Measurement

Part:BBa_K3971010

Designed by: Misaal Bedi   Group: iGEM21_IISER-Pune-India   (2021-08-12)
Revision as of 18:46, 20 October 2021 by Misaalbedi (Talk | contribs)

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cscB (sucrose permease) transport efficiency characterization with different promoters.

This composite part has been designed to characterize the sucrose transport efficiency through the cscB (sucrose permease) transporter in S. elongatus UTEX 2973. The cassette contains two homology arms to the Neutral Site 1 of the S. elongatus UTEX 2973 genome which allows for the homology-repair-based insertion of the cassette into the genome. This cassette is to be cloned into the pSL2680 plasmid [1], which is a Cpf1 based CRISPR plasmid and can be used to insert the cassette into the Neutral Site 1 using a gRNA target within that region.

The promoter in this part is PcpcB-m6 (BBa_K3971000). In order to allow for modularity in the promoter region of the cassette, two restriction sites Nde1 and BamH1 flank it, so that the promoter can be easily excised out and replaced with another. Thus one can study the effect of different promoters on the cscB transport efficiency.

Both ends of the composite part contain Kpn1 restriction sites since this cassette is designed to be cloned into the pSL2680 plasmid which has a Kpn1 site and a Sal1 site to allow for cloning of cassettes.

Figure of the composite construct for characterizing cscB transport efficiencies under different promoters. NS1a and NS1b are the two homology arms to Neutral Site 1.


References

1: Cpf1 Is A Versatile Tool for CRISPR Genome Editing Across Diverse Species of Cyanobacteria. Ungerer J, Pakrasi HB. Sci Rep. 2016 Dec 21;6:39681. doi: 10.1038/srep39681. 10.1038/srep39681 PubMed 28000776

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 2764
    Illegal PstI site found at 362
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 2577
    Illegal PstI site found at 362
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1103
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 2764
    Illegal PstI site found at 362
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 2764
    Illegal PstI site found at 362
    Illegal NgoMIV site found at 1589
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1633
    Illegal BsaI.rc site found at 152


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