Part:BBa_K3332042
Formaldehyde_derivative-1 promoter
An improvement of BBa_K1334002 by enhancing the strength of the component weak promoter. It's more sensitive to formaldehyde. We use it to test whether it has any improvement compared with BBa_K1334002.
Usage and Biology
We use this part to characterize the sensitivity of formaldehyde_derivative-2 promoter to formaldehyde by observing the fluorescence intensity/OD.
As a DNA binding protein, hxlR is the transcriptional activator of the hxlAB operon from Bacillus subtilis. The possible mechanism of formaldehyde induced expression is that the formaldehyde changes the conformation of hxlR which can stimulate RNA polymerase to open the transcription.
Therefore, we increased the amount of binding sites to improve the sensitivity of the formaldehyde promoter.There are two BRH1 and two BRH2 in the formaldehyde_derivative-2 promoter, both of which only has one copy in the registry promoter (BBa_K1334002).
To construct this part, we inserted formaldehyde_derivative-2 promoter (BBa_K3332043) and RBS-ECFP-T(BBa_E0420) into the expression vector pSB1C3 by standard assembly. Then the ligation mixture was transformed into E. coli BL21(DE3) to compare the sensitivity of formaldehyde with other promoters.( formaldehyde_derivative-1 promoter, formaldehyde_derivative-3, formaldehyde promoter)
Characterization
The agarose gel electrophoresis images are below：
Fig 2. Formaldehyde_derivative-2 promoter_B0034_E0020_B0015_pSB1C3(BBa_K3332091) digested by Xba I and Pst I (about 1557 bp)
Protocol:
1.Culture glycerol bacteria containing the corresponding plasmid in test tube for 12h.
2.Add 4ml of the above bacterial solution into 200 mL LB medium and maintain the culture condition at 37 ℃ and 180 rpm.
3.Add 0.8mM formaldehyde into each group when OD600 increased to 0.6 and the culture condition is the same as before.
4.Then, sampling culture in 96-well plate reader every 3 hours to measure the fluorescence intensity (ECFP) and corresponding OD600, then calculate the fluorescence / OD value of each group.
Here is the result:
Fig 3. The curve of fluorescence intensity (ECFP) /OD by pHCHO (BBa_K1334002) and formaldehyde_derivative-2 promoter
In this figure,by comparing formaldehyde_derivative-2 promoter with the other two derivative promoters and the registry one, we can see that formaldehyde_derivative-2 promoter is more sensitive to formaldehyde than the registry one and formaldehyde_derivative-1 promoter. So increasing the amount of binding sites is a more effective way to improve formaldehyde promoter even than replacing weak promoter with strong promoter.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 422
Illegal NheI site found at 445 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Reference
[1]Yurimoto, H., Hirai, R., Matsuno, N., Yasueda, H., Kato, N. and Sakai, Y. (2005), HxlR, a member of the DUF24 protein family, is a DNA‐binding protein that acts as a positive regulator of the formaldehyde‐inducible hxlAB operon in Bacillus subtilis. Molecular Microbiology, 57: 511-519. doi:10.1111/j.1365-2958.2005.04702.x
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