Difference between revisions of "Part:BBa K3332090"

Revision as of 21:05, 27 October 2020

Formaldehyde promoter-ECFP-terminator

It can expresse cyan fluorescent protein according to the concentration of formaldehyde.It is used to characterize the function of the improved promoter.

Usage and Biology

We use this part to be the negative control to compare with the three improved formaldehyde promoters as followed pictures by observing the fluorescence intensity/OD.

Fig 1. Different improvements of formaldehyde promoter.

Characterization

The agarose gel electrophoresis images are below：

Fig 2. pHCHO_B0034_E0020_B0015_pSB1C3 (BBa_K3332090) digested by Xba I and Pst I(about 1507 bp).

Protocol:

1.Culture glycerol bacteria containing the corresponding plasmids 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. The curve of fluorescence intensity (ECFP) /OD by pHCHO (BBa_ K1334002) and formaldehyde_derivative promoters.

We discovered that the three improved formaldehyde promoters are more sensitive than the registry formaldehyde promoter (BBa_K1334002). What’s more, formaldehyde_derivative-3 promoter(BBa_K3332044) is the most sensitive promoter for formaldehyde.

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