Team:LCG-UNAM-Mexico/Wet Lab/Experiments
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====Toxins==== | ====Toxins==== | ||
The colicines (E3 and E9) will be under the transcriptional regulation of the promoter induced by IPTG. We will do another curve using the same procedure described in the section [[Team:LCG-UNAM-Mexico/Wet_Lab/Experiments#T3 and T7 infection plot|phage infection curve]] but without phages and with the induction with IPTG in the time zero. We expect the growth curve obtained to decay before than that of the phages infection curve because we want the toxic activity of the toxins will be faster enough to kill the bacteria before the phages. We will feedback the model with these results. | The colicines (E3 and E9) will be under the transcriptional regulation of the promoter induced by IPTG. We will do another curve using the same procedure described in the section [[Team:LCG-UNAM-Mexico/Wet_Lab/Experiments#T3 and T7 infection plot|phage infection curve]] but without phages and with the induction with IPTG in the time zero. We expect the growth curve obtained to decay before than that of the phages infection curve because we want the toxic activity of the toxins will be faster enough to kill the bacteria before the phages. We will feedback the model with these results. | ||
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We will follow the same procedure described in the last section. But in this case all the cells will be prepared with the whole system including the [[Team:LCG-UNAM-Mexico/Wet_Lab/Objectives#General objectives|defense, gossip and paranoia system]]. This is the last experiment is one of the most importants because we will be able to conclude that all our design works as expected. We predict that most of the times bacteria will beat phage's infection and the population will survive. So, if we will plate after two hours (it is the experiment's duration time) we would see viable colonies after eight hours. | We will follow the same procedure described in the last section. But in this case all the cells will be prepared with the whole system including the [[Team:LCG-UNAM-Mexico/Wet_Lab/Objectives#General objectives|defense, gossip and paranoia system]]. This is the last experiment is one of the most importants because we will be able to conclude that all our design works as expected. We predict that most of the times bacteria will beat phage's infection and the population will survive. So, if we will plate after two hours (it is the experiment's duration time) we would see viable colonies after eight hours. | ||
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+ | === Helper P2 strain Development === | ||
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+ | ====Functionality of Control Construction==== | ||
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+ | The [[Team:LCG-UNAM-Mexico/Wet_Lab/Objectives#Uriel Urquiza|control construction]] built is IPTG-inducible under a lac promoter. The experiment intends to qualitatively assess the induction of such construction. When P2 lysogenic strains are transformed with this plasmid, one coud expect an effect on P2 lysis due to equilibrium disruption of the mutual negative regulation of native COX and C proteins in favor of COX. Such disruption would result in P2 late gene activation, and hence lysis will appear. The assay will check the quality of bacterial lawns by addition of different concentrations of IPTG stocks to part of the plate surface, so internal negative controls can be contrasted. | ||
+ | Semi-quantitative Northern blot analysis is planned once the P2 helper lacking native cox and ogr genes is finished. Controls would be: non-induced construction (basal transcription), induced construction (induced transcription), native gene transcription, no construction. | ||
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+ | ====Removal of Native P2 control genes==== | ||
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Revision as of 01:59, 22 October 2009