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Team:Warsaw/Stage2/Results
From 2010.igem.org
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<img src = "https://static.igem.org/mediawiki/2010/5/5f/OD2.jpg"heigth=420 width=850> | <img src = "https://static.igem.org/mediawiki/2010/5/5f/OD2.jpg"heigth=420 width=850> | ||
| - | <br><h4><i>Fig.3</i>Stationary measurements of OD</h4> <br><br> | + | <br><h4><i>Fig.3</i> Stationary measurements of OD</h4> <br><br> |
<img src = "https://static.igem.org/mediawiki/2010/4/45/CFU2.jpg"heigth=420 width=850> | <img src = "https://static.igem.org/mediawiki/2010/4/45/CFU2.jpg"heigth=420 width=850> | ||
| - | <br><h4><i>Fig.4</i>Stationary measurements of CFU</h4> <br><br> | + | <br><h4><i>Fig.4</i> Stationary measurements of CFU</h4> <br><br> |
Stationary measurements proves the dosage-dependent mode in which MinC affects the bacterial cell. Like before, the effects are more visible when CFUs are considered. For the IPTG concentration of 2,5 uM the number of CFUs was below 1000000 per ml. For concentrations of 5, 10 and 20 uM it was below 10000 CFU/ml (over 10000-fold difference compared to uninduced culture) | Stationary measurements proves the dosage-dependent mode in which MinC affects the bacterial cell. Like before, the effects are more visible when CFUs are considered. For the IPTG concentration of 2,5 uM the number of CFUs was below 1000000 per ml. For concentrations of 5, 10 and 20 uM it was below 10000 CFU/ml (over 10000-fold difference compared to uninduced culture) | ||
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Revision as of 16:10, 24 October 2010
Results
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)) were transformed with the following plasmids:
Approximately 100 ul 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 C 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 was induced by adding IPTG to the final concentration of 10 uM. Cultures were again placed on a shaker at 37 C. 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:
The obtained results are in agreement with theoretical predictions. 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 had no 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 agrees well with the expected effects of MinC overexpression, which is supposed to inhibit cell division.
Cultures for stationary measurement were grown in identical conditions as above, with increasing concentrations of IPTG. Measurements were taken 3h after induction. Results are shown below
Stationary measurements proves the dosage-dependent mode in which MinC affects the bacterial cell. Like before, the effects are more visible when CFUs are considered. For the IPTG concentration of 2,5 uM the number of CFUs was below 1000000 per ml. For concentrations of 5, 10 and 20 uM it was below 10000 CFU/ml (over 10000-fold difference compared to uninduced culture)
- pSB1A2 containing MinC under T7 promoter and B0032 RBS
- pSB1A2 containing MinC without a promoter or RBS
Approximately 100 ul 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 C 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 was induced by adding IPTG to the final concentration of 10 uM. Cultures were again placed on a shaker at 37 C. 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)
The obtained results are in agreement with theoretical predictions. 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 had no 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 agrees well with the expected effects of MinC overexpression, which is supposed to inhibit cell division.
Stationary measurement of OD and CFU
Cultures for stationary measurement were grown in identical conditions as above, with increasing concentrations of IPTG. Measurements were taken 3h after induction. Results are shown below
Fig.3 Stationary measurements of OD
Fig.4 Stationary measurements of CFU
Stationary measurements proves the dosage-dependent mode in which MinC affects the bacterial cell. Like before, the effects are more visible when CFUs are considered. For the IPTG concentration of 2,5 uM the number of CFUs was below 1000000 per ml. For concentrations of 5, 10 and 20 uM it was below 10000 CFU/ml (over 10000-fold difference compared to uninduced culture)
