Future Work
Future work
As the previous biosensor we designed did not work, after consulting the experts and teachers, we changed the new promoter, but it did not work
Inspired by the iGEM Peking team in 2010, we tried to reconstruct our biosensor by using the co-transformation system
Our future design is as follows
Expression of PbrR protein under the control of different strength of constituve promoters
Expression of RFP under the control of lead-responsive promoter pbrA
The genetic circuits constructed two plasmids that co-transformed into E. coli.
And the two plasmids have different copy Numbers,
We selected two plasmid backbones with different copy Numbers, a high-copy plasmid pSB1C3(100-300 copies) and a low-copy plasmid pSB3K3(20-30 copies), and placed these two genetic circuits into these two plasmids
Our future designs was as follows
1. PbrR was expressed under the control of different strength of promoters(S/M/W) in low copy plasmid backone pSB3K3, while mCherry was expressed under the control of pbrA promoter in high copy plasmid backbone pSB1C3.
2. PbrR was expressed under the control of different strength of promoters(S/M/W) in high copy plasmid backone pSB1C3, while mCherry was expressed under the control of pbrA promoter in low copy plasmid backbone pSB3K3
These two types of plasmids were co-transformed into E. coli DH5α to build whole-cell bacteria biosensor with different sensitivity to lead (Pb2+) ion.
Our design will include the following steps
1. Preparation of pSB3K3 low-copy plasmid backbone
1) The pSB3K3 backbone were obtained from iGEM distribution kit at the position of (plate6, 6E), add 10ul ddH20 into the corres hole to dissolve and mixed evenly
2) Take 1ul of plasmid, transform E. coli DH5a competent cells, and smear the plate containing kanamycin (K+) resistance for overnight culture at 37℃
3) The next day, a single colony was selected and inoculated with LB broth containing K kanamycin resistance for overnight culture
4) The next day, the plasmid was extracted and double digested by EcoR I and Pst I enzymes. The enzyme digestion product was taken as a large fragment, about 2700bp, and the fragment was cut and recovered for later use. This fragment was the skeleton of pSB3K3 low-copy plasmid
2. Preparation of inserted fragments of PbrR-S/M/W and pbrA-mCherry
1) The synthesized genes psb1c3-PbrR-s, M, W and psb1c3-pbrA-mCherry plasmid were found and the glycerin bacteria were inoculated into LB broth containing chloramphenicol resistance and cultured overnight
2) The next day, the plasmid was small, the concentration was measured, and the EcoR I/ Pst I double digestion was adopted. The enzyme digestion products were taken into small fragments with the sizes of 600bp, 600bp, 600bp and 1000bp respectively, and the gel was cut and recovered for later use
3) These fragments are the inserted fragments of PbrR-s/ M/W and pbrA-mCherry.
3. Ligation and transformation
1) the previous inserted fragments PbrR-s/ M/W and pbrA-mCherry.were ligated with the low-copy plasmid skeleton using T4 ligase to transform e. coli DH5a competent cells and then coated with a plate containing kanamycin resistance for overnight culture at 37℃
2) the positive colonies on the plate were selected on the next day, and the bacterial liquid PCR was performed. The positive results were re-inoculated into LB broth containing kanamycin resistance and cultured overnight
3) on the next day, the glycerin bacteria were first stored, the plasmid was small, the concentration was measured and sent to the company for sequencing. The sequencing primer was VF2/VR
4) the plasmid successfully sequenced was stored for later use
4. Co-transformation
These two different plasmid containing different combination, 1ul each, co-transformed into E. coli DH5a competent cells, and then coated on a plate containing kanamycin (K+) and chloramphenicol (Cl+) double resistance, overnight culture
3) The next day, bacterial colonies growing on the double-resistance (K+ and Cl+) plate were selected and two pairs of specific primers were taken to amplify the specific fragments on the two plasmids respectively for colony PCR verification
4) Re-inoculate the pcr-positive bacteria solution into LB broth containing kanamycin and chloramphenicol resistance, overnight culture, and store the glycerin bacteria for later use or directly for the next experiment.
5. Effects of different concentrations of lead solution on fluorescence intensity induced by co-recombinant bacteria
5.1 qualitative experiment
1) will the overnight train bacteria liquid to 1:100 scale vaccination fresh containing kanamycin and chloramphenicol in LB broth of the double resistance, stay OD600 0.4-0.6, adding different lead concentration in solution, the concentration of Pb2 + ions eventually reached 0, 1 PPM, 4 h, observe whether bacteria precipitation is red, if red, into the next phase of the experiment, if there is no change, give up the experiment.
5.2 quantitative experiment
1) the method is alexandrine, transfer training for the night, a total of recombinant bacteria to 1:100 scale vaccination fresh containing kanamycin and chloramphenicol resistance of double in LB broth, stay OD600 to 0.4-0.6, respectively, adding different concentration of lead in the bacteria liquid solution, make its final concentration to 0, 10ppb, 50ppb, 100ppb, 200ppb, 500ppb, 1ppm, 2ppm, 5ppm and 10ppm, set up three repeat each concentration
2) 100ul of each sample was taken and added to the 96-well plate (black plate), and the OD600 and red fluorescence intensity (585nm/610nm) were dynamically detected.
3) test every 20min, a total of 300min (5h).
4) centrifuge the remaining liquid and observe whether the precipitate turns red.
6. Effects of different concentrations of lead solution on the growth curve of co-recombinant bacteria
1) inoculate the bacteria solution cultured overnight with 1:100 into fresh LB broth with dual resistance of kanamycin and chloramphenicol, and add different concentrations of lead solution into the bacteria solution respectively to make the final concentration reach 0, 10ppb, 50ppb, 100ppb, 200ppb, 500ppb, 1ppm, 2ppm, 5ppm and 10ppm, with 3 replays for each concentration
2) 100ul was taken from each sample and added to the 96-well enzyme plate (white plate), and its OD600 was dynamically detected
3) Test every 1h for a total of 10h.
4) draw the growth curve according to the results