Team:Goettingen/Parts
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The plate reader experiments allowed deeper insights into the strength of the different promoters. In these measurements, the observed fluctuations and elevated RFP/OD<sub>600nm</sub> values during initial growth might result from the low cell number in LB medium and might therefore be not significant (compare: [https://2012.igem.org/Team:LMU-Munich/Data/Anderson#Luminescence_measurements click here]). The RFP fluorescence remained low during exponential growth. After 5 h, there was a strong increase in RFP fluorescence. At the same time, cell growth slowed down suggesting that both, reduced cell growth and stronger fluorescence might be correlated. In order to fluoresce, fluorescent proteins like GFP and RFP need oxygen (Remington, 2006). The oxygen RFP needs for fluorophore maturation could not have been available during log phase, since the cells consume the oxygen for enhanced cell growth. This possibility underlines the advantage of promoter characterization via transcript levels over promoter characterization via fluorophores. Still, fluorophore-depended characterization might be useful during stationary phase. | The plate reader experiments allowed deeper insights into the strength of the different promoters. In these measurements, the observed fluctuations and elevated RFP/OD<sub>600nm</sub> values during initial growth might result from the low cell number in LB medium and might therefore be not significant (compare: [https://2012.igem.org/Team:LMU-Munich/Data/Anderson#Luminescence_measurements click here]). The RFP fluorescence remained low during exponential growth. After 5 h, there was a strong increase in RFP fluorescence. At the same time, cell growth slowed down suggesting that both, reduced cell growth and stronger fluorescence might be correlated. In order to fluoresce, fluorescent proteins like GFP and RFP need oxygen (Remington, 2006). The oxygen RFP needs for fluorophore maturation could not have been available during log phase, since the cells consume the oxygen for enhanced cell growth. This possibility underlines the advantage of promoter characterization via transcript levels over promoter characterization via fluorophores. Still, fluorophore-depended characterization might be useful during stationary phase. | ||
- | By qRT-PCR we were able to observe a tendency for the strength of the different promoters, though the exact fold changes seemed not to be reproducible. This lack of reproducibility might have resulted from the clones which differed in age. While the clones from replicate 1 were from the first transformation of the Biobrick vectors, the clones from replicate 2 originated from a new re-transformation of the plasmids. In general, our results suggested that Promoter 1 was the weakest, followed by Promoter 2, which was slightly stronger. Most interestingly, in E. coli DH5α, Promoter 3 seemed to be even stronger than Promoter 4, though it was shown to be weaker than promoter 4 before. However, our observations regarding Promoter 4 are difficult to interpret. First, this promoter led to contradicting results regarding the ''rfp'' fold changes during stationary phase, while the plate reader results independent of the clone indicated that it is weaker than Promote 3. Second, qRT-PCR replicate 2 suggested that it is almost as weak as promoter 4, which is in contrast to all other results obtained for this promoter. There are two possible explanations: (a) An experimental mistake resulted in insignificant fold changes; (b) Promoter 4 is subject to a complex regulation depending on the exact growth conditions. Considering this, it has to be noted that it is difficult to harvest the cells for RNA preparation at exactly the same OD<sub>600nm</sub>. Hence, we took the samples for all strains at the same time when they had reached the approximate OD<sub>600nm</sub> we were interested in. Since none of the two explanations could be ruled out, further characterization is necessary to obtain a more detailed view of the promoters and their regulation under different growth conditions. All in all, our results showed that it is useful to apply several methods for characterization. Taking the majority of the results of the three methods into account, we suggest that the order of the promoter activity in ''E. coli'' DH5α grown in broth LB<sup>Amp</sup> is as follows: Promoter 1 BBa_J23117 < Promoter 2 BBa_J23116 < Promoter 4 BBa_J23118 < Promoter 3 BBa_J23110. | + | By qRT-PCR we were able to observe a tendency for the strength of the different promoters, though the exact fold changes seemed not to be reproducible. This lack of reproducibility might have resulted from the clones which differed in age. While the clones from replicate 1 were from the first transformation of the Biobrick vectors, the clones from replicate 2 originated from a new re-transformation of the plasmids. In general, our results suggested that Promoter 1 was the weakest, followed by Promoter 2, which was slightly stronger. Most interestingly, in E. coli DH5α, Promoter 3 seemed to be even stronger than Promoter 4, though it was shown to be weaker than promoter 4 before. However, our observations regarding Promoter 4 are difficult to interpret. First, this promoter led to contradicting results regarding the ''rfp'' fold changes during stationary phase, while the plate reader results independent of the clone indicated that it is weaker than Promote 3. Second, qRT-PCR replicate 2 suggested that it is almost as weak as promoter 4, which is in contrast to all other results obtained for this promoter. There are two possible explanations: (a) An experimental mistake resulted in insignificant fold changes; (b) Promoter 4 is subject to a complex regulation depending on the exact growth conditions. Considering this, it has to be noted that it is difficult to harvest the cells for RNA preparation at exactly the same OD<sub>600nm</sub>. Hence, we took the samples for all strains at the same time when they had reached the approximate OD<sub>600nm</sub> we were interested in. Since none of the two explanations could be ruled out, further characterization is necessary to obtain a more detailed view of the promoters and their regulation under different growth conditions. All in all, our results showed that it is useful to apply several methods for characterization. Taking the majority of the results of the three methods into account, we suggest that the order of the promoter activity in ''E. coli'' DH5α grown in broth LB<sup>Amp</sup> is as follows: Promoter 1 [http://partsregistry.org/Part:BBa_J23117 BBa_J23117]< Promoter 2 [http://partsregistry.org/Part:BBa_J23116 BBa_J23116] < Promoter 4 [http://partsregistry.org/Part:BBa_J23118 BBa_J23118] < Promoter 3 [http://partsregistry.org/Part:BBa_J23110 BBa_J23110]. |
Revision as of 22:30, 2 October 2013