This part contains GvpA with a constitutive promoter, [https://parts.igem.org/Part:BBa_J23100 J23100], and a strong RBS, [https://parts.igem.org/Part:BBa_B0034 B0034], in [https://parts.igem.org/Part:BBa_J61002 J61002]. This part was used in a composite part with GvpC ([https://parts.igem.org/Part:BBa_K1463210 K1463210]) to make the construct [https://parts.igem.org/Part:BBa_K1463345 K1463345]. This construct, under the control of an araC (arabinose-inducible) promoter was transformed into DH5α in order to determine if the problem of producing gas vesicle proteins in E.coli (as detailed by Ocean University China iGEM 2012) could be circumvented. The use of an inducible plasmid would allow the control of GvpA and GvpC expression; this would hopefully allow a level of expression that would cause the formation of gas vesicles. The aim was to induce E.coli to produce gas vesicles which would enable them to float to the top of a water column.
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This part contains GvpA with a constitutive promoter, [https://parts.igem.org/Part:BBa_J23100 J23100], and a strong RBS, [https://parts.igem.org/Part:BBa_B0034 B0034], in [https://parts.igem.org/Part:BBa_J61002 J61002]. This construct was created to allow for the easy insertion of the GvpC biobrick downstream by restriction digest. The plasmid containing this construct would be digested using SpeI and PstI, whereas, the biobrick would be digested with XbaI and PstI to allow the formation of a scar site between the end of GvpA and the RBS of GvpC.
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Cells containing the plasmid (pBAD33) with the composite part and araC promoter were cultured in either glucose (a repressor of the araC promoter) or arabinose.
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<br>Cells were first grown in LB until mid exponential phase had been reached, and then grown a further 2 hours with 0.2% glucose, or 0.2% arabinose to induce GvpA and GvpC expression. Cells were harvested by centrifugation and re-suspended in filter sterilised 0.15% NaCl.
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<br>The samples were observed at intervals over 5 days but no difference was visible between the induced and non-induced cell samples.
<br> <b> Figure 3</b> Comparison of control and arabinose induced cells after 120 hours
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<br>The floating assays above show that there is no floating visible in the induced cells; there was also no obvious difference between the rate of sinking between the non-induced or induced.
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<br>The cells were then investigated using microscopy. The induced cells appear to be noticeably elongated when compared to the non-induced cells. There also appeared to be evidence of an inclusion body in the induced cells; suggesting that the production of GvpA and GvpC proteins causes some form of stress response in E.coli – this is consistent with the findings of OUC.
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Latest revision as of 00:14, 2 November 2014
GvpA in J61002 vector
This part contains GvpA with a constitutive promoter, J23100, and a strong RBS, B0034, in J61002. This construct was created to allow for the easy insertion of the GvpC biobrick downstream by restriction digest. The plasmid containing this construct would be digested using SpeI and PstI, whereas, the biobrick would be digested with XbaI and PstI to allow the formation of a scar site between the end of GvpA and the RBS of GvpC.
