Difference between revisions of "Part:BBa K3332043"
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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 changes the conformation of hxlR which stimulating RNA polymerase to open the transcription.
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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 start the transcription.
  
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There are two binding sites (BRH1, BRH2) of hxlR, which are necessary for formaldehyde induced expression. 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, as opposed to one BRH1 and one BRH2 on the registry formaldehyde promoter (BBa_ K1334002).
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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, while there is only one BRH1 and one BRH2 in the registry formaldehyde promoter (BBa_ K1334002).
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    <figure>
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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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        <figcaption>
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        <p style="font-size:1rem">
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        </p>
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        </figcaption>
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    </figure>
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</html>
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'''Fig 1.''' Different improvements of formaldehyde promoter.
  
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[[File:2042.fig.1.png|none|500px|caption]]
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To construct this part, we moved 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).
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Fig.1 Circuit
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To construct this part, we moved formaldehyde_derivative-1 promoter (BBa_ BBa_K3332042) 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-2 promoter, formaldehyde_derivative-3, formaldehyde promoter)
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===Characterization===
 
===Characterization===
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We use Formaldehyde_derivative-2 promoter _B0034_E0020_B0015_pSB1C3 to characterize Formaldehyde_derivative-2 promoter.
 
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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]]
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Fig.2 Formaldehyde_derivative-2 promoter _B0034_E0020_B0015_pSB1C3(BBa_K3332093) digested by ''Xba'' I and ''Pst'' I (about 1557 bp)
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'''Fig 2.''' Formaldehyde_derivative-2 promoter_B0034_E0020_B0015_pSB1C3 (BBa_K3332093) digested by ''Xba'' I and ''Pst'' I (about 1557 bp).
  
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Protocol:
 
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1.Culture glycerol bacteria containing the corresponding plasmid in test tube for 12h.
 
  
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'''Protocol: '''
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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.
 
2.Add 4mL of the above bacterial solution into 200 mL LB medium and maintain the culture condition at 37 ℃ and 180 rpm.
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3.Add 0.8mM formaldehyde into each group when OD<sub>600</sub> increased to 0.6 and the culture condition is the same as before.
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3.Add 0.8mM formaldehyde into each group when OD600 increased to 0.6 and the culture condition is the same as before.
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4.Then, sampling culture in 96-well plate reader every 3 hours to measure the fluorescence intensity (ECFP) and corresponding OD<sub>600</sub> , then calculate the fluorescence / OD value of each group.
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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.
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Here is the result:
 
Here is the result:
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'''Fig 3. ''' The curve of fluorescence intensity (ECFP) /OD of pHCHO (BBa_ K1334002) and formaldehyde_derivative-2 promoter, respectively.
  
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[[File:2042.fig.4.png|none|500px|caption]]
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'''Note:''' Formaldehyde_derivative-2 promoter is equal to pHCHO(modified-2).
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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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In this figure, we can compare formaldehyde_derivative-2 promoter with the other two derivative promoters and the registry formaldehyde promoter. And we can conclude that formaldehyde_derivative-2 promoter is more sensitive to formaldehyde than the registry formaldehyde promoter 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.
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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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===Reference===
 
===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
 
[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
  
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===Sequence and Features===
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K3332043 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3332043 SequenceAndFeatures</partinfo>
  

Revision as of 17:31, 27 October 2020


Formaldehyde_derivative-2 promoter

An improvement of BBa_K1334002 by 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 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 start 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, while there is only one BRH1 and one BRH2 in the registry formaldehyde promoter (BBa_ K1334002).

Fig 1. Different improvements of formaldehyde promoter.

To construct this part, we moved 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_K3332093) digested by Xba I and Pst I (about 1557 bp).


Protocol: 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. The curve of fluorescence intensity (ECFP) /OD of pHCHO (BBa_ K1334002) and formaldehyde_derivative-2 promoter, respectively.

Note: Formaldehyde_derivative-2 promoter is equal to pHCHO(modified-2). In this figure, we can compare formaldehyde_derivative-2 promoter with the other two derivative promoters and the registry formaldehyde promoter. And we can conclude that formaldehyde_derivative-2 promoter is more sensitive to formaldehyde than the registry formaldehyde promoter 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]