Part:BBa_K3332044
Formaldehyde_derivative-3 promoter
An improvement of BBa_K1334002 by replacing weak promoter with strong promoter BBa_J23100 and adding the binding sites. It's more sensitive to formaldehyde than BBa_K1334002.
Usage and Biology
As a DNA binding protein, hxlR serves as a transcriptional activator of the hxlAB operon from Bacillus subtilis. The formaldehyde changes the conformation of hxlR, stimulating RNA polymerase to open the transcription.
It is reported that the reaction intensity of complex binding becomes stronger and stronger with the increasing of the concentration of hxlR. Similar to formaldehyde_derivative-1 promoter, the strong promoter BBa_J23100 is used in the formaldehyde_derivative-3 promoter rather than the weak promoter to express hxlR. Meanwhile, there are two BRH1 and two BRH2 in the formaldehyde_derivative-3 promoter, both of which only has one copy in the registry promoter (BBa_K1334002).Thus, the improvement of formaldehyde_derivative-3 promoter is the combination of formaldehyde_derivative-1 promoter and formaldehyde_derivative-2 promoter.
To construct this part, we inserted formaldehyde_derivative-3 promoter (BBa_K3332044) 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-2 promoter)
Characterization
The agarose gel electrophoresis images are below：
Fig 2. Formaldehyde_derivative-3 promoter _B0034_E0020_B0015_pSB1C3(BBa_K3332092) digested by Xba I and Pst I (about 1543 bp)
Protocol:
Part one: to compare the strength of weak promoter on the registry formaldehyde promoter (BBa_K1334002) and BBa_J23100
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. Measure the fluorescence intensity (GFP) and corresponding OD600, then calculate the fluorescence / OD value of each group.
Part two: to compare the sensitivity to formaldehyde of formaldehyde_derivative-3 promoter and formaldehyde promoter (BBa_K1334002)
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. Fluorescence intensity (GFP) /OD expressed by weak promoter, J23100 and blank. Data are collected and analyzed according to iGEM standard data analysis form after 6 hours of induction.
Note: weak promoter is the promoter on the registry formaldehyde promoter (BBa_K1334002)
Fig 4. The curve of fluorescence intensity (eCFP) /OD by pHCHO (BBa_K1334002) and formaldehyde_derivative-3 promoter.
In the former figure, we can see that the strong promoter BBa_J23100 group has a higher relative fluorescence intensity than the weak promoter present in the registry formaldehyde promoter, and in the latter figure, we can compare formaldehyde_derivative-3 promoter with the other two derivative promoters. We can conclude from the latter figure that formaldehyde_derivative-3 promoter is more sensitive to formaldehyde than the registry one. What’s more, formaldehyde_derivative-3 promoter is the most sensitive promoter among them. The detail about formaldehyde_derivative-1 promoter and formaldehyde_derivative-2 promoter can be seen in BBa_K3332042 and BBa_K3332043.
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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