Difference between revisions of "Part:BBa K3924045"

 
Line 12: Line 12:
 
<partinfo>BBa_K3924045 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3924045 SequenceAndFeatures</partinfo>
  
 +
==Profile==
 +
Name: mcmKL<br/>
 +
Base Pairs: 2417bp<br/>
 +
Origin: Escherichia coli, merge mcmK and mcmL<br/>
 +
Properties: A polycistron can express both McmK and McmL. MchK and MchL can modify the precursor of MccH47.<br/>
 +
==Usage and Biology==
 +
The mcmK and mcmL are both from E.coli CA46. 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.<sup>[1]</sup> The polycistron aim to make the engineering bacteria have the ability to modify precursor of MccH47.<br/>
 +
==Design and Construction==
 +
To construct the plasmid, we use endonuclease SpeI to cut RGP-mcmK at 2bp after mcmK open reading frame(ORF), then we used NEB Hifi DNA assembly to add RBS(ribosome binding site)-mcmL between mcmK and rrnB terminator.<br/>
 +
[[Image: T--Tsinghua--part_mcmKL_construct.png|center|600px|thumb|'''Figure 1: The design of mcmKL''']]
 +
==Functional Verification==
 +
We did western blotting(WB) to test whether the polycistron can express mcmL and mcmK successfully and we succeed.The time was the boiling sample time in WB.<br/>
 +
[[Image: T--Tsinghua--part_mcmKL_WB.png|center|600px|thumb|'''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. <br/>
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Revision as of 09:45, 21 October 2021


mcmK-mcmL polycistrons with Ptac lacO promoter

mcmK-mcmL polycistrons with Ptac lacO promoter

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 1663
    Illegal PstI site found at 2368
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1663
    Illegal PstI site found at 2368
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1663
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 1663
    Illegal PstI site found at 2368
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1663
    Illegal PstI site found at 2368
    Illegal AgeI site found at 1838
  • 1000
    COMPATIBLE WITH RFC[1000]

Profile

Name: mcmKL
Base Pairs: 2417bp
Origin: Escherichia coli, merge mcmK and mcmL
Properties: A polycistron can express both McmK and McmL. MchK and MchL can modify the precursor of MccH47.

Usage and Biology

The mcmK and mcmL are both from E.coli CA46. 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 aim to make the engineering bacteria have the ability to modify precursor of MccH47.

Design and Construction

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

Figure 1: The design of mcmKL

Functional Verification

We did western blotting(WB) to test whether the polycistron can express mcmL and mcmK successfully and we succeed.The time was the boiling sample time in WB.

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.