Reagent Preparation

LB culture medium

For solid medium, an additional 1-1.5% agarose needs to be added.

Media was sterilized in an autoclave.

CaCl2-MgCl2 (80 mM MgCl2, 20 mM CaCl2)

Solution was sterilized in an autoclave and stored in 4℃ fridge.

glycerol-CaCl2 (include 0.1mM CaCl2 and 15% glycerol)

Solution was sterilized in an autoclave and stored in 4℃ fridge.

40% glycerol

Solution was sterilized in an autoclave.

Kanamycin/Streptomycin sulfate (50mg/mL)

Solution was sterilized by filtration.

The filtrate was divided into 1mL and stored at -20℃.

Chloramphenicol (50mg/mL)

Solution was sterilized by filtration.

The filtrate was divided into 1mL and stored at -20℃.

Ampicillin (100mg/mL)

The working concentration was 100 mg/L.

The sample was diluted with double distilled water to the appropriate volume, and then filtered with a 0.22 μm sterile aqueous filter in an ultra-clean bench to remove the bacteria, and then stored at -20℃ .

Isopropyl-β-thiogalactopyranoside (IPTG, 1M)

Solution was sterilized by filtration.

The filtrate was stored at -20℃.

L-Ara (0.4g/L)

Plasmid Extraction

1. Transfer well-grown bacterial culture to a 2mL centrifuge tube.

2. Centrifuge the tube at 12000rpm for 60s to pellet the cells and discard the supernatant completely.

3. Add 200μL of FAPD1 Buffer (RNase A added) to the cell pellet and resuspend the cells completely by pipetting.

4. Add 200μL of FAPD2 Buffer and gently invert the tube 5-10 times.

5. Add 300μL of FAPD3 Buffer and invert the tube 5-10 times immediately to neutralize the lysate.

6. Centrifuge the tube at 12000rpm for 10mins to clarify the lysate. During centrifugation, place a FAPD Column in a Collection Tube.

7. Transfer the supernatant carefully to the FAPD Column and centrifuge the column at 12000rpm for 60s. Discard the flow-through and place the column back to the Collection Tube.

8. Add 400μL of W1 Buffer to the FAPD Column and centrifuge the column at 12000rpm for 60s. Discard the flow-through and place the column back to the Collection Tube.

9. Add 700μL of Wash Buffer to the FAPD Column and centrifuge the column at 12000rpm for 60s. Discard the flow-through and place the column back to the Collection Tube. Repeat the step.

10. Centrifuge the column at 12000rpm for 60s to dry the column. Put the column into a new 1.5mL microcentrifuge tube.

11. Add 80μL elution buffer (heated at 65℃ in advance) to the membrane center of the FAPD Column. Stand the column for 60s.

12. Centrifuge the tube at 12000rpm for 60s to get plasmid DNA and store the DNA at -20 ℃. Detect the concentration of the DNA.

PCR

Prepare reaction mixture in PCR tubes:

Set PCR program:

X and time in Annealing depend on the product length.

Gel Electrophoresis

1. Remove the comb from the cast gel.

2. Place the gel into an electrophoresis system and make sure that the volume of 1×TAE buffer is sufficient.

3. After TAE submerging the wells of the gel, load DNA marker and samples.

4. Load 5-10μL of DNA size marker.

5. Mix 3μL of the samples with 3μL loading buffer and then load the mixture. The volume depends on the purpose of the gel (confirmation or purification).

6. Place the electrodes with the cover over the gel system and start the electrophoresis.

7. Set current to 190mA. Set the appropriate gel run time as 15-30 minutes. After the electrophoresis is over, switch off the system.

8. Take the gel and place it over a UV or Blue-light illuminator to visualize DNA bands for either confirmation of experimental results or gel DNA extraction.

Gel Extraction

1. Excise the agarose gel withh a clean scalpel.

2. Transfer up to 300mg of the gel slice into a microcentrifuge tube.

3. Add 500μL of FADF Buffer to the sample and mix by vortexing.

4. Incubate at 55℃ for 5-10 minutes and vortex the tube every 2-3 minutes until the gel slice dissolved completely.

5. Cool down the sample mixture to room temperature. And place a FADF Column into a Collection Tube.

6. Transfer 800μL of the sample mixture to the FADF Column. Centrifuge at 12000rpm for 60s, then discard the flow-through.

