Sensing part
The sensing part is to achieve the awareness of the engineering bacteria on the rare earth ions in the environment.
①Amplify
pmrA-B-Fe、pmrC/GFP gene through the pcr protocol
②Plasmid Construction
Coding sequences pmrA/pmrB and pmrC-GFP were cloned into the expression vector pBAD30 by restriction enzyme digestion and DNA ligation.
Then we transformed these vectors into Ecoli BL21 cells.
③Detection
Arabinose was used to induce expression of pmrABC system.
We use the original pmrA-pmrB / Fe-pmrC-GFP circuit to test whether our
pmrABC system works in our plasmid, then the expression and efficacy
of LBT1-12 were tested .
gene circuit |
vector |
description |
pmrA-pmrB/Fe-pmrC-GFP |
pBAD30 |
Test whether our pmrABC system works in our plasmid |
pmrA-pmrB/LBT1-pmrC-GFP |
pBAD30 |
Detecting effectiveness of protein LBT1 |
pmrA-pmrB/LBT2-pmrC-GFP |
pBAD30 |
Detecting effectiveness of protein LBT2 |
pmrA-pmrB/LBT3-pmrC-GFP |
pBAD30 |
Detecting effectiveness of protein LBT3 |
pmrA-pmrB/LBT4-pmrC-GFP |
pBAD30 |
Detecting effectiveness of protein LBT4 |
pmrA-pmrB/LBT5-pmrC-GFP |
pBAD30 |
Detecting effectiveness of protein LBT5 |
pmrA-pmrB/LBT6-pmrC-GFP |
pBAD30 |
Detecting effectiveness of protein LBT6 |
pmrA-pmrB/LBT7-pmrC-GFP |
pBAD30 |
Detecting effectiveness of protein LBT7 |
pmrA-pmrB/LBT8-pmrC-GFP |
pBAD30 |
Detecting effectiveness of protein LBT8 |
pmrA-pmrB/LBT9-pmrC-GFP |
pBAD30 |
Detecting effectiveness of protein LBT9 |
pmrA-pmrB/LBT10-pmrC-GFP |
pBAD30 |
Detecting effectiveness of protein LBT10 |
pmrA-pmrB/LBT11-pmrC-GFP |
pBAD30 |
Detecting effectiveness of protein LBT11 |
pmrA-pmrB/LBT12-pmrC-GFP |
pBAD30 |
Detecting effectiveness of protein LBT12 |
Recycling part
The recycling part is to capture the rare earth ions in the environment and to anchor our cells over the silicon net.
①With appropriate primers, PCR was carried out to amplify target gene oprf-LBT1-12 and oprf-Si tag.
②Then we constructed two vectors to achieve expression of LBT and Si-tag.
We cloned the sequence of oprf-LBT and Si-tag into the vector pET28α in order to make the sequence be tagged N-terminally with a 6×His tag. 6×His tag is an affinity tag which allows the tagged recombinant protein to be purified in the process of affinity purification.
③Detection
In the meanwhile,we added the FLAG tag to achieve that whether the LBTs and the Si tag was expressed on the cell membrane.