Sensing part
The sensing part is to achieve the awareness of the engineering bacteria on the rare earth ions in the environment.
①Amplify
PmrA-PmrB(Fe)、 PmrC-GFP gene through the pcr protocol.
②Plasmid Construction
Coding sequences PmrA-PmrB(Fe) 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 PmrA-PmrB-PmrC system.
We used the original PmrA-PmrB(Fe) - PmrC-GFP circuit to test whether our
PmrA-PmrB-PmrC 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 |
Enrichment part and recycling part
The Capture 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-Sitag.
②Then we constructed two vectors to achieve expression of LBT and Sitag.
We cloned the sequence of oprF-LBT and Sitag 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 test whether the LBTs and the Sitag was expressed on the cell membrane.
Construction of the whole circuit
PmrA-PmrB(Fe)-PmrC-GFP 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-Sitag.
Detection of sensing part
We use our medium containing a small amount of rare earth ions to culture our E. coli. When our engineered E. coli sense the existence of rare earth ions, the PmrC promoter will activate the transcription of the GFP gene. Through the detection of GFP expression, we can know the efficacy of the sensing part.
Detection of adsorption capacity of LBTs
A method of precipitation titration is used to detect the level of rare earth ions adsorption. We add a certain concentration of bacteria to the solution containing different concentrations of rare earth ions. Then centrifuge to remove the bacteria in the solution.
In order to make the terbium ions sufficiently precipitated, we add excess base. Rinse the precipitate and dissolve the precipitate with 0.1 M hydrochloric acid titration. In this way, we can know the amount of terbium ions which have already adsorbed on the surface of the bacteria.
