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The efficiency of the kill-switch was measured by following means:
  • dynamic measurement of OD
  • dynamic measurement of CFU (colony-forming units)
  • stationary measurement of OD
  • stationary measurement of CFU (colony-forming units)

Dynamic measurement of OD and CFU

For the measurement BL21 RIL strain of E. coli (F− ompT hsdS(rB −mB −) dcm +Terr gal λ (DE3) endA Hte (argU ileY leuW Camr)) was transformed with the following plasmids:
  • pSB1A2 containing MinC under T7 promoter and B0032 RBS
  • pSB1A2 containing MinC without a promoter or RBS

Approximately 100 μl of over-night culture were used to inoculate 50 ml of LB medium with ampicilin and chloramphenicol (volume of inoculate was adjusted to ensure equal initial OD values). One flask was inoculated with pSB-MinC and two more with pSB-pT7-B0032-MinC. Cultures were incubated in 37 oC (with shaking) for 30 min. Following this initial incubation, OD was measured and a small volume of culture was plated (on LA medium with amp and cm) for CFU measurement. pSB-MinC and one of the pSB-pT7-B0032-MinC cultures were induced by addition of IPTG to the final concentration of 10 μM. Cultures were again placed on a shaker at 37 oC. Samples for OD and CFU measurements were then taken every 30 min until the OD began to exceed 1,5. Results of the measurements are shown on the plots below:

Fig.1 OD measurements taken in 30 min intervals (contr.+IPTG - pSB-MinC induced with IPTG; MinC+IPTG - pSB-pT7-B0032-MinC induced with IPTG; MinC-IPTG - pSB-pT7-B0032-MinC without induction)

Fig.2 CFU measurements taken in 30 min intervals (contr.+IPTG - pSB-MinC induced with IPTG; MinC+IPTG - pSB-pT7-B0032-MinC induced with IPTG; MinC-IPTG - pSB-pT7-B0032-MinC without induction)

Obtained results fit the theoretical predictions very well. pSB-pT7-B0032-MinC displays severely decreased growth rate. Growth in the samples, where pSB-pT7-B0032-MinC was not induced and where the construct did not include a promoter, was not visibly affected. Therefore we can conclude, that neither the potentially leaky promoter nor the toxicity of IPTG cause any unspecific decrease in the growth rate. The difference in growth of induced construct and the controls is also much more pronounced when measures by the number of colony forming units (rather than by OD). This in agreement with the expected effects of MinC overexpression, which is supposed to inhibit cell division.

Agar plate during 150 minute of IPTG induction shows striking difference between the experiment (right side of the plate) and our two negative controls (down and left).

Stationary measurement of OD and CFU

Cultures for stationary measurement were grown in identical conditions as described above, only with increasing concentrations of IPTG. Measurements were taken 3h following induction. Results are displayed below:

Fig.3 Stationary measurements of OD

Fig.4 Stationary measurements of CFU

Stationary measurements prove the dosage-dependent mode in which MinC affects the bacterial cell. Like previously, the effects are more drastical when CFUs are considered. For the IPTG concentration of 2,5 μM the number of CFUs was below 106 per ml. For concentrations of 5, 10 and 20 μM it was below 104 CFU/ml (over 106-fold decrease in the number of viable cells compared to uninduced culture).

Filamentous cells

During our experiment, we were able to see under the microscope filamentous E.coli cells formation. This observation is consistent with theoretical basis of MinC function. MinC stops cell division even though bacterium can grow and enlarge its size (within certain limits).

Picture taken under the microscope showing filamentous cells E.coli formation. Bacteria is longer as a result of division cell inhibition. Picture on the left shows cell not expressing MinC. One on the right displays cells where MinC-containing construct was expressed

Additional observations

An alternative construct for MinC expression was obtained. Unlike the one measured above, it contained a B0034 RBS. Cloning was preformed in DH5α cells. However, all attempts to transform BL21 cells failed, even when the bacteria were plated on LA medium without IPTG and with glucose (for additional repression of T7 polimerase). This observation confirmed the significance of modulating kill-switch expression. B0034 RBS is over 3-fold stronger than B0032. Apparently, even a highly specific promoter, such as the T7 promoter, can display sufficient leaking to inhibit baterial growth. The reason why this effect was not observed in DH5α was the lack of T7 polimerase gene in this particular strain.