Team:University College London/Module 4/Design

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== Design==
== Design==
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<html><div align="center"><img src="http://2012.igem.org/wiki/images/c/cb/UcligemBuoyancy_BioBrick.png" /></div></html>
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The following circuit is the one on which studies were carried out:
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<html><img src="http://2012.igem.org/wiki/images/7/7b/UCL2012BOUYANCYGFP.png"></html>
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<div> Gas vesicles are found largely in cyanobacteria and halophillicarchea, where they are necessary to access oxygen and light. Their  formation is driven by a number of genes required for production and regulation. We are using the gas vesicle gene to attribute buoyancy to our cells so that they float on to the surface of the ocean. Our particular cluster -known as GVP is extracted from Bacillus Megaterium which contains 14 putative gens gvp-A,-P,-Q,-B,-R,-N,-F,-G,-L,-S,-K,-J,-T and -U of which the last 11 genes, in a 5.7-kb gene cluster are the maximum required for gas vesicle synthesis and function in E.coli.
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The following circuit will be implemented in the future:
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<html><img src="http://2012.igem.org/wiki/images/4/46/Ucl2012BOUYANCY.png"></html>
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The following 3 requirements were identified for the Buoyancy module:
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'''Requirement 1: The sinking of the cell should trigger the expression of the buoyancy gene.'''
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For the purpose our project we are using our designed device that (cstA promoter, T7 RNA polymerase and T7 promoter) induces the production of GFP. Our future goal is to replace the GFP gene with gas vesicle protein(BBa_I750016). This will optimise the position of the cell in the water column. As the  cell sinks there will be a significant continuous reduction of nutrients this change in environmental conditions will induce the promoter and subsequently the gas vesicle formation.
   
   
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Requirements:
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'''Requirement 2: The amount of gas vesicles must be high.'''
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1. The sinking of the cell should trigger the expression of the buoyancy gene.
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For the purposes of our project we are using a constitutive promoter for the expression of the gas vesicle (BBa_I750016). However, our plan is to have 2 promoters one is the original starvation promoter from the partsregistry and in addition  we are planning to have a heat shock promoter. This will optimise the position of the cell in the water column. As the  cell sinks there will be a significant reduction of nutrients and decrease in temperature consequently this change in environmental conditions will induce the promoter and subsequently the gas vesicle formation.
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Large amounts of gas vesicles are necessary to keep the cells afloat as well, so using a starvation sensitive promoter to control the production of gas vesicles requires a mechanism to amplify the expression of the buoyancy vesicles cluster. Therefore T7 RNA polymerase and a the promoter T7 are included in this module, as very large amounts of mRNA are generated from small amounts of T7 RNA polymerase (1) when genes are placed under the control of the T7 promoter, since T7 RNA polymerize is very selective for T7-like promoters (2), hence we believe that this will produce larger amounts of gas vesicle gene cluster.
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2. The size and the strength of the gas vesicle must be enough to keep the cell afloat.
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'''Requirement 3: The size and the strength of the gas vesicle must be enough to keep the cell afloat.'''
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The size and strength of the vesicle are predetermined genetically and varies from species to species. Also it is very important to say that there is a trade-off between the size of the vesicle and its strength. The smaller the  vesicle the less it is prone to collapsing and vice-a-versa.
The size and strength of the vesicle are predetermined genetically and varies from species to species. Also it is very important to say that there is a trade-off between the size of the vesicle and its strength. The smaller the  vesicle the less it is prone to collapsing and vice-a-versa.
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The genes we are missing in our construct are gvp-A,-P and -Q and it is worth saying that the gvp-A gene is  coding for a very hydrophobic 70-amino acid long proteing that is responsible for stregthen the vesicle so the lack of this proteinmay affect the strength of our vesicle negatively. Another concern about this biobrick is its size - 5.7 kb by removing some of 11 open reading frames that may play a regulatory role.There are only 2 proteins that are known to be responsible for the structure of the gas vesicle. However some of the other open reading frames may dertermine the protein folding and the Biobrick must be carefully analysed to specify those open reading frames that are the minimum necessary for the successful formation of the gas vesicle.<div>
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==References==
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1. Studier FW, Moffatt BA. (1986) Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 189(1):113-30
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2. Studier FW, Rosenberg AH, Dunn JJ, Dubendorff JW. (1990) Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol.185:60-89
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{{:Team:University_College_London/templates/foot}}

Latest revision as of 02:43, 27 September 2012

Module 4: Buoyancy

Description | Design | Construction | Characterisation | Modelling | Results | Conclusions

Design

The following circuit is the one on which studies were carried out:



The following circuit will be implemented in the future:



The following 3 requirements were identified for the Buoyancy module:


Requirement 1: The sinking of the cell should trigger the expression of the buoyancy gene.

For the purpose our project we are using our designed device that (cstA promoter, T7 RNA polymerase and T7 promoter) induces the production of GFP. Our future goal is to replace the GFP gene with gas vesicle protein(BBa_I750016). This will optimise the position of the cell in the water column. As the cell sinks there will be a significant continuous reduction of nutrients this change in environmental conditions will induce the promoter and subsequently the gas vesicle formation.


Requirement 2: The amount of gas vesicles must be high.

Large amounts of gas vesicles are necessary to keep the cells afloat as well, so using a starvation sensitive promoter to control the production of gas vesicles requires a mechanism to amplify the expression of the buoyancy vesicles cluster. Therefore T7 RNA polymerase and a the promoter T7 are included in this module, as very large amounts of mRNA are generated from small amounts of T7 RNA polymerase (1) when genes are placed under the control of the T7 promoter, since T7 RNA polymerize is very selective for T7-like promoters (2), hence we believe that this will produce larger amounts of gas vesicle gene cluster.


Requirement 3: The size and the strength of the gas vesicle must be enough to keep the cell afloat.

The size and strength of the vesicle are predetermined genetically and varies from species to species. Also it is very important to say that there is a trade-off between the size of the vesicle and its strength. The smaller the vesicle the less it is prone to collapsing and vice-a-versa.

References

1. Studier FW, Moffatt BA. (1986) Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 189(1):113-30

2. Studier FW, Rosenberg AH, Dunn JJ, Dubendorff JW. (1990) Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol.185:60-89