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Team:Warsaw/Stage2/Results
From 2010.igem.org
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<img src = "https://static.igem.org/mediawiki/igem.org/d/da/OD1.jpg" height=420 width=850> | <img src = "https://static.igem.org/mediawiki/igem.org/d/da/OD1.jpg" height=420 width=850> | ||
| - | <br><br><br> | + | <br><i>Fig.1</i> OD measurements taken in 30 min intervals<br><br> |
<img src = "https://static.igem.org/mediawiki/igem.org/2/21/CFU1.jpg"heigth=420 width=850> | <img src = "https://static.igem.org/mediawiki/igem.org/2/21/CFU1.jpg"heigth=420 width=850> | ||
| - | <br><br><br> | + | <br><i>Fig.2</i> CFU measurements taken in 30 min intervals<br><br> |
| + | 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 | ||
| + | <br><br> | ||
<div class="note">Stationary measurement of OD and CFU</div> | <div class="note">Stationary measurement of OD and CFU</div> | ||
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Revision as of 14:42, 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:
Fig.1 OD measurements taken in 30 min intervals
Fig.2 CFU measurements taken in 30 min intervals
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
- 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
Fig.2 CFU measurements taken in 30 min intervals
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
