be lost as metal containers. Our population model simulates whole cell populations using inputs from single cellular models and will be used to determine the required increase in sporulation rate.
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We will also design our own stochastic switch, which will be based on the decision of being a metal container or not. This will be regulated by an invertible segment of DNA using the hin/hix system [3]. In this way our artificial stochastic switch will be a ‘biased heads or tails’ which we can control. This stochastic switch will control expression of our ‘metal sponge’.
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==Stochastic switch==
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Our artificial stochastic switch in a ‘biased heads or tails’ which we can tune. The stochastic switch determines the cell's decision to become a metal container or not. This switch will be regulated by an invertible segment of DNA using the hin recombinase/hix [3]. We favoured the use of an invertable DNA segment since the choice will be heritable and maintain its expression inside a spore. This stochastic switch will either induce the expression of the genes which switch on the ‘metal sponge’ phenotype or will direct the cell to a wild-type lifestyle.
== Metal Containers ==
== Metal Containers ==
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To make our spores metal containers we will be using the metallothionein protein smtA which is a relatively small cysteine rich protein known to ‘soak up’ metals such as cadmium [4]. We will guide this protein to the spore whilst sporulating by creating a fusion with the spore coat protein cotC, which will coat the spore in cadmium bound metallothionein [5].  
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To make our spores into metal containers we will be using the metallothionein protein SmtA, which is a relatively small cysteine rich protein known to ‘soak up’ metals such as cadmium [4]. We will guide this protein to the spore by creating a fusion with the spore coat protein CotC, which will embed cadmium bound metallothionein throughout the spore [5].  
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Finally, to ensure that metal containing spores will not germinate again, releasing their bound cadmium, we will ‘knock-out’ important germination genes using our invertible sequence system.
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Finally, to ensure that metal containing spores will not germinate again, releasing their bound cadmium, we will ‘knock-out’ essential germination genes ''sleB'' and ''cwlJ'' and selectively re-complement the resulting mutation.
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==Sporulation tuning==
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In order to tune sporulation, our team is proposing the idea of inducing the synthesis of KinA, with IPTG as a sporulation initiation signal. KinA is a major kinase which provides phosphate input to the phosphorelay, which in turn, activates the sporulation pathway upon starvation via the phosphorylated Spo0A transcription factor,[2] which governs entry into the sporulation pathways of the bacterium ''Bacillus subtilis''.[3]
== Novelty ==
== Novelty ==
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Our design has many components which we believe are novel, such as our control over the sporulation cycle, and our synthetic stochastic switch, both systems we believe are reusable concepts within synthetic biology.</div>
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Our design has many components which we believe are novel, such as our control over the sporulation cycle, and our synthetic stochastic switch, both systems we believe are reusable concepts within synthetic biology.
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{|style="color:DarkBlue;background-color:#ffffcc;" cellpadding="20" cellspacing="0" border="1"
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! colspan="2" |<font size=4> <center>'''Summary of achievements:'''</center></font>
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|-
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|<span style="color:Sienna">'''Bronze:'''</span>
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|<span style="color:SaddleBrown">We have designed our project together as a team and shared our ideas on the iGEM wiki, as well as registering new standard BioBrick parts on the Registry of Standard Biological Parts. Our BioBricks are novel and easily reusable within synthetic biology. We have submitted DNA for the new BioBrick Parts and Devices which can be viewed [[Team:Newcastle/Parts|here]].</span>
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|-
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|<span style="color:Silver">'''Silver:'''</span>
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|<span style="color:DimGray">Our new BioBrick Part BBa_K174011 works as expected and so we have characterised its operation and documented our results on the [[Team:Newcastle/Characterisation |iGEM wiki]] and the [http://partsregistry.org/Part:BBa_K174011 Registry]</span>
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|-
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|<span style="color:Goldenrod">'''Gold:'''</span>
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|<span style="color:SaddleBrown">We have improved the existing BioBrick Part [http://partsregistry.org/Part:BBa_K090501 Pspac promoter] and documented our new information back on the Registry: [http://partsregistry.org/Part:BBa_K174004 BBa_K174004].We have also helped another iGEM team by sending UQ a  [[Team:Newcastle/Helping_other_teams |mercury sensing model ]] for their project along with a tutorial. </span>
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|-
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|}
== References ==
== References ==

Latest revision as of 11:37, 9 November 2009

GOLD Medal winners 2009

NewcastleBac-Man bacs.png


In 2009 the Newcastle team are tackling environmental issues using Bacillus subtilis. We are a team of eight with wide ranging backgrounds in the fields of Bioinformatics, Computing Science, Chemical Engineering, Genetics and Medical Sciences. This is Newcastle's second year in the iGEM competition; last year our team designed BugBuster, which achieved a Gold Medal.
In 2009 we matched our Gold medal from the previous year, and hope to be back in 2010 to participate in iGEM again.