7. Add 750μL of Wash Buffer to the FADF Column. Centrifuge at 12000rpm for 60s, then discard the flow-through.

8. Centrifuge at 12000rpm for additional 60s, then discard the flow-through.

9. Place the FADF Column to a new microcentrifuge tube.

10. Add 40μL of Elution Buffer to the membrane center of the FADF Column. Stand the FADF Column for 60s.

11. Centrifuge at 12000rpm for 60s. The flow-through is the DNA we need.

DNA Digestion

Prepare reaction mixture:

Incubate the mixture at 37℃ for 1.5-2 hours. Then use Gel Electrophoresis and Gel Extraction to purify digestion products.

DNA Ligation

Prepare reaction mixture:

Incubate the mixture at 22℃ for 1 hour.

Gibson Assembly

1. Prepare linearized vector by DNA digestion or reverse PCR. Perform a gel extraction to purify samples.

2. Design the primers and amplify the insert segments. Perform a gel extraction to purify samples.

3. Prepare Seamless Cloning reaction mixture:

X, Y depend on the molar ratio the Insert to the linearized vector (usually 2:1, or 5:1 when the Insert is smaller than 200bp)

4. Mix up and react at 50℃ for 15 minutes. When the reaction is over, put the mixture onto ice.

5. Chemical transformation and Colony PCR Confirmation.

Competent cell preparation

1. Use spread plate method to culture E.coil overnight and single colonies are picked for amplification in a test tube with 5mL LB culture medium.

2. Add 1 ml of the bacterial solution to a shake flask containing 50 ml of LB medium for incubation until the OD reaches between 0.3-0.4.

3. Centrifuge the bacterial solution at 4000g, 4℃ for 10 minutes.

4. Remove the liquid and add 15mL CaCl2-MgCl2. Mix up and repeat step 3.

5. Remove the liquid and add 1mL CaCl2-glycerol. Mix up and divide into ten portions of 100μL each.

6. Store at -80℃.

Chemical transformation

1. Take 100μL competent bacteria out of the 80 °C freezer and melt on ice.

2. Turn on the UV sterilization function in the clean bench for about 15 minutes to sterilize the working environment before opening the test tube with the bacteria to avoid any risk of contamination.

3. Take out petri dishes with required medium and antibiotic out of the refrigerator.

4. Put 0.5-1μL of plasmid into 100μL of competent cells culture.

5. Hold the samples on ice for 15 minutes.

6. Perform a heat shock for 90s at 42 ℃.

7. Incubate samples on ice for 3 minutes.

8. Suspend the bacteria with 1mL of LB medium and incubate samples in the 37 ℃ heat block for 40-60 minutes.

9. Centrifuge the sample and remove 1mL liquid. Mix up.

10. Plate out this mixture on an LB petri plate with the correct antibiotic.

11. Incubate the plates overnight at an optimal temperature for selected bacteria.

Colony PCR Confirmation

Prepare samples by picking up colony and mixing up with 10μL double distilled water. Prepare reaction mixture:

Perform PCR.

Another 9μL of sample is used to amplify for afterward sequencing and storage.

Salidroside Fermentation

1. Use streak plate to gain single colony of the suitable strain. After confirmation, amplify in test tubes overnight.

2. Detect the OD and add the bacterial culture above to 50mL LB medium (let OD = 0.05).Start culturing.

3. After 1-2 hours, if the OD reaches between 0.6-0.8, add IPTG (1/1000)to it. If not, continue culturing.

4. Take samples every 12 hours. Detect OD and detect products by HPLC.

CRISPR Cas9 Genome Edition of E.coli[1]

1. Design primers to amplify 500bp sequences upstream and downstream of the target gene as homology arms. Gel extraction.

2. Splice the upstream and the downstream as donor DNA by fusion PCR. Gel extraction. Link to T vector as pDonor-T.

3. Perform PCR amplification of donor DNA using pDonor-T as a template. Gel extraction to 10μL.

4. Design pTarget by using primers pTarget-F and pTarget-R-com with mutation of 20bp before the PAM site of primer pTarget-F.

5. Prepare competent cells of pCas. Chemical transformation of pDonor-T and pTarget and confirmation.

6. Elimination of pCas and pTarget and confirmation.

HPLC

1. Use Diamonsil C18 column (Diamonsil 5μm, 250*4.6mm)

2. Centrifuge the sample at 12000rpm for 2 minutes and filter the supernatant through a 22μm membrane into required bottles.

