Team:Exeter/Experiments
Experiments
18th July
A Taq polymerase PCR procedure was run to amplify the fim operon in E.coli strains MG1655 (as a reference) and DH5ɑ (if present). The primers were based on the known sequence for the operon and several were used in order to separate the three genes fimD, fimH and fimA into different bands on the subsequent electrophoresis.
19th July
The products of the PCR from the previous day were run down an agarose gel. As shown on the image, DH5ɑ contains all three of the fim genes which were tested for. If DH5ɑ were to be used as a chassis for our product, the fim genes would have had to have been knocked out prior to the insertion of the operon and modified fimH gene containing plasmids. The decision was made to look for an alternative strain of E.coli that does not contain the genes for pilus biosynthesis.
25th July
With a genomic extraction having been done at the beginning of the day, another PCR was carried out with the same primers previously used in order to amplify any present fimA,D or H genes in MG1655(as a reference) and Top10.
26th July
An electrophoresis was carried out using the amplified DNA from the previous day. As shown, the MG1655 reference is positive for all three fim genes that were amplified, while Top10 is negative for all three. This confirmed that Top10 was a more suitable candidate with regards to its genome.
28th July
Overnight cultures of MG1655 and Top10 were removed from the incubator and were taken to the Bioimaging department. The samples were prepared with a negative stain and were placed on a small, circular copper grid before being inserted into the transmission electron microscope. Several exposures were taken, as shown and these are demonstrated both that MG1655 does indeed produce pilus structures and that pili are absent from the surface of the Top10 strain. This, combined with the electrophoresis evidence, confirmed that the Top10 strain is the suitable chassis for our project.
TEM
Add 100μl(?) of a bacterial liquid culture to a circular copper grid with a parafilm cover. Leave for 2mins to allow binding. Prepare 3 droplets of deionised water on parafilm and transfer grid to each one, with five minutes in between transfer. Add 100μl(?) uranyl acetate to grid and leave for 2mins. Dry grid with filter paper and transfer to dish for analysis.
2nd August
The fim operon + pAra MoClo products were cut using restriction endonucleases before being run down an agarose gel. The result gave confirmation that the fim operon had been transformed wholly into dh5ɑ in pSB1A3.
