Difference between revisions of "Part:BBa K4468006"
Zhichao Li (Talk | contribs) |
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<h1>'''SDS-PAGE'''</h1> | <h1>'''SDS-PAGE'''</h1> | ||
− | After confirming through colony PCR and sequencing, we used the successfully transformed E. coli BL21 (DE3) for expression. We induced with IPTG and | + | After confirming through colony PCR and sequencing, we used the successfully transformed E. coli BL21 (DE3) for expression. We induced with IPTG and Tb<sup>3+</sup> or IPTG and Cu<sup>2+</sup> then followed by cell disruption to detect membrane proteins, as our fusion proteins would be expressed on the cell membrane. |
[[File:HUST-China-06-2.png|400px|thumb|center|Fig.2 SDS-PAGE result of membrane protein oprf-Sitag-LanM(PmrCAB) induced by different lanthanides.<br><br>After induction using different lanthanide ions, we obtained several strains that successfully expressed the oprf-Sitag-LanM(PmrCAB). All their membrane proteins were detected by SDS-PAGE. The band of oprf-Sitag-LanM(PmrCAB) is about 65kDa, identical to the theoretical length of 62.82kDa and still within explainable and acceptable range of glycosylation or phosphorylation modification. Oprf-Sitag-LanM(PmrCAB) could be confirmed as successfully expressed. Besides, following elution result also could verify it.]] | [[File:HUST-China-06-2.png|400px|thumb|center|Fig.2 SDS-PAGE result of membrane protein oprf-Sitag-LanM(PmrCAB) induced by different lanthanides.<br><br>After induction using different lanthanide ions, we obtained several strains that successfully expressed the oprf-Sitag-LanM(PmrCAB). All their membrane proteins were detected by SDS-PAGE. The band of oprf-Sitag-LanM(PmrCAB) is about 65kDa, identical to the theoretical length of 62.82kDa and still within explainable and acceptable range of glycosylation or phosphorylation modification. Oprf-Sitag-LanM(PmrCAB) could be confirmed as successfully expressed. Besides, following elution result also could verify it.]] | ||
Revision as of 11:05, 30 September 2022
Oprf-Sitag-LanM
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 463
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1393
Illegal XhoI site found at 805 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 247
- 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
Oprf-Sitag-LanM is a protein composed of oprf, Sitag and LanM peptides. It is the main element for adsorption and recovery of lanthanides. Oprf has a membrane-binding domain, which helps the protein binding on the cell membrane of our engineered bacteria. Sitag is a tag that can connect with silicon element. It allows us to easily fix our protein just using a silica column. LanM has great characteristics of efficient and specific absorbing lanthanides which can effectively absorb the free lanthanides in the environment. Through GS linker to combine them in a whole, we have created a new protein that can stick on our E. coli membrane and fix to silica column with its engineered bacteria together. When the mining wastewater flows through the column, the lanthanides can be effectively adsorbed, so as to achieve the purpose of rare earth element recovery.
Molecular cloning
First of all, we need to amplificated all the commercially synthesized plasmid to acquire enough amount for further study. After transformation, colony PCR is applied for confirmation. Then we go for plasmid extraction.
Using E. coli to extraction. Through designed primers, we have obtained different high copies linearized fragments from our plasmids by PCR. These fragments are then connected together by homologous recombination to form a complete plasmid. After transformed into E. coli, colony PCR was applied for confirmation. Then we go for extracting plasmids again.
Finally we transformed our recombinant plasmids into E. coli BL21(DE3) competent cells. Correct as checked by colony PCR.
SDS-PAGE
After confirming through colony PCR and sequencing, we used the successfully transformed E. coli BL21 (DE3) for expression. We induced with IPTG and Tb3+ or IPTG and Cu2+ then followed by cell disruption to detect membrane proteins, as our fusion proteins would be expressed on the cell membrane.