Team:Tsinghua/Experiments
EXPERIMENTS
lux pR Mutation Analysis
With the hope to find an optimal promotor for our NEON system, we designed 9 mutations on sites -35, -36, -37 near the luxR binding site and tested another on -10 site that TUST 2017 reported to show decreased leakage. We conducted experiments to evaluate these lux pR mutants' reaction to AHL stimulation and their leakage level. The test devices we designed include a constantly expressed luxR and a lux pR (or mutant) driven sfGFP (like BBa_K2558211 with original lux pR promotor , and BBa_K2558212 with lux pR-HS promotor).The protocol is as follows:
1. Transform the plasmids into E. coli DH5α.
2. Pick a single colony by a sterile tip from each of the LB plates for all the experimental and control groups. Add the colony into 5ml LB medium with ampicillin at 100 ng/µl. Incubate for 6-8 h at 37℃ in a shaker.
3. Measure OD600 of the culture medium with photometer. Dilute the culture medium until OD600 reaches 0.6.
4. Add 100 µl bacteria culture medium into a sterile 96-well plate. Add AHL to final concentrations of 0, 10-10,10-9,10-8 M. Fresh LB medium serves as blank control. Positive control is colony constantly expressing sfGFP and negative control is colony without sfGFP expression. Place the 96-well plate into an automatic microplate reader. Incubate at 16℃ overnight and measure the fluorometric value at 510 nm and OD600 of each well every 30 minutes.
5. Each group should be repeated for at least 3 times..
lacI Dosage & IPTG Induction
During pilot studies we found that high level of lacI expression severely decrease IPTG induction efficiency. In order to investigate how lacI dosage affects IPTG induction, we used Anderson promotor J23100, J23110 and J23114 to design three constitutive lacI generators of different intensities. The three lacI generators were then ligated with Ptac driven reporter sfGFP to make three IPTG induction devices (BBa_K2558203, BBa_K2558204,BBa_K2558205). By measuring sfGFP fluorescence we tested how these devices react to IPTG.The protocol is as follows:
1. Transform the plasmids into E. coli DH5α.
2. Pick a single colony by a sterile tip from each of the LB plates for all the experimental and control groups. Add the colony into 5ml M9 medium with ampicillin at 100 ng/µl. Incubate for 6-8 h at 37℃ in a shaker.
3. Measure OD600 of the culture medium with photometer. Dilute the culture medium until OD600 reaches 0.6.
4. Add 100 µl bacteria culture medium into a sterile 96-well plate. Add IPTG to final concentrations of 0, 1, 5, 10, 20 mM. Fresh M9 medium serves as blank control. Positive control is colony constantly expressing sfGFP and negative control is colony without sfGFP expression. Place the 96-well plate into an automatic microplate reader. Incubate at 16℃ overnight and record the fluorometric value at 510 nm and OD600 for each well every 30 minutes.
5. Each group should be repeated for at least 3 times.
IPTG Induced CRISPRi
After we clarified the correlation between lacI dosage and its induction efficiency, we furthered the experiments by adding CRISPRi to the system. Instead of sfGFP, the IPTG induction devices with different levels of lacI generators would produce a gRNA that targets the promotor of a constitutive sfGFP generator (BBa_K2558206, BBa_K2558207, BBa_K2558208). By adding IPTG, we induced the transcription of gRNA. Binding with constantly expressed dCas9, gRNA could inhibit the expression of sfGFP. We were able to observe how this process proceeded under different levels of lacI expression. This is the experiment design: (Table.1)Table.1. IPTG induced CRISPRi experiment
| Plasmid 1 (Amp+) | Plasmid 2 (Chl+) | Group | |
|---|---|---|---|
| Bacteria 1 | sfGFP generator | dCas9 generator | Positive control |
| Bacteria 2 | sfGFP generator | CRISPRi device with Ptac expressed gRNA and Anderson strong (J23100)- expressed lacI | Test 1 |
| Bacteria 3 | sfGFP generator | CRISPRi device with Ptac expressed gRNA and Anderson medium (J23110)- expressed lacI | Test 2 |
| Bacteria 4 | sfGFP generator | CRISPRi device with Ptac expressed gRNA and Anderson weak (J23114)- expressed lacI | Test 3 |
| Bacteria 5 | Empty backbone | CRISPRi device with Ptac expressed gRNA and Anderson strong (J23100)- expressed lacI | Negative control 1 |
| Bacteria 6 | Empty backbone | dCas9 generator | Negative control 2 |
The protocol is as follows:
