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

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 change 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-1 promoter to express the hxlR, as opposed to weak promoter on the registry formaldehyde promoter (BBa_ K1334002)

Fig.1 Circuit

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

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 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 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 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.

ps: 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

We discovered that formaldehyde_derivative-1 promoter is more sensitive to the registry formaldehyde promoter (BBa_ K1334002).

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