3. Set flow rate = 1mL/min and temperature = 40℃.

4. Start HPLC with samples and standard salidroside.

Liposome

1. Add phosphatidylcholine and cholesterol (w/w = 7:2 or 7:4 or 7:6, 90mg in total) in an 100mL round-bottomed flask. Add 14mL chloroform to dissolve.

2. Use a rotary evaporator to slowly remove the organic solvent at 40°C, 140rpm, resulting in the formation of a very thin film of dry lipid on the inner surface of the flask.

3. Slowly hydrate the dried lipid membrane with 10 mL of PBS (pH 7.4) plus 5 mL of water (with medium and bacteriophage added to the water). Heterogeneous suspensions of liposomes of different sizes are produced by spinning at atmospheric pressure and 50℃ for 60 minutes.

4. If the solution is cloudy, centrifuge it appropriately into emulsion at a low speed. Store the liquid at -4℃.

5. Use dynamic light scattering to probe liposome particle size distribution and use microscopy to observe liposome size and bacterial encapsulation.

5' UTR thermosensor elements[2]

1. Design the individual modules of the thermosensor . Start by selecting an SD sequence (e.g., 5'-AAGGAG-3';) of appropriate strength and designing a stem structure based on a complementary ASD sequence.

2. Design the loop module .

3. Include unique restriction sites . Insert a spacer of appropriate length between the SD sequence and the translation initiation codon. (The optimal distance between the SD sequence and the start codon in E. coli is generally believed to be 4-9 base pairs.)

4. Design a series of different synthetic 5' UTR constructs that differ in loop size and/or the extent of complementarity between ASD and SD .

5. Include a 5' UTR element that is not expected to fold into a temperature-responsive structure to be used as a control ). To exclude fortuitous hairpin formation, run the sequence through a secondary structure prediction program as described in Step 6.

6. Determine the size of the full-length 5' UTRs (from the transcriptional start site to the translation initiation codon). Predict the secondary structures and their theoretical stabilities (free energies) using, for example, the Mfold web server.

7. Compare the predicted free energies of the different 5' UTR constructs. Choose 5' UTR constructs covering a suitable range of theoretical stabilities (depending on the desired switch temperature). Evaluate the probability of alternative secondary-structure formation by comparing the free energy of predicted alternative structures with that of the desired structure.

8. Order the synthetic oligodeoxynucleotides (HPLC purified) required for assembly of the thermometers .

9. Mix the two complementary single-stranded oligonucleotides at equimolar concentrations (150 pmol each) in a volume of 20 μl .

10. Denature the reaction mixture at 95 °C for 5 min in a heating block and subsequently let the samples slowly cool down to 25 °C.

11. Ligate the annealed synthetic oligonucleotides into the expression vector (carrying, e.g., gfp as a reporter gene) that was digested using the restriction enzymes corresponding to the 5' and 3' overhangs of the annealed synthetic oligonucleotides. Set up the ligation reaction as follows and incubate at 16 °C overnight:

12. To identify correct clones, inoculate 6-12 colonies into individual aliquots of 4 ml LB medium supplemented with the selective antibiotic and incubate the cultures overnight at 37 °C.

13. Isolate the plasmids according to standard protocols.

14. Carry out test digests using suitable combinations of unique restriction enzymes flanking the 5' UTR fragment using the reaction buffers and conditions recommended by the supplier. Separate the restriction enzyme-digested samples by electrophoresis in 1.5-2% agarose gels and determine the fragment sizes using a suitable DNA size marker.

15. To exclude DNA synthesis errors, confirm correctness of the 5' UTR sequence by DNA sequencing.

16. Inoculate one or two colonies for each of the different thermometer constructs and the non-inducible control (from Step 16) in liquid growth medium in a standard culture tube. Incubate each clone at different physiological temperatures (for E. coli, 17 °C, 22 °C, 30 °C and 37 °C are recommended) under continuous shaking (180 r.p.m.).

17. Proceed with expression analysis using one or more of options A-C, depending on the reporter gene(s) used. Option A is used to detect GFP reporter expression, option B can be used to detect the expression of any protein for which a suitable antibody is available and option C is used to detect lacZ reporter expression. Note that only options B and C are quantitative assays. Ideally, each RNA thermometer should be tested in the context of two different reporter genes

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References