Team:Glasgow/Project/Measurements/Gas Vesicles

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What are Gas Vesicles?

GvpA and GvpC

Before any work could be done with GvpA and GvpC, the genes had to be isolated from the constructs found in the distribution. In order to do this, PCR primers were designed to isolate the GV genes as well as being designed to add the BioBrick prefix and suffix to the genes. To allow the genes to be translated, a RBS was added into the primers between the prefix and the coding sequence. The GvpA forward primer contained the sequence for RBS 0034 (a relatively strong RBS) and the GvpC forward primer contained the sequence for RBS 0032 – comparatively weak RBS. GvpA and GvpC were then isolated and amplified by PCR; this was that confirmed by gel electrophoresis of a sample of the PCR.
The DNA was then digested with EcoRI and PstI to allow the construct to be inserted into pSB1C3, the constructs were then transformed successfully into TOP10 and DH5α. The resultant tranformant DNA was isolated by miniprep; the presence of the constructs was confirmed by running the DNA on a gel.

In an attempt to cause GV formation, GvpA and GvpC would need to be placed under the control of a promoter. The first step taken was to insert GvpA into an Ampr plasmid containing a strong constitutive promoter (J23100); to insert GvpA upstream of the promoter, the gene had to be isolated by restriction digest using XbaI and PstI to allow the ends to be compatible with the J23100 plasmid – which has also been digested with SpeI and PstI. GvpC was then inserted upstream of GvpA using the same process. GvpC/pSB1C3 was disgested with XbaI and PstI and the J23100/GvpA plasmid was digested with SpeI and PstI – this caused the formation of a scar sequence between GvpA and GvpC upon ligation of the correct fragments. Again, the constructs were transformed into TOP10 and DH5α. The DNA was isolated by miniprep to be digested and run on the gel to check the presence of the inserts. The DNA was also sent for sequencing.
A similar process was carried out to place GvpC upstream of GvpA in pSB1C3. The results of the sequencing showed that the construct had mutated in the RBS and would therefore be unable to function.

In order to resolve the mutant construct, new PCR primers were designed to isolate the mutated construct from the plasmid by PCR – as well as correct the mutation – and reinsert it into a new plasmid.
The construct was reinserted into the plasmid containing the J23100 promoter. The corrected construct was also inserted into plasmids containing promoters of varying strengths (J23103, J23106, J23112 and J23116). Again the constructs were confirmed by transforming into TOP10 and DH5α, the transformants were minipreped and restricted to run on a gel. Liquid cultures of the different transformants were left for 3 days in 30C and 37C to give the cultures time to settle – this would allow any floating to be observed. Unfortunately, there appeared to be no difference between the different transformants and floating was not observed.

The construct was also placed in a Chlorr plasmid under the control of an arabinose inducible promoter (araC). The resultant DNA from the transformants was sent for sequencing to confirm the presence of an unmutated construct. The confirmed transformants were then observed for their ability to float.
Cells were suspended in NaCl, one sample of cells was given arabinose and another sample was given glucose. The samples were observed at intervals over 5 days but no difference was visible between the induced and non-induced cell samples.

Using microscopy, the cells containing the GvpA/GvpC construct appear to be elongated suggesting the cells were under stress; it may be logical to assume that GvpA/GvpC is detrimental to the cells.

Efficiency of Switch Promoter

In order to determine how efficient the switch promoter is, in comparison to the J23100 promoter without the att sites, a plasmid was generated with J23100 promoter upstream of the GFP with the BB0034 RBS. The fluorescence of transformants containing this plasmid was compared with the fluorescence of transformants containing K1463000 (RFP/switch/GFP) exposed to integrase which fluoresce green. Measuring the fluorescence levels revealed that the switch promoter is less efficient compared to the J23100 promoter. This suggests that the att sites and intervening sequences between the promoter and the RBS interferes with the strength of the promoter.

This experiment was also repeated using the J23100/RFP and comparing its level of fluorescence to K1463000 which was not exposed to integrase – therefore fluoresces red. This yielded the same result; further validating that the att sites and intervening sequences in the switch effects the activity of the switch promoter.

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