Difference between revisions of "Part:BBa K3332090"

 
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===Usage and Biology===
 
===Usage and Biology===
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[[File:2042.fig.1.png|none|500px|caption]]
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Fig.1 Circuit
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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 BH21 to compare the sensitivity of formaldehyde with other promoter.( formaldehyde_derivative-1 promoter, formaldehyde_derivative-2, formaldehyde promoter)
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===Characterization===
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The agarose gel electrophoresis images are below：
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[[File:T--XMU-2090.fig.2 ger.png|none|500px|caption]]
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Fig.2 pHCHO_B0034_E0020_B0015_pSB1C3 (BBa_K3332090) digested by ''Xba'' I and ''Pst'' I
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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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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 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 OD600, then calculate the fluorescence / OD value of each group.
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Here is the result:
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[[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-3 promoter
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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.
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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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Revision as of 02:39, 25 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.

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]