Part:BBa_K808000:Experience

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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.

User Reviews

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• DTU-Denmark

We experienced the promoter to be very leaky. We improved the part and the new promoter is considerably more tight (Part:BBa_K1067007) also see the library of different pBAD promoters that can be use to improve and model BBa_K808000.

••• CLSB-UK

Overview: We aimed to test our constructs in a cell free system following amplification in E.coli. However, we found that the transcripts from our constructs were produced in toxic concentrations to E.coli under the control of the constitutive promoter BBa_J23111. Thus, to prevent transcription during amplification, we made new constructs containing the promoter BBa_K808000, as it was reported to have very low leakage. We characterised BBa_K808000 in a cell free system by measuring the amount of GFP produced in response to varying concentrations of arabinose. We also determined if this promoter is suitable for regulating the expression of lethal parts in E. coli. Methods: For characterisation of the leakage of the promoter, we amplified our constructs containing BBa_k80800 in E.coli and performed plasmid minipreps to extract circular DNA for expression in a cell free system. We then sequenced our plasmid DNA. For characterisation of BBa_K808000 in response to arabinose concentrations, we prepared a cell free system containing BBa_K808000 from the parts registry and left it to incubate for 10 hours at 37°C. The cell free system we used was the E.coli S30 extract system for circular DNA from Promega. We then added 1 μl of arabinose at the concentrations of: 0.05%, 0.1%, 0.5%, 1% and 2% and ran the experiment for 10 hours at 37°C, recording fluorescence intensity every 10 minutes. Results: Characterisation during amplification of our constructs: Sequencing analysis showed that we successfully amplified the part BBa_K2206006 (this part contains BBa_K2206000, BBa_E0040 and BBa_K808000) with no fidelity errors. Therefore BBa_K808000 was suitable for preventing toxic levels of our part BBa_K2206006 in E.coli, demonstrating that BBa_K808000 has low levels of leakage. However, we found some mutagenesis in the promoter (BBa_K808000) for part BBa_K2206007 (which contains BBa_K2206001, BBa_E0040 and BBa_K808000), meaning toxic levels of BBa_K2206007 were still produced. This indicates that the promoter has some leakage and may therefore be unsuitable for regulating the expression of lethal parts. Characterisation using GFP: We found that fluorescence increased in as little as one hour and maximum fluorescence was reached after ~8 hours for all the concentrations. We found that the fluorescence increase occurred with as little as 0.05% arabinose (we did not measure lower than this) and increased with arabinose concentrations up to 0.5%. Interestingly, we saw a decline in fluorescence at 1% and 2% arabinose concentrations. This may be explained by the fact that in a cell free system there is a limited amount of nucleoside triphosphates (NTP) and ribosomes. At 1% and 2% arabinose concentrations we speculate that lots of GFP mRNA was produced in a short period of time. Consequently, the NTP pool was rapidly depleted and there was an excess of mRNA relative to the number of ribosomes. This led to degradation of the excess mRNA, which otherwise would have been produced later when there would be free ribosomes. Additionally, the excess production of mRNA in early stages resulted in only a few NTPs remaining for further mRNA production later on. Therefore, less mRNA was translated, so GFP production was reduced, leading to decreased fluorescence at higher concentrations.

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