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.
To prevent transcription during amplification, we made new constructs containing the promoter BBa_K808000. BBa_K808000 is an arabinose inducible promoter that was reported to have very low leakage. We characterized 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.
We used Promega's E. coli S30 extract system for circular DNA as our cell free system.
We characterized BBa_K808000, an arabinose inducible promoter. We measured both its leakiness and response to varying arabinose concentrations.
To characterize the promoter's leakiness, we amplified our constructs containing BBa_K808000 in E. coli. We then performed plasmid minipreps to extract circular DNA and sequenced this DNA.
Sequencing analysis showed that we successfully amplified our part BBa_K2206006 with no fidelity errors. This composite part contains BBa_K808000 (the promoter), BBa_K2206000 (our 15b-5p toehold switch) and BBa_E0040 (GFP). Therefore BBa_K808000, the promoter, 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 mutations in the promoter for part BBa_K2206007. This composite part contains BBa_K808000 (the promoter), BBa_K2206001 (our 27b-3p toehold switch) and BBa_E0040 (GFP). Therefore 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.
Response to varying arabinose concentrations
To characterize the promoter's response to varying arabinose concentrations, we prepared a cell free system containing BBa_K808000 and incubated it for 10 hours at 30°C. We then added 1 μl of arabinose at the concentrations of: 0.05%, 0.1%, 0.5%, 1% and 2% and measured fluorescence every 10 minutes for 10 hours at 30°C.
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.
We used this GFP part as a reporter protein in a cell free system. We found that there was observable fluorescence from this part, and experienced no issues with expressing it in our cell free system.