Plasmid

Part:BBa_K4808011

Designed by: Zhao Guichun   Group: iGEM23_AIS-China   (2023-10-10)

pEcCas

pEcCas plasmid carry the cas 9 protein which can cut the target gene.

Characterization

pEcCas plasmid play an important role in our CRISPR-CAS 9 knocking out experiment.

f49dac1bd8ad0ec8913a15418c6e2f4d.png


Step1: Transformation of the pEcCas plasmid(carry cas9 protein) into E. coli AIS-0 (CICC20905). Step2: construction of the pTarget plasmid and donor DNA. Step3: transformation of pTarget plasmid and donor DNA into the AIS-0 with pEsCas inserted. Step4: Incubation of the transformed strains.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 4334
    Illegal EcoRI site found at 8435
    Illegal EcoRI site found at 9944
    Illegal EcoRI site found at 11128
    Illegal EcoRI site found at 12594
    Illegal XbaI site found at 7132
    Illegal SpeI site found at 1124
    Illegal PstI site found at 1805
    Illegal PstI site found at 2760
    Illegal PstI site found at 9439
    Illegal PstI site found at 9686
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 4334
    Illegal EcoRI site found at 8435
    Illegal EcoRI site found at 9944
    Illegal EcoRI site found at 11128
    Illegal EcoRI site found at 12594
    Illegal NheI site found at 4093
    Illegal NheI site found at 14303
    Illegal SpeI site found at 1124
    Illegal PstI site found at 1805
    Illegal PstI site found at 2760
    Illegal PstI site found at 9439
    Illegal PstI site found at 9686
    Illegal NotI site found at 1443
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 4334
    Illegal EcoRI site found at 8435
    Illegal EcoRI site found at 9944
    Illegal EcoRI site found at 11128
    Illegal EcoRI site found at 12594
    Illegal BglII site found at 13274
    Illegal BamHI site found at 6372
    Illegal BamHI site found at 8374
    Illegal XhoI site found at 2613
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 4334
    Illegal EcoRI site found at 8435
    Illegal EcoRI site found at 9944
    Illegal EcoRI site found at 11128
    Illegal EcoRI site found at 12594
    Illegal XbaI site found at 7132
    Illegal SpeI site found at 1124
    Illegal PstI site found at 1805
    Illegal PstI site found at 2760
    Illegal PstI site found at 9439
    Illegal PstI site found at 9686
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 4334
    Illegal EcoRI site found at 8435
    Illegal EcoRI site found at 9944
    Illegal EcoRI site found at 11128
    Illegal EcoRI site found at 12594
    Illegal XbaI site found at 7132
    Illegal SpeI site found at 1124
    Illegal PstI site found at 1805
    Illegal PstI site found at 2760
    Illegal PstI site found at 9439
    Illegal PstI site found at 9686
    Illegal AgeI site found at 8209
    Illegal AgeI site found at 9866
    Illegal AgeI site found at 13006
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 8191


References：

Cheng L, Wang J, Zhao X, et al. An antiphage Escherichia coli mutant for higher production of L-threonine obtained by atmospheric and room temperature plasma mutagenesis. Biotechnol Prog. 2020;36(6):e3058. doi:10.1002/btpr.3058

Li Q, Sun B, Chen J, Zhang Y, Jiang Y, Yang S. A modified pCas/pTargetF system for CRISPR-Cas9-assisted genome editing in Escherichia coli. Acta Biochim Biophys Sin (Shanghai). 2021;53(5):620-627. doi:10.1093/abbs/gmab036

Restrepo-Pineda S, O Pérez N, Valdez-Cruz NA, Trujillo-Roldán MA. Thermoinducible expression system for producing recombinant proteins in Escherichia coli: advances and insights. FEMS Microbiol Rev. 2021;45(6):fuab023. doi:10.1093/femsre/fuab023

Chen L, Chen Z, Zheng P, Sun J, Zeng AP. Study and reengineering of the binding sites and allosteric regulation of biosynthetic threonine deaminase by isoleucine and valine in Escherichia coli. Appl Microbiol Biotechnol. 2013;97(7):2939-2949. doi:10.1007/s00253-012-4176-z

Zhang C, Qi J, Li Y, et al. Production of α-ketobutyrate using engineered Escherichia coli via temperature shift. Biotechnol Bioeng. 2016;113(9):2054-2059. doi:10.1002/bit.25959

Park JH, Oh JE, Lee KH, Kim JY, Lee SY. Rational design of Escherichia coli for L-isoleucine production. ACS Synth Biol. 2012;1(11):532-540. doi:10.1021/sb300071a Hao R, Wang S, Jin X, Yang X, Qi Q, Liang Q. Dynamic and balanced regulation of the thrABC operon gene for efficient synthesis of L-threonine. Front Bioeng Biotechnol. 2023;11:1118948. Published 2023 Mar 2. doi:10.3389/fbioe.2023.1118948

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