GvpA and B0034
Construct containing GvpA and strong RBS, B0034 (efficiency of 1). This part was used as a composite part with GvpC (K1463210) to make the construct 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.
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.
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12COMPATIBLE WITH RFC
- 21COMPATIBLE WITH RFC
- 23COMPATIBLE WITH RFC
- 25COMPATIBLE WITH RFC
- 1000COMPATIBLE WITH RFC