Difference between revisions of "Part:BBa K5520004"
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===Usage and Biology===
 
===Usage and Biology===
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Plasmid construction
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==Plasmid construction==
 
PET-28a linearized vector (5371 bp) was amplified using PET-28a plasmid from the laboratory conserved as template and ZT-F and ZT-R as primers. Using CDA-F and CDA-R as primers, we amplified PET-28a-CDA as a vector using Gibson assembly. The T7 promoter、RBS、 lac operator, CDA gene, 6×His tag, kana antibiotic gene, and T7 terminator on the vector PET-28a were already inserted before experiments.
 
PET-28a linearized vector (5371 bp) was amplified using PET-28a plasmid from the laboratory conserved as template and ZT-F and ZT-R as primers. Using CDA-F and CDA-R as primers, we amplified PET-28a-CDA as a vector using Gibson assembly. The T7 promoter、RBS、 lac operator, CDA gene, 6×His tag, kana antibiotic gene, and T7 terminator on the vector PET-28a were already inserted before experiments.
  
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<center><img src = "https://static.igem.wiki/teams/5208/parts/00-2.png" style = "width:300px"></center>
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<center><img src = "https://static.igem.wiki/teams/5520/parts/1.png" style = "width:300px"></center>
 
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<figcaption><center>Figure 2. Gel electrophoresis of PCR product of AiiA. </center></figcaption>
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<h2>Detection of PET-28a-CDA</h2>
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<h3>1. SDS-PAGE analysis of CDA protein</h3>
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<center><img src = "https://static.igem.wiki/teams/5520/parts/1.png" style = "width:300px"></center>
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<figcaption><center>Figure 1. Gel electrophoresis of PCR product of AiiA. </center></figcaption>
 
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Revision as of 04:10, 30 September 2024


pT7-LacO-His-CDA

This part contains BBa_K2406020, BBa_B0034, BBa_K5520002 and BBa_K731721. BBa_B0034 contains a natural ribosome binding site (RBS) to facilitate the expression of downstream genes. By inducing efficient expression of the CDA protein through the addition of IPTG, the protein can subsequently be purified using the His tag for further enzyme activity tests and product polymerization.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 149
    Illegal NheI site found at 897
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 182
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 641


Usage and Biology

Plasmid construction

PET-28a linearized vector (5371 bp) was amplified using PET-28a plasmid from the laboratory conserved as template and ZT-F and ZT-R as primers. Using CDA-F and CDA-R as primers, we amplified PET-28a-CDA as a vector using Gibson assembly. The T7 promoter、RBS、 lac operator, CDA gene, 6×His tag, kana antibiotic gene, and T7 terminator on the vector PET-28a were already inserted before experiments.

Detection of PET-28a-CDA

1. SDS-PAGE analysis of CDA protein

Figure 1. Gel electrophoresis of PCR product of AiiA.