Difference between revisions of "Part:BBa K3924048"
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==Design and Construction== | ==Design and Construction== | ||
To construct the plasmid, we use endonuclease NcoI to cut RGP-mcmKL polycistron at 2bp after mcmL open reading frame(ORF), then we used NEB Hifi DNA assembly to add RBS(ribosome binding site)-mchIB between mcmL and rrnB terminator.<br/> | To construct the plasmid, we use endonuclease NcoI to cut RGP-mcmKL polycistron at 2bp after mcmL open reading frame(ORF), then we used NEB Hifi DNA assembly to add RBS(ribosome binding site)-mchIB between mcmL and rrnB terminator.<br/> | ||
| − | [[Image: T--Tsinghua--part_mchIB_mcmKL_construct.png|'''Figure 1: The design of mchIB-mcmKL''']] | + | [[Image: T--Tsinghua--part_mchIB_mcmKL_construct.png|center|600px|thumb|'''Figure 1: The design of mchIB-mcmKL''']] |
==Functional Verification== | ==Functional Verification== | ||
We did western blotting(WB) to test whether the polycistron can express mchI, mchB, mcmK and mcmL successfully. mcmK and mcmL succeed. However, mchI and mchB maybe fail for some reason.<br/> | We did western blotting(WB) to test whether the polycistron can express mchI, mchB, mcmK and mcmL successfully. mcmK and mcmL succeed. However, mchI and mchB maybe fail for some reason.<br/> | ||
Revision as of 09:08, 21 October 2021
mchI-mchB-mcmK-mcmL polycistrons with Ptac lacO promoter
mchI-mchB-mcmK-mcmL polycistrons with Ptac lacO promoter
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1663
Illegal EcoRI site found at 5058
Illegal SpeI site found at 2422
Illegal PstI site found at 2368
Illegal PstI site found at 3293
Illegal PstI site found at 5269
Illegal PstI site found at 5278 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1663
Illegal EcoRI site found at 5058
Illegal SpeI site found at 2422
Illegal PstI site found at 2368
Illegal PstI site found at 3293
Illegal PstI site found at 5269
Illegal PstI site found at 5278 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1663
Illegal EcoRI site found at 5058 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1663
Illegal EcoRI site found at 5058
Illegal SpeI site found at 2422
Illegal PstI site found at 2368
Illegal PstI site found at 3293
Illegal PstI site found at 5269
Illegal PstI site found at 5278 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1663
Illegal EcoRI site found at 5058
Illegal SpeI site found at 2422
Illegal PstI site found at 2368
Illegal PstI site found at 3293
Illegal PstI site found at 5269
Illegal PstI site found at 5278
Illegal NgoMIV site found at 3779
Illegal AgeI site found at 1838
Illegal AgeI site found at 4501 - 1000COMPATIBLE WITH RFC[1000]
Profile
Name: mchIB-mcmKL
Base Pairs: 5423bp
!!!!!!!!
Origin: Escherichia coli, merge mchI, mchB, mcmK and mcmL
Properties: A polycistron can express MchI, MchB, McmK and McmL. MchB is the precursor of microcin H47(MccH47) and MchI is the immunity protein of MccH47. McmK and McmL can modify the precursor of MccH47.
Usage and Biology
The mchI, mchB, mcmK and mcmL 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 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 is the part that try to simply the MccH47 expressing system and endow the Nissle 1917 with the ability of anti-bacteria activity.
Design and Construction
To construct the plasmid, we use endonuclease NcoI to cut RGP-mcmKL polycistron at 2bp after mcmL open reading frame(ORF), then we used NEB Hifi DNA assembly to add RBS(ribosome binding site)-mchIB between mcmL and rrnB terminator.
Functional Verification
We did western blotting(WB) to test whether the polycistron can express mchI, mchB, mcmK and mcmL successfully. mcmK and mcmL succeed. However, mchI and mchB maybe fail for some reason.
We used FACS to test the anti-bacteria activity of the polycistron. The result suggested that the basic polycistron had significant anti-bacteria activity.
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.