1. Transform the plasmids into E. coli DH5α according to the design in Table 1.
2. Pick a single colony by a sterile tip from each of the LB plates for all the experimental and control groups. Add the colony into 5ml M9 medium with ampicillin at 100 ng/µl and chloramphenicol at 34 ng/µl. Incubate for 6-8 h at 37℃ in a shaker.
3. Measure OD600 of the culture medium with photometer. Dilute the culture medium until OD600 reaches 0.6.
4. Add 100 µl bacteria culture medium into a sterile 96-well plate. Add IPTG to final concentrations of 0, 1, 3, 5, 10 mM. Fresh M9 medium serves as blank control. Place the 96-well plate into an automatic microplate reader. Incubate at 16℃ overnight and record the fluorometric value at 510 nm and OD600 of each well every 30 minutes.
5. Each group should be repeated for at least 3 times.
NEON System Characterization
Assisted with the experience we gained form the experiments above, we built and tuned the NEON system. We designed this experiment to characterize how Neon the positive feedback plasmid (BBa_K2558214), and Safety Catch the CRISPRi plasmid (BBa_K2558215,BBa_K2558216) work together. This is the experiment design: (Table.2)Table.2. NEON system characterization experiment design.
| Plasmid 1 (Amp+) | Plasmid 2 (Chl+) | Group | |
|---|---|---|---|
| Bacteria 1 | Neon | dCas9 generator | Positive control |
| Bacteria 2 | Empty backbone | dCas9 generator | Negative control (to see how dCas9 interfere with bacteria growth) |
| Bacteria 3 | lux pR test device | dCas9 generator | Non-positive feedback control 1 |
| Bacteria 4 | lux pR-HS test device | dCas9 generator | Non-positive feedback control 2 |
| Bacteria 5 | Neon | CRISPRi Safety Catch device with lux pR promotor | Test 1 |
| Bacteria 6 | Neon | CRISPRi Safety Catch device with lux pR-HS promotor | Test 2 |
| Bacteria 7 | lux pR test device | CRISPRi Safety Catch device with lux pR promotor | Non-positive feedback test 1 |
| Bacteria 8 | lux pR-HS test device | CRISPRi Safety Catch device with lux pR-HS promotor | Non-positive feedback test 2 |
The protocol is as follows:
1. Transform the two plasmids into E. coli DH5α according to the design in Table 2.
2. Pick a single colony by a sterile tip from each of the LB plates for all the experimental and control groups. Add the colony into 5ml LB medium with ampicillin at 100 ng/µl and chloramphenicol at 34 ng/µl. Incubate for 6-8 h at 37℃ in a shaker.
3. Measure OD600 of the culture medium with photometer. Dilute the culture medium until OD600 reaches 0.6.
4. Add 100 µl bacteria culture medium into a sterile 96-well plate. Add IPTG to final concentrations of 0 or 10 mM and AHL to final concentrations of 0, 10-9, 10-8 M. Fresh LB medium serves as blank control. Place the 96-well plate into an automatic microplate reader. Incubate at 16 ℃ overnight and record the fluorometric value at 510 nm and OD600 of each well every 30 minutes. Or Fix sample with 1.5 mg/ml kanamycin at one hour intervals. Then use flow cytometry to measure the fluorescent intensity at 488 nm of each sample.
5. Each group should be repeated for at least 3 times.