「Experiment」
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.
Construction of the whole circuit
All of our part should be built in one plasmid. Therefore, we use endonuclease and ligase to replace the GFP by Oprf-LBT and Oprf-Si tag.