Difference between revisions of "Part:BBa K3332090"

Revision as of 19:22, 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 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

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

@@ Line 6: / Line 6: @@
 ===Usage and Biology===
-[[File:2042.fig.1.png|none|500px|caption]]
+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 Circuit
+[[File:2042.fig.1.png|none|600px|caption]]
-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 promoter, formaldehyde_derivative-2, formaldehyde promoter)
+'''Fig 1.''' Different improvements of formaldehyde promoter.
 ===Characterization===