Difference between revisions of "Part:BBa K3924043"
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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)-mchB between mchI 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)-mchB between mchI and rrnB terminator.<br/> | ||
| − | [[Image: T--Tsinghua--part_mchIB_construct.png|'''Figure 1: The design of mchIB''']] | + | [[Image: T--Tsinghua--part_mchIB_construct.png|center|600px|thumb|'''Figure 1: The design of mchIB''']] |
==Functional Verification== | ==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/> | 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/> | ||
Revision as of 08:57, 21 October 2021
mchI-mchB polycistrons with Ptac lacO promoter
mchI-mchB polycistrons with Ptac lacO promoter
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 99
Illegal PstI site found at 310
Illegal PstI site found at 319 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 99
Illegal PstI site found at 310
Illegal PstI site found at 319 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 99
- 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 99
Illegal PstI site found at 310
Illegal PstI site found at 319 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 99
Illegal PstI site found at 310
Illegal PstI site found at 319 - 1000COMPATIBLE 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.
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.
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.
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.