Project Description

Cadmium contamination can be a serious problem in countries where polluting industries are located close to agricultural sites. Our team developed a design to address this problem using the resiliant spore-forming bacterium Bacillus subtilis. We engineered B. subtilis to sense and sequester cadmium from the environment into metallothionein containing spores, rendering it bio-unavailable. Germination of the spores can be disabled, making retrieval of the cadmium unnecessary since they can persist intact for thousands of years. We computationally simulated the life cycles of individual cells and entire cell populations, to estimate the parameter values necessary to maintain sustainable populations of sporulating, germinating and vegetative cells. Our design required us to engineer stochastic differentiation processes at a single cell level. A sporulation rate tuner was developed and we also engineered a tuneable stochastic invertase switch to stochastically control cell differention and fate.

The following diagram gives an overview of our design, click on different areas of the diagram to go to the appropriate section of our project:

Cadmium Sensing

Our design allows bacteria to intake cadmium through the manganese channel MntH, as cadmium also leaks through these channels in addition to other metals [1]. The metal sensing proteins we intend to use are CzrA and ArsR which both bind cadmium as well as zinc cobalt and nickel, and arsenic, silver and copper respectively [2]. These proteins are repressor proteins which also bind DNA preventing transcription of downstream CDS. The repressor proteins however release the DNA to preferentially bind cadmium [2] allowing transcription to occur allowing selective sensing of cadmium in the form of a logic AND gate.

Population Dynamics

As well as sensing cadmium we plan to engineer our bacteria’s normal population dynamics, by nudging the natural stochastic sporulation decision in favour of higher sporulation rates, to account for the spores that will be lost as metal containers. Our population model simulates whole cell populations using inputs from single cellular models and will be used to determine the required increase in sporulation rate.

Stochastic switch

Our artificial stochastic switch in a ‘biased heads or tails’ which we can tune. The stochastic switch determines the cell's decision to become a metal container or not. This switch will be regulated by an invertible segment of DNA using the hin recombinase/hix [3]. We favoured the use of an invertable DNA segment since the choice will be heritable and maintain its expression inside a spore. This stochastic switch will either induce the expression of the genes which switch on the ‘metal sponge’ phenotype or will direct the cell to a wild-type lifestyle.

Metal Containers

To make our spores into metal containers we will be using the metallothionein protein SmtA, which is a relatively small cysteine rich protein known to ‘soak up’ metals such as cadmium [4]. We will guide this protein to the spore by creating a fusion with the spore coat protein CotC, which will embed cadmium bound metallothionein throughout the spore [5]. Finally, to ensure that metal containing spores will not germinate again, releasing their bound cadmium, we will ‘knock-out’ essential germination genes sleB and cwlJ and selectively re-complement the resulting mutation.

Sporulation tuning

In order to tune sporulation, our team is proposing the idea of inducing the synthesis of KinA, with IPTG as a sporulation initiation signal. KinA is a major kinase which provides phosphate input to the phosphorelay, which in turn, activates the sporulation pathway upon starvation via the phosphorylated Spo0A transcription factor,[2] which governs entry into the sporulation pathways of the bacterium Bacillus subtilis.[3]

Novelty

Our design has many components which we believe are novel, such as our control over the sporulation cycle, and our synthetic stochastic switch, both systems we believe are reusable concepts within synthetic biology.


Summary of achievements:
Bronze: We have designed our project together as a team and shared our ideas on the iGEM wiki, as well as registering new standard BioBrick parts on the Registry of Standard Biological Parts. Our BioBricks are novel and easily reusable within synthetic biology. We have submitted DNA for the new BioBrick Parts and Devices which can be viewed here.
Silver: Our new BioBrick Part BBa_K174011 works as expected and so we have characterised its operation and documented our results on the iGEM wiki and the [http://partsregistry.org/Part:BBa_K174011 Registry]
Gold: We have improved the existing BioBrick Part [http://partsregistry.org/Part:BBa_K090501 Pspac promoter] and documented our new information back on the Registry: [http://partsregistry.org/Part:BBa_K174004 BBa_K174004].We have also helped another iGEM team by sending UQ a mercury sensing model for their project along with a tutorial.

References

  1. Que, Q. and J.D. Helmann, Manganese homestasis in Bacillus subtilis is regulated by MntR, a bifunctional regulator related to the diphtheria toxin repressor family of proteins. Molecular Microbiology, 2000. 35(6): p. 1454-1468.
  2. Harvie, D.R., et al., Predicting metals sensed by ArsR-SmtB repressors: Allosteric interference by a non-effector metal. Molecular Microbiology, 2006. 59(4): p. 1341-1356.
  3. Haynes, K.A., et al., Engineering bacteria to solve the Burnt Pancake Problem. Journal of Biological Engineering, 2008. 2.
  4. Blindauer, C.A., et al., Multiple bacteria encode metallothioneins and SmtA-like zinc fingers. Molecular Microbiology, 2002. 45(5): p. 1421-1432.
  5. Mauriello, E.M.F., et al., Display of heterologous antigens on the Bacillus subtilis spore coat using CotC as a fusion partner. Vaccine, 2004. 22(9-10): p. 1177-1187.

External links

  • [http://www.ncl.ac.uk Newcastle University]



News

Events

Social Net

  • Newcastle iGEM Twitter
  • [http://www.facebook.com/home.php#/group.php?gid=131709337641 Newcastle on Facebook]
  • [http://www.youtube.com/user/newcastle2009igem Newcastle Youtube Channel]
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