Part:BBa_K3332044

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 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 start the transcription.

Through in vitro experiments, the reaction intensity of the binding becomes stronger and stronger as the increasing of the concentration of hxlR. As the same as formaldehyde_derivative-1 promoter, the strong promoter BBa_J23100 is used in the formaldehyde_derivative-3 promoter to replace the weak promoter to express hxlR. But at the same time, there are two BRH1 and two BRH2 in the formaldehyde_derivative-3 promoter, while there is only one BRH1 and one BRH2 in the registry formaldehyde promoter (BBa_ K1334002). So the formaldehyde_derivative-3 promoter is the combination of formaldehyde_derivative-1 promoter and formaldehyde_derivative-2 promoter.

Fig 1. Different improvements of formaldehyde promoter.

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 promoters(formaldehyde_derivative-1 promoter, formaldehyde_derivative-2, formaldehyde promoter).

Characterization

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:

Part one: to compare the strength of weak promoter on the registry formaldehyde promoter (BBa_K1334002) and BBa_J23100

1.Culture glycerol bacteria containing the corresponding plasmids 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 compare the sensitivity to formaldehyde of formaldehyde_derivative-3 promoter and formaldehyde promoter (BBa_K1334002)

1.Culture glycerol bacteria containing the corresponding plasmids 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, repectively. Data are collected and analyzed according to iGEM standard data analysis form after 6 hours of induction.

Note: 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-3 promoter.

Note: Formaldehyde_derivative-3 promoter is equal to pHCHO(modified-3).

In the former figure, we can see that the strong promoter BBa_J23100 group has a higher relative fluorescence intensity than the weak promoter in the registry formaldehyde promoter group, and in the latter figure, we can compare formaldehyde_derivative-3 promoter with the other two derivative promoters. And we can conclude from the latter figure that formaldehyde_derivative-3 promoter is more sensitive to formaldehyde than the registry formaldehyde promoter. 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.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 422
  Illegal NheI site found at 445
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

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