Difference between revisions of "Part:BBa K3332042"

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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

As a DNA binding protein, hxlR is the transcriptional activator of 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 start the transcription.

There are two binding sites (BRH1, BRH2) of hxlR, which are necessary for formaldehyde induced expression. Through in vitro experiments, the reaction intensity of the binding becomes stronger and stronger as the increasing of the concentration of hxlR. Therefore, we enhanced the expression of hxlR to improve the sensitivity of formaldehyde promoter. The strong promoter BBa_J23100 is used in the formaldehyde_derivative-1 promoter to replace the weak promoter to express hxlR.

To construct this part, we moved formaldehyde_derivative-1 promoter (BBa_K3332042) 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-2 promoter, formaldehyde_derivative-3 formaldehyde promoter).

Characterization

The agarose gel electrophoresis images are below：

Protocol:

Part one: to the compare the strength of weak promoter on the registry formaldehyde promoter (BBa_K1334002) and J23100

1.Culture glycerol bacteria containing the corresponding plasmids 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-1 promoter and formaldehyde promoter (BBa_K1334002)

1.Culture glycerol bacteria containing the corresponding plasmids 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, respectively. 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 of pHCHO (BBa_ K1334002) and formaldehyde_derivative-1 promoter.

Note: Formaldehyde_derivative-1 promoter is equal to pHCHO(modified-1).

In the former figure, we can see that the strong promoter BBa_J23100 group has a higher relative fluorescence intensity than the weak promoter in the registry formaldehyde promoter group. And in the latter figure, we can compare formaldehyde_derivative-1 promoter with the other two derivative promoters. And we can conclude from the latter figure that formaldehyde_derivative-1 promoter is more sensitive to formaldehyde than the registry formaldehyde promoter. So replacing weak promoter with strong promoter BBa_J23100 is an effective way to improve formaldehyde promoter.

Sequence and Features

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