Part:BBa_K737010:Experience

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http://2014.igem.org/wiki/index.php?title=Team:Glasgow/Project/Measurements/Gas_Vesicles

BBa_K1463340
This part contains GvpA with a strong ribosome binding site (relative strength of 1) and GvpC with a comparatively weaker RBS. The construct does not contain individual promoters for the GvpA and GvpC; it also does not contain the two promoters found at the end of Part:BBa_K737010. This composite part was placed 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 OUC 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. 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.
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.
The samples were observed at intervals over 5 days but no difference was visible between the induced and non-induced cell samples.

Figure 1 Comparison of control and arabinose induced cells after 72 hours

Figure 2 Comparison of control and arabinose induced cells after 96 hours

Figure 3 Comparison of control and arabinose induced cells after 120 hours

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.
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.