- Human Practices
As mentioned in the design page, the formaldehyde promoter is one of the parts in our kill switch system. However, during our experiments, we found that the sensitivity of the current formaldehyde promoter is not good enough. Therefore, we are trying to find a way to improve the promoter.
The formaldehyde promoter BBa_K1334002 contains hxlR coding sequence, a weak promoter, the formaldehyde promoter and two hxlR protein binding sites. Therefore, we figured out three ways below to improve its function.
Firstly, we changed the weak promoter to J23100, which is considered as a strong promoter in the Anderson family, and built BBa_K3332042.
Secondly, we added two more hxlR binding sites, creating BBa_K3332043.
Thirdly, we combined those two methods mentioned above and built BBa_K3332044, whose promoter has been changed and the binding sites are added.
Fig 1. Gene circuit of formaldehyde promoter(BBa_K1334002).
After designing different improvement of Formaldehyde promoters, we verified together with the
original formaldehyde promoter.
Fig 2. Different improvement of Formaldehyde promoters
Three types of promoter improvements were designed as above. Firstly we’ve characterized the strength of J23100 and weak promoter by measuring the fluorescence/OD600 of J23100-B0034-eCFP and weak promoter-B0034-eCFP, promoter J23100 is far more stronger than weak promoter.
Fig 3. Comparison of the strength of J23100 promoter and weak promoter by measuring the fluorescence/OD600
As the figure shown above, J23100 promoter can be used to enhance the strength of formaldehyde promoter as a strong promoter. However, the result of J23100 alternation was not significantly stronger than that compared to the original pHCHO. Therefore, we came out the other two improved version of pHCHO by adding extra binding site as well as both extra binding site and J23100, as the figure shown below.
Table1. Three version of modified pHCHO.
Then, the three version of promoters were jointed with fluorescence protein ECFP, and we detected the fluorescence of ECFP as an indication of promoter strength after formaldehyde induction, which activated the promoters and downstream gene expression.
Fig 4. Fluorescence intensity/OD600 after inducing with 0.8 mM HCHO in 18 hours. Data are collected and analyzed according to iGEM standard data analysis.
From this figure, we can see that the fluorescence/OD600 of the induction group increased, but the improved promoters(modified-1~3) are more obvious than the registry formaldehyde promoter (BBa_ K1334002).
Among the three modified promoters, the third modified version appeared to be the strongest one, showed the highest ECFP fluorescence intensity. That is to say the improved Formaldehyde promoters has a higher responsiveness to HCHO, which means that our three kinds of improvements work indeed.