Team:BHSF ND/Lab
2019 IGEM BHSF_NDProtocol
PCR
PCR Mix System（Q5® High-Fidelity 2× Master Mix）
Methods:
1. In a PCR tube on ice, combine 1-10 ng of template DNA, 2.50 μl of 10 μM forward primer, 2.50 μl of 10 μM reverse primer, 25μl of Q5® High-Fidelity 2× Master Mix, and sterile water to 50 μl.
2. Gently mix the reaction.
3. Collect all liquid to the bottom of the tube if necessary.
4. Transfer PCR tubes to a PCR machine and begin thermocycling.
PCR Reaction Condition (Q5® High-Fidelity 2× Master Mix):
Colony PCR
Methods:
1. Mix the materials mentioned except E. coli colony in PCR tubes
2. Pick one E. coli colony and add it to the reaction mixture
3. Gently mix the reaction
4. Transfer the PCR tubes to a PCR machine and begin thermocycling
Thermocycling:
PCR Reaction Condition（2×Taq PCR Star Mix with Loading Dye）
Note:
If loading on a gel, the Taq Mix contains loading dye, so don’t add anything else.
Gibson Assembly
Methods:
1. Keep 5µL Gibson mix on ice.
2. Add 5µL DNA fragment.
3. Incubate at 50℃ to 60min.
4. Transformation.
Gibson Assembly Reaction System:
Golden Gate Assembly
Methods:
Oligo Annealing
Denaturation at 95 ℃ for 3 minutes, then hold at 4 ℃ (0.1℃/s) indefinitely.
Oligo phosphorylation
Note:
Use the Oligo mentioned above directly.
React at 37 ℃ for 1 hour, then hold at 4 ℃ indefinitely.
Thermocycling conditions for a cycle of Golden Gate Assembly:
Note:
Transfer the product immediately.
Electrophoresis
Agarose Gel DNA Electrophoresis
Materials:
1× TAE buffer
agarose powder
DNA loading dye (6×)
DNA ladder
Agarose Gel DNA Electrophoresis
Methods:
1. Prepare 1% w/v solution of agarose powder in 1× TAE buffer .Weigh agarose powder and TAE buffer and add them to a flask.
2. Melt the mixture in a microwave until the agarose is completely dissolved.
3. Add SYBR（1/10000 v/v solution of the mixture）to the solution.
4. Pour the solution into the gel casting tray with appreciate comb.
5. Let the gel cool until it is solid and then pull out the comb.
6. Place the gel in the electrophoresis chamber and add enough TAE Buffer.
7. Pipette DNA samples mixed with appreciate amount of loading dye (6×) into wells on the gel and add appropriate DNA ladder.
8. Run the gel at 180V for about 15min.
Plasmid Extraction:
It is performed according to GenStar D201 StarPrep Plasmid Miniprep Kit
Gel Extraction:
It is performed according to the GenStar D205 StarPrep Gel Extraction Kit StarPrep
Preparation of LB Broth and LB Agar culture medium
LB Broth:
Methods:
1. Mix the components listed above
2. Autoclave
LB Agar:
Preparation of LB Broth, LB Ager, and M9 culture medium
Methods:
1. Mix the components listed above
2. Autoclave
Chemical Transformation
Materials：
50µL DH5a competent E. coli cells
5 µl DNA or 10-100ng DNA of a plasmid
200 µl LB media
Ice
Methods:
1. Thaw 50µL DH5a competent E. coli cells on ice.
2. Add 5 µl DNA from reaction mix or 10-100ng DNA of a plasmid.
3. Place the mixture on ice for 20-30 minutes.
4. Heat shock at 42°C for 60-90 seconds.
5. Place on ice for 5 minutes.
6. Pipette 200 µl of room temperature LB media into the mixture.
7. Incubate at 37°C and 200 rpm for 60 minutes.
8.Add 50-100 µl of the transformed cells to the selection plate.
Growing Overnight cultures
Materials:
5ml LB broth
5μl antibiotic
Tips
12 ml culture tube
Methods:
Overnight cultures are prepared under sterile conditions using a Bunsen burner.
1. Add 5ml LB broth into 12 ml culture tubes
2. Add 5μl antibiotic into the broth.
3. Pick a single colony by a sterile tip and inoculate the culture by dipping the tip into the LB broth.
4. Seal the tubes and incubate overnight at 37℃ shaking at 200 rpm.
Quantitative measurement
Microplate Reader:
100μl of E. coli culture was harvested and resuspended in 100 μl of PBS (phosphate-buffered saline). OD600 and GFPuv fluorescence (excitation 485 nm and emission 515 nm) was measured using microplate reader (Thermo). As for the data analysis, OD600 and fluorescence intensity of PBS measured in the same way was subtracted as blank. Fluorescence intensity of each well was normalized by OD600 of the same well. The average of fluorescence intensity was obtained from three replicates performed on different 96-well plates.
