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Team:University College London/Module 4
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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. | 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. | ||
In order to test this module we used '''GFP''' as a reporter gene instead of the gas vesicle gene cluster. | In order to test this module we used '''GFP''' as a reporter gene instead of the gas vesicle gene cluster. | ||
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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. | ||
== References== | == References== | ||
Revision as of 14:37, 26 September 2012
Module 4: Buoyancy
Description | Design | Construction | Characterisation | Modelling | Results | Conclusions
Description
The Buoyancy module is key to both the Degradation and the Aggregation systems. Buoyancy is required to position our bacteria in the water column, and also to enable them to buoy the plastic aggregates.
This module requires driving the expression of a gas vesicle gene cluster. Gas vesicles are formed within the cell, and are hollow spaces surrounded by a wall of hydrophobic protein. These gas vesicles are permeable to gases, which diffuse into the gas vesicles, increasing its partial pressure, thereby increasing buoyancy.
The Buoyancy system is subject to the control of a glucose-repressible promoter, cstA (BBa_K118011). During carbon starvation, ATP is transformed into cAMP and then it binds the cAMP receptor protein, this complex activates the cstA promoter. Studies suggest that cstA promoter can be used to induce the expression of reporter genes in cultures with different glucose concentrations (Schultz and Matin, 1990). Free glucose concentration varies in the upper 300 m of the seawater (Skoog et al., 1999), so this gives us a gradient of activation which allows us to control the transcription of gas vesicle genes in different glucose concentrations. Since the plastic particles are mostly found in the upper 25 m, bacteria needs to be positioned in this water column. This is very important for our system therefore we designed a mechanism to amplify the expression of gene under the control of the cstA promoter, such design includes 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. In order to test this module we used GFP as a reporter gene instead of the gas vesicle gene cluster.
References
-Schultz JE, Matin A. (1991) Molecular and functional characterization of a carbon starvation gene of Escherichia coli. J Mol Biol.; 218(1):129-40.
-Skoog, A., Biddanda B, Benner R. (1999) Bacterial utilization of dissolved glucose in the upper water column of the Gulf of Mexico. Limnol. Oceanogr. 44:1625-1633.
-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.
-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
