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From 2012.igem.org

Requirements

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)to induce the production of GFP. Our future goal would be to replace the GFP gene with gas vesicle protein(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.

2. 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.

3. 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 we designed such mechanism includes the cstA promoter, T7 RNA polymerase and a second promoter (pT7). When genes are placed under the control of the T7 promoter, very large amounts of RNA are generated from small amounts of T7 RNA polymerase (Studier and Moffatt, 1986) since the enzyme is very selective for T7-like promoters (Studier et al., 1990), hence we believe that this will produce larger amounts of gas vesicle gene cluster. 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.