Difference between revisions of "Part:BBa K3020001:Design"

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===Design Notes===
 
===Design Notes===
 +
The recA promoter fragment was cloned from the genome of E.coli K12 MG1655 strain as a template.
  
−
   
+
====Primers For PCR====
 +
{| class="wikitable" style="margin: 1em auto 1em auto;"
 +
|align="center"|PCR amplification of upstream primers, addition of EcoRI restriction sites 
 +
|5’-AGGAgaattcCAGATGATCGGCGTACGCG-3’
 +
|-
 +
|align="center"|PCR amplification of downstream primers, addition of XbaI restriction sites
 +
|5’-CATGtctagaTTTTACTCCTGTCATGCCGGG-3’
 +
|}
  
 +
{| class="wikitable" style="margin: 1em auto 1em auto;" |
 +
!align="center" colspan=2|PCR system
 +
|-
 +
|align="center"|enzyme 
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|align="center"|2xPCR Mastermix（Tiangen）
 +
|-
 +
|align="center"|Upstream and downstream primers 
 +
|align="center"|2μL
 +
|-
 +
|align="center"|Genomic template 
 +
|align="center"|2μL
 +
|-
 +
|align="center"|ddH2O
 +
|align="center"|to 50μL
 +
|}
  
 +
====PCR Time Protocol====
 +
{| class="wikitable" style="margin: 1em auto 1em auto;" |
 +
!align="center"|Temperature(°C)
 +
!align="center"|Time(sec)
 +
!align="center"|Number of Cycles
 +
|-
 +
|align="center"|94
 +
|align="center"|180
 +
|align="center"| --
 +
|-
 +
|align="center"|94
 +
|align="center"|30
 +
|align="center" rowspan=3|30
 +
|-
 +
|align="center"|56
 +
|align="center"|30
 +
|-
 +
|align="center"|72
 +
|align="center"|60
 +
|-
 +
|align="center"|72
 +
|align="center"|300
 +
|align="center"| --
 +
|}
  
 
===Source===
 
===Source===
Line 17: Line 64:
  
 
===References===
 
===References===
 +
Norman A , Hestbjerg Hansen L , Sørensen, Søren J. Construction of a ColD cda Promoter-Based SOS-Green Fluorescent Protein Whole-Cell Biosensor with Higher Sensitivity toward Genotoxic Compounds than Constructs Based on recA, umuDC, or sulA Promoters[J]. Appl Environ Microbiol, 2005, 71(5):2338-2346.

Latest revision as of 06:45, 8 September 2019


recA promoter-Respond to sos response and initiate expression of downstream repair proteins


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]


Design Notes

The recA promoter fragment was cloned from the genome of E.coli K12 MG1655 strain as a template.

Primers For PCR

PCR amplification of upstream primers, addition of EcoRI restriction sites 5’-AGGAgaattcCAGATGATCGGCGTACGCG-3’
PCR amplification of downstream primers, addition of XbaI restriction sites 5’-CATGtctagaTTTTACTCCTGTCATGCCGGG-3’
PCR system
enzyme 2xPCR Mastermix（Tiangen）
Upstream and downstream primers 2μL
Genomic template 2μL
ddH2O to 50μL

PCR Time Protocol

Temperature(°C) Time(sec) Number of Cycles
94 180 --
94 30 30
56 30
72 60
72 300 --

Source

The recA promoter fragment was cloned from the genome of E.coli K12 MG1655 strain as a template.Please note that the recA promoter sequences from different genomes are not consistent.

References

Norman A , Hestbjerg Hansen L , Sørensen, Søren J. Construction of a ColD cda Promoter-Based SOS-Green Fluorescent Protein Whole-Cell Biosensor with Higher Sensitivity toward Genotoxic Compounds than Constructs Based on recA, umuDC, or sulA Promoters[J]. Appl Environ Microbiol, 2005, 71(5):2338-2346.