Difference between revisions of "Part:BBa K3924043"

 
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<partinfo>BBa_K3924043 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3924043 SequenceAndFeatures</partinfo>
  
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==Profile==
 +
Name: mchIB<br/>
 +
Base Pairs: 461bp<br/>
 +
Origin: Escherichia coli, merge mchI and mchB<br/>
 +
Properties: A polycistron can express both MchI and MchB. MchB is the precursor of microcin H47(MccH47) and MchI is the immunity protein of MccH47.<br/>
 +
==Usage and Biology==
 +
The mchB and mchI are both 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.<sup>[1]</sup>The polycistron has two roles. First, it is the most basic part to increase the anti-bacteria activity of Nissle 1917. Second, it is the basis of constructing a big plasmid which contains the whole MccH47 expressing system.<br/>
 +
==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)-mchB between mchI and rrnB terminator.<br/>
 +
[[Image: T--Tsinghua--part_mchIB_construct.png|'''Figure 1: The design of mchIB''']]
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==Functional Verification==
 +
We did western blotting(WB) to test whether the polycistron can express MchI and MchB successfully. Maybe because both of the them are small peptide, the bands can not be separated successfully in the WB result.<br/>
 +
[[Image: T--Tsinghua--part_mchIB_WB.png|center|600px|thumb|'''Figure 2: The Western Blotting result of mchIB''']]
 +
We used FACS to test the anti-bacteria activity of the polycistron. The result suggested that the basic polycistron had anti-bacteria activity to a degree.<br/>
 +
[[Image: T--Tsinghua--part_mchIB_FACS.png|center|600px|thumb|'''Figure 3: The FACS result of mchIB''']]
 +
 +
==Reference==
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[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. <br/>
  
 
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Revision as of 08:56, 21 October 2021


mchI-mchB polycistrons with Ptac lacO promoter

mchI-mchB 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: mchIB
Base Pairs: 461bp
Origin: Escherichia coli, merge mchI and mchB
Properties: A polycistron can express both MchI and MchB. MchB is the precursor of microcin H47(MccH47) and MchI is the immunity protein of MccH47.

Usage and Biology

The mchB and mchI are both 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.[1]The polycistron has two roles. First, it is the most basic part to increase the anti-bacteria activity of Nissle 1917. Second, it 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)-mchB between mchI and rrnB terminator.
Figure 1: The design of mchIB

Functional Verification

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

Figure 2: The Western Blotting result of mchIB

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

Figure 3: The FACS result of mchIB

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.