Difference between revisions of "Part:BBa K3924044"
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==Design and Construction==
 
==Design and Construction==
 
To construct the plasmid, we use endonuclease SpeI to cut RGP-mchI at 2bp after mchI open reading frame(ORF), then we used NEB Hifi DNA assembly to add RBS(ribosome binding site)-mchX between mchB and rrnB terminator.<br/>
 
To construct the plasmid, we use endonuclease SpeI to cut RGP-mchI at 2bp after mchI open reading frame(ORF), then we used NEB Hifi DNA assembly to add RBS(ribosome binding site)-mchX between mchB and rrnB terminator.<br/>
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[[Image: mchIBX.png|center|600px|thumb|'''Figure 1: The design of mchIBX''']]
+
[[Image: T--Tsinghua--part_mchIBX_construct.png|center|600px|thumb|'''Figure 1: The design of mchIBX''']]
 
==Functional Verification==
 
==Functional Verification==
 
We did western blotting(WB) to test whether the polycistron can express MchI and MchB successfully. Maybe because all of the them are small peptide, the bands can not be separated successfully in the WB result.<br/>
 
We did western blotting(WB) to test whether the polycistron can express MchI and MchB successfully. Maybe because all of the them are small peptide, the bands can not be separated successfully in the WB result.<br/>

Revision as of 09:38, 21 October 2021


mchI-mchB-mchX polycistrons with Ptac lacO promoter

mchI-mchB-mchX polycistrons with Ptac lacO promoter

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 99
    Illegal PstI site found at 310
    Illegal PstI site found at 319
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 99
    Illegal PstI site found at 310
    Illegal PstI site found at 319
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 99
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 99
    Illegal PstI site found at 310
    Illegal PstI site found at 319
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 99
    Illegal PstI site found at 310
    Illegal PstI site found at 319
  • 1000
    COMPATIBLE WITH RFC[1000]

Profile

Name: mchIBX
Base Pairs:607bp
Origin: Escherichia coli, merge mchI，mchB， and mchX
Properties: A polycistron can express MchI, MchB and MchI. MchB is the precursor of microcin H47(MccH47).MchI is the immunity protein of MccH47.MchX's function is unsure, which might play a role in regulating the expression of mchB and mchI.

Usage and Biology

The mchB, mchI and mchX 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 mchx encodes MchX, whose's function is unsure[1]The polycistron is the basis of constructing a big plasmid which contains the whole MccH47 expressing system.

Design and Construction

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

Figure 1: The design of mchIBX

Functional Verification

We did western blotting(WB) to test whether the polycistron can express MchI and MchB successfully. Maybe because all of the them are small peptide, the bands can not be separated successfully in the WB result.

File:Western Blotting.png
Figure 3: The Western Blotting result

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.