Difference between revisions of "Part:BBa K3332044"

Revision as of 10:27, 27 October 2020

Formaldehyde_derivative-3 promoter

An improvement of BBa_K1334002 by both enhancing the strength and adding the binding sites. 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 the hxlAB operon from Bacillus subtilis. The possible mechanism of formaldehyde induced expression is that the formaldehyde changes the conformation of hxlR which stimulating RNA polymerase to open the transcription.

There are two binding sites (BRH1, BRH2) of hxlR, which are necessary for formaldehyde induced expression. Through in vitro experiments, the higher the concentration of hxlR, the stronger the reaction intensity of the binding. Therefore, we enhanced the expression of hxlR to improve the sensitivity of formaldehyde promoter. J23100 is used in the formaldehyde_derivative-3 promoter to express the hxlR, as opposed to weak promoter on the registry formaldehyde promoter (BBa_ K1334002). In addition, there are two BRH1 and two BRH2 in the formaldehyde_derivative-3 promoter, which is only one in the registry formaldehyde promoter (BBa_ K1334002).

Fig.1 Circuit

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

Characterization

We use Formaldehyde_derivative-3 promoter_B0034_E0020_B0015_pSB1C3 to characterize Formaldehyde_derivative-3 promoter.

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:

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-3 promoter

We discovered that formaldehyde_derivative-3 promoter is more sensitive to the registry formaldehyde promoter (BBa_K1334002). What’s more, formaldehyde_derivative-3 promoter is the most sensitive promoter for formaldehyde. The detail about formaldehyde_derivative-1 promoter and formaldehyde_derivative-2 promoter can be seen in BBa_K3332042 and BBa_K3332043.

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

Sequence and Features