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USP-Brazil 2019 Characterization

Methods

In our project we aimed to compared 3 different promoters (pBad, pLac and pT3). For analysing the promoters, we choose to utilise the 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. Utilise this reporter allows us to have a better compassion between different promoters, since has the same "reporter backbone" the comparison is normalised by the eCfp, providing a more accurate comparasion. As shown in the Figure 1, the 3 plasmids were correctly constructed.

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.

Results

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.

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 standardise this assay.

Figure 2: Expression of yfp induced by IPTG or (L)-Arabinose with Glucose or Glycerol as carbon source. –Control is DH10B strain carrying characterization reporter (BBa_K2771020) without promoter for yfp expression.

From the induction experiment we could contribute with accurate data by plotting our graphs with normalised expression of the reporter. Also, we compared the induction between Glucose or Glycerol as carbon source. As we can see in the graphic, the pLac showed a very higher expression then the pBad. However a normalisation of the inducer molecule is necessary for a more accurate analysis.

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