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Speed Control - Experiments

CheZ knockout of E. coli 2443 and Salmonella SL7207

Two methods of detection of whether the CheZ gene were combined to make sure the success of recombination for CheZ knock out.

Swimming Test

After 8 hours’ bacteria swimming, the patterns three strains formed below showed that the ineffectiveness of the swimming ability of the bacteria, which was an indication of the successful knockout of CheZ gene in E.coli 2443 and Salmonella SL7207. The third panel of E.coli 2443 acted as a positive control in this test.

Salmonella SL7207 (delta CheZ)

E. coli 2443 (delta CheZ)

‎ E. coli 2443 (positive control)

PCR test

The expected DNA size of this test was about 400bp, which showed the success recombination procedure.

Regulation of CheZ expression

Swimming test

Test plac-his-cheZ-cm under 0.0005, 0.001, 0.002, 0.004, 0.008, 0.012mmol/L IPTG in MG3 stain.

No Chloramphenicol

Chloramphenicol added

The results above are ambiguous, and we tried to verify if we were successful in achieving differential levels of CheZ in MG3 by doing western blotting.

Western Blotting

From the results above, it can be seen that the CheZ concentration in MG3 was proportional to the concentration of IPTG added to the cultures when the induced time was about 24 hours. The samples with induced time less than 24 hours showed no obvious bands in the Western Blotting.

According to the result of Western Blotting, we could explain the similar swimming abilities of bacteria with different IPTG concentrations. As the speeds of swimming were tested within 9 hours, which were too short for the induced reactions to undergo, the speeds were more or less the same. Another problem was that even negative control strain (no IPTG added) swam, which was probably caused by leaky expression.