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

(→‎Design Notes)
(→‎Design Notes)
 
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===Design Notes===
 
===Design Notes===
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We delete the dsbG coding and following sequence next to dsbG reverse primer, making the functional sequence shorter, so that PCR can be more easy and efficient. The length of remained parts is about half of the original  part: [https://parts.igem.org/Part:BBa_K362001 K362001].
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The recA promoter fragment was cloned from the genome of E.coli K12 MG1655 strain as a template.
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+
−
Besides, AhpCp2D1 has a Pst1 site in the sequence. We succesfully mutated the Pst1 site and make Assembly Compatibility better.
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====Primers For PCR====
 
====Primers For PCR====
 
{| class="wikitable" style="margin: 1em auto 1em auto;"  
 
{| class="wikitable" style="margin: 1em auto 1em auto;"  
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|align="center"|AhpCp2_DsbGp_AhpCp_FP  
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|align="center"|PCR amplification of upstream primers, addition of EcoRI restriction sites  
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|gcttctagag caatcgcttttactggagcaggaa
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|5’-AGGAgaattcCAGATGATCGGCGTACGCG-3’
 
|-
 
|-
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|align="center"|AhpCp_RP
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|align="center"|PCR amplification of downstream primers, addition of XbaI restriction sites
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|gga ctgcagcggccgctactagta ctatacttcctccgtgttttcgatgaga
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|5’-CATGtctagaTTTTACTCCTGTCATGCCGGG-3’
 
|}
 
|}
  
 
{| class="wikitable" style="margin: 1em auto 1em auto;" |
 
{| class="wikitable" style="margin: 1em auto 1em auto;" |
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!align="center" colspan=2|ahpC promoter([https://parts.igem.org/Part:BBa_K362001 K362001])has a Pst1 site in the sequence. <br />We designed this part AhpCp2D1, and succesfully mutated the Pst1 site (ctgcag->ctacag).
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!align="center" colspan=2|PCR system
 
|-  
 
|-  
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|align="center"|ahpCp_mut_FP  
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|align="center"|enzyme  
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|cacctcagcctacaggcaggcactgaa
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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
 
|-
 
|-
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|align="center"|ahpCp_mut_RP
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|align="center"|ddH2O
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|ttcagtgcctgcctgtaggctgaggtg
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|align="center"|to 50μL
 
|}
 
|}
  
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|-  
 
|-  
 
|align="center"|94
 
|align="center"|94
−
|align="center"|120
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|align="center"|180
 
|align="center"| --
 
|align="center"| --
 
|-  
 
|-  
 
|align="center"|94
 
|align="center"|94
−
|align="center"|15
+
|align="center"|30
−
|align="center" rowspan=3|35
+
|align="center" rowspan=3|30
 
|-  
 
|-  
−
|align="center"|53
+
|align="center"|56
 
|align="center"|30
 
|align="center"|30
 
|-  
 
|-  
 
|align="center"|72
 
|align="center"|72
−
|align="center"|30
+
|align="center"|60
 
|-  
 
|-  
 
|align="center"|72
 
|align="center"|72

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.