Difference between revisions of "Part:BBa K3219002"

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===Usage===
 
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This part can be used by transforming it directly into Microcystis Aeruginosa UTEX 2388 via electroporation or naturual transformation. Our team used the protocols provided by Au Yang Qin <ref>章军, 徐虹, 楼士林, 欧阳青. Blue-green alga shuttle plasmid expression vector and method for expressing thymison 'alpha' 1. Thesis. Xia Men: Xia Men University, 1999. </ref> Nermin Adel El Semary<ref>, Semary, Nermin Adel El. “Optimized electroporation-induced transformation in Microcystis aeruginosa PCC7806.” Biotechnol. Agron. Soc. Environ (2010): 149-152 . Journal.</ref> and Elke Dittmann<ref>Dittmann, Elke. “Insertional mutagenesis of a peptide synthetase gene that is responsible for hepatotoxin production in the cyanobacterium Microcystis Aeruginosa PCC 7806.” Molecular Microbiology (1997): 779–787. Journal.</ref>. After the expression of dCas9 and sgRNA will result in the repression of the McyB gene. Thus, no Microcystin will be produced.  
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This part can be used by transforming it directly into Microcystis Aeruginosa UTEX 2388 via electroporation or naturual transformation. Our team used the protocols provided by Au Yang Qin <ref>章军, 徐虹, 楼士林, 欧阳青. Blue-green alga shuttle plasmid expression vector and method for expressing thymison 'alpha' 1. Thesis. Xia Men: Xia Men University, 1999. </ref>, Nermin Adel El Semary<ref> Semary, Nermin Adel El. “Optimized electroporation-induced transformation in Microcystis aeruginosa PCC7806.” Biotechnol. Agron. Soc. Environ (2010): 149-152 . Journal.</ref> and Elke Dittmann<ref>Dittmann, Elke. “Insertional mutagenesis of a peptide synthetase gene that is responsible for hepatotoxin production in the cyanobacterium Microcystis Aeruginosa PCC 7806.” Molecular Microbiology (1997): 779–787. Journal.</ref>. After the expression of dCas9 and sgRNA will result in the repression of the McyB gene. Thus, no Microcystin will be produced.  
  
 
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Revision as of 13:35, 12 October 2019


Plasmid for in vivo expression of dCas9-sgRNA targeting mcyB

This plasmid consists of a cyanobacteria shuttle vector, ribosome binding sites, a dCas9-GFP complex and a sgRNA targeting mcyB of Microcystis Aeruginosa UTEX McyB.

The shuttle vector is from team 2016 iGEM team Nanjing_NFLS, part BBa_K1894001. It consists of two origin of replications, f1 ori and pUC ori, which allows it to replicate in both E.coli cells and cyanobacteria cells. It also consists of a t7 promoter, CaMV35S promoter, and Kanamycin resistance gene. The T7 promoter allows expression of the dCas9-GFP-sgRNA in E.coli and the CaMVS promoter allows expression in cyanobacteria.

The dCas9, also known as a catalytically dead Cas9 enzyme, is a mutated Cas9 enzyme without endonuclease activity. With the help of a single-guide RNA (sgRNA), it specifically binds to the target sequences and blocks transcript elongation by RNA polymerase. The GFP is added to the C-terminus of the dCas9, connected using a linker. There is also a 7x His-Tag, allowing for protein purification.

The sgRNA consists of a handle, a base-pairing region and a terminator. The base-pairing region is designed to target 25 base pairs of the McyB gene of Microcystis Aeruginosa UTEX2388.

Usage

This part can be used by transforming it directly into Microcystis Aeruginosa UTEX 2388 via electroporation or naturual transformation. Our team used the protocols provided by Au Yang Qin [1], Nermin Adel El Semary[2] and Elke Dittmann[3]. After the expression of dCas9 and sgRNA will result in the repression of the McyB gene. Thus, no Microcystin will be produced.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 3964
    Illegal NheI site found at 12275
    Illegal NotI site found at 777
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1685
    Illegal XhoI site found at 716
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 7979
    Illegal NgoMIV site found at 8139
    Illegal NgoMIV site found at 11186
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 5721
    Illegal SapI site found at 9059


  1. ↑ 章军, 徐虹, 楼士林, 欧阳青. Blue-green alga shuttle plasmid expression vector and method for expressing thymison 'alpha' 1. Thesis. Xia Men: Xia Men University, 1999.
  2. ↑ Semary, Nermin Adel El. “Optimized electroporation-induced transformation in Microcystis aeruginosa PCC7806.” Biotechnol. Agron. Soc. Environ (2010): 149-152 . Journal.
  3. ↑ Dittmann, Elke. “Insertional mutagenesis of a peptide synthetase gene that is responsible for hepatotoxin production in the cyanobacterium Microcystis Aeruginosa PCC 7806.” Molecular Microbiology (1997): 779–787. Journal.