Difference between revisions of "Part:BBa K3332093"
 
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===Usage and Biology===
 
===Usage and Biology===
  
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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.
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We use this part to characterize the sensitivity of formaldehyde_derivative-2 promoter to formaldehyde by observing the fluorescence intensity/OD.
  
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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)
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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.
  
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[[File:2042.fig.1.png|none|500px|caption]]
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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).
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Fig.1 Circuit
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To construct this part, we moved formaldehyde_derivative-2 promoter (BBa_ 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 BH21 to compare the sensitivity of formaldehyde with other promoter.( formaldehyde_derivative-1 promoter, formaldehyde_derivative-3, formaldehyde promoter)
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        <img src="https://2020.igem.org/wiki/images/0/0d/T--XMU-China--XMU-China_2020-pHCHO_improve_1%2C2%2C3.png" width="60%" style="float:center">
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'''Fig 1.''' Different improvements of formaldehyde promoter
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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===
 
===Characterization===
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[[File:2043.fig.2.png|none|500px|caption]]
 
[[File:2043.fig.2.png|none|500px|caption]]
  
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Protocol:  
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'''Fig 2.''' Formaldehyde_derivative-2 promoter_B0034_E0020_B0015_pSB1C3(BBa_K3332091) digested by ''Xba'' I and ''Pst I (about 1557 bp)
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'''Protocol: '''
  
 
1.Culture glycerol bacteria containing the corresponding plasmid in test tube for 12h.
 
1.Culture glycerol bacteria containing the corresponding plasmid in test tube for 12h.
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Here is the result:
 
Here is the result:
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[[File:2042.fig.4.png|none|500px|caption]]
 
[[File:2042.fig.4.png|none|500px|caption]]
  
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Fig.3 The curve of fluorescence intensity (ECFP) /OD by pHCHO (BBa_ K1334002) and formaldehyde_derivative-2 promoter
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'''Fig 3.''' The curve of fluorescence intensity (ECFP) /OD by pHCHO (BBa_K1334002) and formaldehyde_derivative-2 promoter
  
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We discovered that formaldehyde_derivative-2 promoter is more sensitive to the registry formaldehyde promoter (BBa_ K1334002).
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In this figure,by comparing formaldehyde_derivative-2 promoter with the other two derivative promoters and the registry one, we can see
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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.
  
 
===Reference===
 
===Reference===

Latest revision as of 19:59, 27 October 2020


Formaldehyde_derivative-2 promoter-ECFP-terminator

An improvement of BBa_K1334002 by adding the binding sites.It's more sensitive to formaldehyde.It is used to test whether it has any improvement compared with BBa_K1334002

Usage and Biology

We use this part to characterize the sensitivity of formaldehyde_derivative-2 promoter to formaldehyde by observing the fluorescence intensity/OD.

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


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：

caption

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:

caption

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.

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]