Team:USP-Brazil/Promoters

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Promoters

Promoter comparisons

As we aim to replace chemical inductors by light in bioproduction we needed to, at first place, compare the promoters induced by led light versus induced by chemical reagents.

For the comparison of promoters we chose pBAD(BBa_K808000), pLac(BBa_K731500) and pT3 (BBa_K2598026). First, we needed to clone these 3 promoters in three individual plasmids. The best backbone for this construction was the vector ratiometric promoter characterization reporter (BBa_K2771020) because it already had the reporter gene (eYfp) for the quantification of expression, and a constitutive reporter (eCfp) for normalization of measurements. Since ratiometric promoter characterization reporter, construction led to the award of Best Measurement in 2018, we thought that this method would provide us with a more accurate data and also, that we could contribute to better characterize this vector.

The promoters fragments were inserted to the backbone vector. Colony PCR was performed to detect successful cloning, using ‘pT3confFw’ and ‘VR’ for pT3 construct, and ‘VF’ and ‘VR’ for the pLac and pBAD construct. As shown in the Figure 1, the 3 plasmids were correctly constructed.

Because LB broth media has a natural fluorescence that hinders the measurement of reporter proteins, we decided to use a Minimal Medium supplemented with Leucine and Vitamin B1, as our strains (DH10B and HST08) were respectively auxotrophic for these components. The carbon sources used for this experiment were Glucose or Glycerol.

Figure 1 (A) Colony PCR: an amplification of about 2000 bp fragment confirms the insertion of pT3 (lane 3). (B) – Colony PCR: a fragment of about 3300 bp confirms the insertion of pLac (lanes 1-3) and pBAD (lane 9). Red arrow indicates the chosen colonies for induction assay.

The ratiometric promoter characterization reporter + pLac or pBAD constructs were cloned in DH10B strain, while ratiometric promoter characterization reporter + pT3 construct was cloned in HST08 carrying single blue light sensor (BBa_K3095003). The following graphs (Figure 2) show the experiment performed with pLac and pBAD constructs. Unfortunately we did not get any results for pT3 construct, since it took a lot of time (still taking) to standardize this assay.

Figure2> Expression of yfp induced by IPTG or (L)-Arabinose with Glucose or Glycerol as carbon source. –Control is DH10B strain carrying ratiometric promoter characterization reporter vector without promoter for yfp expression.

From the induction experiment we could contribute with accurate data by plotting our graphs with normalized expression of the reporter. Also, we compared the induction between Glucose or Glycerol as carbon source.

We conclude that addition of Glucose in the medium decreases the level of expression for pLac and pBAD constructs, probably due to catabolic repression. On the other hand, despite the expression with Glycerol being much higher compared to Glucose, we noticed almost no bacterial growth using the first carbon source, in the end of the experiment.

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