Difference between revisions of "Part:BBa K3332091"

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Formaldehyde_derivative-1 promoter-ECFP-terminator

An improvement of BBa_K1334002 by enhancing the strength of the component "weak promoter". It is used 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-1 promoter to formaldehyde by observing the fluorescence intensity/OD.

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.The strong promoter BBa_J23100 is used in the formaldehyde_derivative-3 promoter rather than the weak promoter to express hxlR.

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.

Characterization

The agarose gel electrophoresis images are below：

Fig 2. Formaldehyde_derivative-1 promoter _B0034_E0020_B0015_pSB1C3(BBa_K3332091) digested by Xba I and Pst I (about 1781 bp).

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 plasmid in test tube for 12h.

2.Add 4 mL 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 the compare the sensitivity to formaldehyde of formaldehyde_derivative-1 promoter and formaldehyde promoter (BBa_ K1334002)

1.Culture glycerol bacteria containing the corresponding plasmid in test tube for 12h.

2.Add 4 mL of the above bacterial solution into 200 mL LB medium and maintain the culture condition at 37 ℃ and 180 rpm.

3.Add 0.8 mM 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 by pHCHO (BBa_ K1334002) and formaldehyde_derivative-1 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 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