Part:BBa_K3924046

mchC-mchD polycistrons with Ptac lacO promoter

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal EcoRI site found at 1830
Illegal XbaI site found at 1017
Illegal PstI site found at 126
12
INCOMPATIBLE WITH RFC[12]
Illegal EcoRI site found at 1830
Illegal PstI site found at 126
21
INCOMPATIBLE WITH RFC[21]
Illegal EcoRI site found at 1830
Illegal BglII site found at 417
Illegal BglII site found at 580
Illegal BamHI site found at 1823
23
INCOMPATIBLE WITH RFC[23]
Illegal EcoRI site found at 1830
Illegal XbaI site found at 1017
Illegal PstI site found at 126
25
INCOMPATIBLE WITH RFC[25]
Illegal EcoRI site found at 1830
Illegal XbaI site found at 1017
Illegal PstI site found at 126
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI.rc site found at 464
Illegal SapI.rc site found at 1384

Profile

Name: mchCD
Base Pairs: 2024bp
Origin: Escherichia coli, merge mchC and mchD
Properties: A polycistron can express both MchC and MchD. MchC and MchD can modify the precursor of MccH47

Usage and Biology

The mchC and mchD are both from E.coli CA46. The mchC encodes MchC, which can modify the precursor of the Microcin H47(MccH47) . The mchD encodes MchD, 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-mchC at 2bp after mchC open reading frame(ORF), then we used NEB Hifi DNA assembly to add RBS(ribosome binding site)-mchD between mchC and rrnB terminator.

Figure 1: The design of mchCD

Functional Verification

We did western blotting(WB) to test whether the polycistron can express mchD and mchC successfully and we succeed.

File:Western Blotting.png

Figure 3: The Western Blotting result of mchCD

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.