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Part:BBa_K3924048

Designed by: Wenda Zhang   Group: iGEM21_Tsinghua   (2021-10-21)
Revision as of 09:08, 21 October 2021 by Songwh19 (Talk | contribs)


mchI-mchB-mcmK-mcmL polycistrons with Ptac lacO promoter

mchI-mchB-mcmK-mcmL polycistrons with Ptac lacO promoter

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 1663
    Illegal EcoRI site found at 5058
    Illegal SpeI site found at 2422
    Illegal PstI site found at 2368
    Illegal PstI site found at 3293
    Illegal PstI site found at 5269
    Illegal PstI site found at 5278
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1663
    Illegal EcoRI site found at 5058
    Illegal SpeI site found at 2422
    Illegal PstI site found at 2368
    Illegal PstI site found at 3293
    Illegal PstI site found at 5269
    Illegal PstI site found at 5278
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1663
    Illegal EcoRI site found at 5058
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 1663
    Illegal EcoRI site found at 5058
    Illegal SpeI site found at 2422
    Illegal PstI site found at 2368
    Illegal PstI site found at 3293
    Illegal PstI site found at 5269
    Illegal PstI site found at 5278
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1663
    Illegal EcoRI site found at 5058
    Illegal SpeI site found at 2422
    Illegal PstI site found at 2368
    Illegal PstI site found at 3293
    Illegal PstI site found at 5269
    Illegal PstI site found at 5278
    Illegal NgoMIV site found at 3779
    Illegal AgeI site found at 1838
    Illegal AgeI site found at 4501
  • 1000
    COMPATIBLE WITH RFC[1000]

Profile

Name: mchIB-mcmKL
Base Pairs: 5423bp
!!!!!!!! Origin: Escherichia coli, merge mchI, mchB, mcmK and mcmL
Properties: A polycistron can express MchI, MchB, McmK and McmL. MchB is the precursor of microcin H47(MccH47) and MchI is the immunity protein of MccH47. McmK and McmL can modify the precursor of MccH47.

Usage and Biology

The mchI, mchB, mcmK and mcmL are all from E.coli CA46. The mchB encodes MchB, which is the precursor of microcin H47(MccH47). The mchI encodes MchI, which is the immunity protein of MccH47.The mcmK encodes McmK, which can modify the precursor of the Microcin H47(MccH47) . The mcmL encodes McmL, which can modify the precursor of MccH47.[1] The polycistron is the part that try to simply the MccH47 expressing system and endow the Nissle 1917 with the ability of anti-bacteria activity.

Design and Construction

To construct the plasmid, we use endonuclease NcoI to cut RGP-mcmKL polycistron at 2bp after mcmL open reading frame(ORF), then we used NEB Hifi DNA assembly to add RBS(ribosome binding site)-mchIB between mcmL and rrnB terminator.

Figure 1: The design of mchIB-mcmKL

Functional Verification

We did western blotting(WB) to test whether the polycistron can express mchI, mchB, mcmK and mcmL successfully. mcmK and mcmL succeed. However, mchI and mchB maybe fail for some reason.

Figure 2: The Western Blotting result of mchIBKL

We used FACS to test the anti-bacteria activity of the polycistron. The result suggested that the basic polycistron had significant anti-bacteria activity.

Figure 3: The FACS result of mchIBKL

Reference

[1] Vassiliadis G, Destoumieux-Garzón D, Lombard C, Rebuffat S, Peduzzi J. Isolation and characterization of two members of the siderophore-microcin family, microcins M and H47. Antimicrobial Agents and Chemotherapy. 2010 Jan;54(1):288-297. DOI: 10.1128/aac.00744-09. PMID: 19884380; PMCID: PMC2798501.


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