Difference between revisions of "Part:BBa K3332091"
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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.
+
We use this part to characterize the sensitivity of formaldehyde_derivative-1 promoter to formaldehyde by observing the fluorescence intensity/OD.
  
−
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)
+
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.
  
 +
There are two binding sites (BRH1, BRH2) of hxlR, which are necessary for formaldehyde induced expression. Through ''in vitro'' experiments, the reaction intensity of the binding becomes stronger and stronger as the increasing of the concentration of hxlR. Therefore, we enhanced the expression of hxlR to improve the sensitivity of formaldehyde promoter. The strong promoter BBa_J23100 is used in the formaldehyde_derivative-1 promoter to replace the weak promoter to express hxlR.
 
[[File:2042.fig.1.png|none|500px|caption]]
 
[[File:2042.fig.1.png|none|500px|caption]]
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Fig.1 Circuit
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'''Fig 1.''' Different improvements of formaldehyde promoter.
  
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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 promoter.( formaldehyde_derivative-2 promoter, formaldehyde_derivative-3, formaldehyde promoter)
+
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).
  
 
===Characterization===
 
===Characterization===
 
The agarose gel electrophoresis images are below：
 
The agarose gel electrophoresis images are below：
 
[[File:2042.fig.2.png|none|500px|caption]]
 
[[File:2042.fig.2.png|none|500px|caption]]
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Fig.2 Formaldehyde_derivative-1 promoter _B0034_E0020_B0015_pSB1C3(BBa_K3332091) digested by ''Xba'' I and ''Pst'' I (about 1781 bp)
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'''Fig 2.''' Formaldehyde_derivative-1 promoter _B0034_E0020_B0015_pSB1C3(BBa_K3332091) digested by ''Xba'' I and ''Pst'' I (about 1781 bp).
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Protocol:
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'''Protocol:'''
 
Part one: to the compare the strength of weak promoter on the registry formaldehyde promoter (BBa_ K1334002) and J23100
 
Part one: to the compare the strength of weak promoter on the registry formaldehyde promoter (BBa_ K1334002) and J23100
  
 
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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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 4 mL 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.Measure the fluorescence intensity (GFP) and corresponding OD600, then calculate the fluorescence / OD value of each group.
+
3.Measure the fluorescence intensity (GFP) and corresponding OD<sub>600</sub> , then calculate the fluorescence / OD value of each group.
  
 
Part two: to the compare the sensitivity to formaldehyde of formaldehyde_derivative-1 promoter and formaldehyde promoter (BBa_ K1334002)
 
Part two: to the compare the sensitivity to formaldehyde of formaldehyde_derivative-1 promoter and formaldehyde promoter (BBa_ K1334002)
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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.
  
−
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 4 mL 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 OD600 increased to 0.6 and the culture condition is the same as before.
+
3.Add 0.8 mM 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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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.
+
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.
  
 
Here is the result:
 
Here is the result:
 
[[File:2042.fig.3.png|none|500px|caption]]
 
[[File:2042.fig.3.png|none|500px|caption]]
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'''Fig 3.''' Fluorescence intensity (GFP) /OD expressed by weak promoter, J23100 and blank, respectively. Data are collected and analyzed according to iGEM standard data analysis form after 6 hours of induction.
  
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Fig.3 Fluorescence intensity (GFP) /OD expressed by weak promoter, J23100 and blank. Data are collected and analyzed according to iGEM standard data analysis form after 6 hours of induction.
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'''note:''' Weak promoter is the promoter on the registry formaldehyde promoter (BBa_ K1334002).
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ps: weak promoter is the promoter on the registry formaldehyde promoter (BBa_ K1334002)
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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.4 The curve of fluorescence intensity (ECFP) /OD by pHCHO (BBa_ K1334002) and formaldehyde_derivative-1 promoter
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'''Fig 4''' The curve of fluorescence intensity (ECFP) /OD by pHCHO (BBa_ K1334002) and formaldehyde_derivative-1 promoter.
  
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We discovered that formaldehyde_derivative-1 promoter is more sensitive to the registry formaldehyde promoter (BBa_ K1334002).
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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-1 promoter with the other two derivative promoters. And we can conclude from the latter figure that formaldehyde_derivative-1 promoter is more sensitive to formaldehyde than the registry formaldehyde promoter. So replacing weak promoter with strong promoter BBa_J23100 is an effective way to improve formaldehyde promoter.
  
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===Reference===
 
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[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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<partinfo>BBa_K3332091 parameters</partinfo>
 
<partinfo>BBa_K3332091 parameters</partinfo>
 
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===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

Revision as of 19:34, 27 October 2020


Formaldehyde_derivative-1 promoter-ECFP-terminator

An improvement of BBa_K1334002 by enhancing the strength of the component "weak promoter". 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-1 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.

There are two binding sites (BRH1, BRH2) of hxlR, which are necessary for formaldehyde induced expression. Through in vitro experiments, the reaction intensity of the binding becomes stronger and stronger as the increasing of the concentration of hxlR. Therefore, we enhanced the expression of hxlR to improve the sensitivity of formaldehyde promoter. The strong promoter BBa_J23100 is used in the formaldehyde_derivative-1 promoter to replace the weak promoter to express hxlR.

caption

Fig 1. Different improvements of formaldehyde promoter.

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).

Characterization

The agarose gel electrophoresis images are below：

caption

Fig 2. Formaldehyde_derivative-1 promoter _B0034_E0020_B0015_pSB1C3(BBa_K3332091) digested by Xba I and Pst I (about 1781 bp).

Protocol: Part one: to the compare the strength of weak promoter on the registry formaldehyde promoter (BBa_ K1334002) and J23100

1.Culture glycerol bacteria containing the corresponding plasmid 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.Measure the fluorescence intensity (GFP) and corresponding OD600 , then calculate the fluorescence / OD value of each group.

Part two: to the compare the sensitivity to formaldehyde of formaldehyde_derivative-1 promoter and formaldehyde promoter (BBa_ K1334002)

1.Culture glycerol bacteria containing the corresponding plasmid 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:

caption

Fig 3. Fluorescence intensity (GFP) /OD expressed by weak promoter, J23100 and blank, respectively. 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).

caption

Fig 4 The curve of fluorescence intensity (ECFP) /OD by pHCHO (BBa_ K1334002) and formaldehyde_derivative-1 promoter.

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-1 promoter with the other two derivative promoters. And we can conclude from the latter figure that formaldehyde_derivative-1 promoter is more sensitive to formaldehyde than the registry formaldehyde promoter. So replacing weak promoter with strong promoter BBa_J23100 is an effective way to improve formaldehyde promoter.


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