Flow cytometry analysis:
Flow cytometer data were obtained using an LSRFortessa flow cytometer (BD Biosciences). All the data were gated by forward and side scatter, and each data consists of at least
50,000 cells. The geometry mean fluorescence was calculated with FlowJo. The average of means was obtained from three replicates performed on different 96-well plates.
Protocols for Test
Strains and Growth Media:
E. coli 5a was used for all the experiments and grown in Luria–Bertani (LB) medium. Kanamycin (100 μg/mL), ampicillin (100 μg/mL) and chloramphenicol (25 μg/mL) were added appropriately.
Method:
E. coli was grown overnight in LB medium at 37 °C and then diluted 100-fold in fresh LB medium in 96-well plates. Then each culture (1000 μL) was induced for 4 hours at 37°C with inducers of different concentrations. Then the fluorescence intensity of cultures was measured by microplate reader (Thermo) or LSRFortessa flow cytometer (BD Biosciences).
Compartmentalization of modules testing
Leakage-inducer module test:
We constructed AraC-pBAD, Xyls-Pm induction systems and tested the expression of fluorescent proteins during same time period under different inducer concentrations and at same inducer concentration after different time periods.
Recombinase module test:
We constructed three recombinase expression systems to test the flipping effect of recombinases.
Bistable system module test:
We constructed a bistable system to reduce the leakage of the induction system and tested the expression of fluorescent protein in inducer's absence.
Toxin module test:
We constructed a toxin expression system to test the effect of the toxin on killing the bacteria which is shown by the OD level of the bacteria after a certain amount of time.
Lab Notebooks
June 2019
Week 1
We collected all the parts from former participants’ groups, confirmed their sequences, designed plasmids, and synthesized needed parts.
Week 2
We restored point mutations in Xyls parts, constructed leakage-inducer module(BN006, BN007, and BN009) through Gibson Assembly.
Week 3
We continued on constructing BN009, tested quantitatively the expression leakage of the plasmid carrying the leakage-inducer module by analyzing through flow cytometry results.
Week 4
We collected up all the synthesized gene fragments and started to design the gene circuit of our bistable system.
July 2019
Week 1
We started to construct the plasmids with our bistable system through two leakage-inducer modules(AraC-pBAD & Xyls-Pm) and plasmids of recombinase.
Week 2
We tested the usability of recombinases both qualitatively and quantitatively. And then we found out the expression leakage of recombinase in inducer’s absence. We gathered up ideas aiming to optimize the function of recombinase through the buildup of RBS library.
Week 3
After improving our circuit for recombinase, we tested the improved function of recombinase, and reflected on our results.
Week 4
We tested the fluorescence of reporter protein in the bistable system in inducer’s presence and absence and found that there is always no expression of the reporter protein. We searched through documents and tried to debug our circuit.
August 2019
Week 1
We replaced RBS1 with B0034 in order to check if there is problems in other sites of our circuit. Experiments results proved to us that there was nothing wrong with else parts. Through later reading, we found another translation-coupling RBS2 mentioned in the document which could be useful and designed the plasmid with B0034 being replaced by RBS2.
Week 2
We constructed the plasmid with RBS2 and qualitatively tested its function. We found that it indeed could reduce expression leakage in inducer’s absence.
Week 3
We quantitatively checked the sequence of RBS2. Through analyzing the whole plasmid’s sequence and comparing the sequence with our previous RBS1 plasmid sequence, we found that the RBS2 we used exclude the terminator(TGA) of the repressor. We reflected upon that and considered to delete the terminator(TGA) of repressor in RBS1 plasmid.
Week 4
We constructed the plasmid of RBS1 with exclusion of the terminator of the repressor and tested their expression leakage both qualitatively and quantitatively.
September 2019
Week 1
Both results proved to us that RBS1 works much more efficiently than RBS2 in reducing expression leakage. Our attempts finally succeeded!
Week 2
We constructed the plasmid with pBAD-toxin and tested the function of the toxin.
Week 3
In order to meet our superior goal of applying this bistable system to plasmids with various copy number, we transferred our system from low-copy-number plasmid(PSB4K5) to a medium-copy-number plasmid(P15A) to check if it also works well in this type of plasmid.
Week 4
We continued to test the function of our bistable system in different induction systems with different repressors (different backbones).
October 2019
Week 1
We tested the function of our bistable system in the new backbone.
Week 2
We incorporated the recombinase and toxin into our double bistable system.
Week 3
We tested the function of toxin in the bistable system. And eventually finished the construction of our plasmid.