Part:BBa_K808000:Experience

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User Reviews

UNIQ3bc70a23593428d4-partinfo-00000000-QINU

• 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.

UNIQ3bc70a23593428d4-partinfo-00000004-QINU