Difference between revisions of "Part:BBa K3332043"

Revision as of 20:01, 27 October 2020 (view source) PolarisBi (Talk | contribs) ← Older edit		Revision as of 21:23, 27 October 2020 (view source) Nico123 (Talk | contribs) Newer edit →
Line 22:		Line 22:
	'''Fig 1.''' Different improvements of formaldehyde promoter		'''Fig 1.''' Different improvements of formaldehyde promoter
		+	===Characterization===
		+	We use ECFP as the reporter gene to characterize the improvement.
−	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.( formaldehyde_derivative-1 promoter, formaldehyde_derivative-3, formaldehyde promoter)
−
−	===Characterization===
	The agarose gel electrophoresis images are below：		The agarose gel electrophoresis images are below：
	[[File:2043.fig.2.png|none|500px|caption]]		[[File:2043.fig.2.png|none|500px|caption]]

---

Revision as of 21:23, 27 October 2020

Formaldehyde_derivative-2 promoter

An improvement of BBa_K1334002 by adding the binding sites to make it be more sensitive to formaldehyde than 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 can stimulate RNA polymerase to open the transcription.

Therefore, we increased the amount of binding sites to improve the sensitivity of the formaldehyde promoter.There are two BRH1 and two BRH2 in the formaldehyde_derivative-2 promoter, both of which only has one copy in the registry promoter (BBa_K1334002).

Fig 1. Different improvements of formaldehyde promoter

Characterization

We use ECFP as the reporter gene to characterize the improvement.

The agarose gel electrophoresis images are below：

Fig 2. Formaldehyde_derivative-2 promoter_B0034_E0020_B0015_pSB1C3(BBa_K3332091) digested by Xba I and Pst I (about 1557 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-2 promoter

In this figure,by comparing formaldehyde_derivative-2 promoter with the other two derivative promoters and the registry one, we can see that formaldehyde_derivative-2 promoter is more sensitive to formaldehyde than the registry one and formaldehyde_derivative-1 promoter. So increasing the amount of binding sites is a more effective way to improve formaldehyde promoter even than replacing weak promoter with strong 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