Plasmid

Part:BBa_K4808009

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

pTarget

pTarget plasmid that carrying specific gRNA sequence which can identity the target gene for the further gene knockout.

Characterization

pTarget 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 330
    Illegal XbaI site found at 336
    Illegal SpeI site found at 221
    Illegal PstI site found at 348
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 330
    Illegal NheI site found at 198
    Illegal SpeI site found at 221
    Illegal PstI site found at 348
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 330
    Illegal BglII site found at 360
    Illegal BamHI site found at 186
    Illegal XhoI site found at 384
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 330
    Illegal XbaI site found at 336
    Illegal SpeI site found at 221
    Illegal PstI site found at 348
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 330
    Illegal XbaI site found at 336
    Illegal SpeI site found at 221
    Illegal PstI site found at 348
    Illegal NgoMIV site found at 1155
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 122


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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