PCR

Set up reaction as follows:

Run PCR program:

Colony PCR

Set up reaction as follows:

Use toothpick to pick out single colony and dip the tip into the reaction system to add template.

Run PCR program:

Gibson assembly

1. After PCR, run gel extraction and determine the concentration of the fragments.
2. Add 2nmol of each linearized fragment and vector into a PCR tube.
3. Adjust the volume to 10ul with ddH2O.
4. Add 10ul of 2× Gibson Mastermix into the system.
5. Incubate in the thermocycler at 37 ℃ for 30min.
6. Perform transformation.

1. Design the first version device for better enteral drug simulation.

HP

1. Visit professor Yi Yu in School of Pharmaceutical Sciences.

Transforming chemically competent cells

1. Thaw chemically competent cells on ice.
2. Add DNA, pipette gently to mix (1μl of prepped plasmid is more than enough).
3. Let sit for 30 minutes on ice.
4. Incubate cells for 30 seconds at 42℃.
5. Incubate cells on ice for 2 min.
6. Add 1 mL SOC or LB at room temp.
7. Incubate for 1 hour at 37℃ on shaker.
8. Spread 100-300 μl onto a plate made with appropriate antibiotic.
9. Grow overnight at 37 °C.
10. Save the rest of the transformants in liquid culture at 4 °C. If nothing appears on your plate, you can spin this down, resuspend in enough medium to spread on one plate and plate it all. This way you will find even small numbers of transformants.

Preparing chemically competent cells

1. Grow a 5 mL seed culture of cells in LB medium to saturation. Dilute this culture back into 25–50 mL fresh LB in a 200 mL conical flask. Dilute the seed culture by at least 1/100.
2. Grow the diluted culture to an OD600 = 0.2–0.5.
3. Put eppendorf tubes on ice now so that they are cold when cells are aliquotted into them later. If your culture is X ml, you will need X tubes.
4. Chill TSS buffer by putting it in an ice bath.
5. Split the culture into two 50 mL falcon tubes and incubate on ice for 10 min.

All subsequent steps should be carried out at 4°C and the cells should be kept on ice wherever possible

6. Centrifuge for 10 min at 3000 rpm and 4°C.
7. Remove supernatant, pipette out any remaining media.
8. Resuspend cells in chilled TSS buffer. The volume of TSS to use is 10% of the culture volume that you spun down. Vortex gently to fully resuspend the culture.
9. Add 100 µL aliquots to your chilled eppendorfs and store at -80°C.

Gel extraction

1. Cut out the target band minimizing extra gel.
2. Put it in an eppendorf tube and add 600uL QG.
3. Incubate at 50°C until the gel is fully dissovled.
4. Pour into the gel extraction column.
5. Spin through and disgard the waste.
6. Wash with 750uL PE buffer.
7. Centrifuge for two minutes to dry.
8. Elute with ddH2O.

Plasmid mini-prep

1. Pick a single colony, cultivate in 5mL LB medium with antibiotic to saturation.
2. Put 1.5mL culture into a 1.5mL EP tube.
3. Centrifuge at 13000rpm for 1min.
4. Discard the medium, make the remaining pellet as dry as possible.
5. Add 240ul Solution I into the 1.5mL EP tube and resuspend the pellet.
6. Add 240ul Solution II into the same EP tube.
7. Mix it by gentle reversal for 5 times until the solution gets clear.
8. Add 350ul Solution III into the same EP tube.
9. Vortex the tube until the solution is uniform.
10. Centrifuge at 12000rpm for 15min.
11. Move the supernatant to a new mini-prep column.
12. Centrifuge at 12000rpm for 1min and discard the flowthrough.
13. Add 750ul Solution PE into the column.
14. Centrifuge at 12000rpm for 1min and discard the flowthrough.
15. Centrifuge again at 12000rpm for 2min to completely dry the column.
16. Put the column into a new EP tube, and add 50ul ddH2O.
17. Centrifuge at 12000rpm for 1min.
18. Use Nanodrop to measure the concentration of the obtained plasmid.

Fluorescence observation using microscopy

1. Grow a seed culture in 5ml LB medium overnight.
2. Dilute 50ul of the culture in another 5ml LB medium and culture for 2h.
3. Prepare solid LB medium. Drop one drop of the melted solid LB medium onto a glass slide and let it cool and solidify.
4. Spread some of the diluted culture onto the prepared glass slide, and cover it with another cover slide.
5. Detect with fluorescence microscopy and take images.

SAMe quantification

I. Sample preparation

1.Inoculate 1µl bacteria solution into 5ml medium and incubate for 12 hours.
2.Transfer the culture into 45ml medium and incubate for 2 hours.
3.Separate 5ml culture into each tube and add corresponding inducer, incubating respectively according to the needed incubation time.
4.Centrifuge at 12000rpm for 2min.

Test for opSam2:

5.Add 100µl RIPA to the sediment, resuspend and incubate on ice for 30min.
6.Centrifuge at 12000rpm for 2min and collect the supernatant as samples.

Test for opPet8p

7.Collect the supernatant as samples.

II. Measurement

1.Add 50µl of each sample and standard to each well.
2.Add 50µl Detection A to each well immediately and incubate for 1 hour at 37˚C.
3.Aspirate and wash with 350µl wash buffer for three times.
4.Add 100µl Detection B to each well and incubate for 30 minutes at 37˚C.
5.Aspirate and wash with 350µl wash buffer for five times.
6.Add 90µl TMB substrate to each well and incubate for 20 minutes at 37˚C.
(Protect from light.)
7.Add 50µl Stop solution to each well and shake to uniform.
8.Run the microplate reader and conduct the measurement at 450nm.

Western Blot

I. Equipment and Instrument:

1. 50 ml flasks
2. Micropipettes
3. Incubator shaker for bacterial cultures
4. Refrigerated centrifuge
5. Thermomixer compact
6. Gel electrophoresis chamber
7. Tank blot device
8. Rotating wheel
9. Developer or WB documentation system
10. Autoradiography Cassette Sonicator

II. Reagent:

1. PVDF, 0.45/0.22 um,
2. Coomassie Blue R250 Methanol
3. Antibodies (e.g., anti-TP serum raised in mouse)
4. Goat anti- mouse horseradish peroxidase-conjugate antibody ECLTM Blotting Reagents
5. Trizma® base
6. Phosphate buffered saline pH 7.5 (PBS)
7. Phosphate buffered saline pH 7.5 with Tween (PBS- -T)
8. Blocking solution
9. Polyacrylamide gel with 5% stacking gel and 15% running gel
10. Transfer buffers
11. Western transfer buffer Standard transfer buffer Modified transfer buffer

III. Protocol:

1. Dip the Gel in a buffer (10 mM CAPS, 20% methanol) for 10 min. Treat PVDF membrane with methanol for a few seconds and dip it in buffer (10 mM CAPS, 20% methanol). Set the gel, membrane, and buffer (10 mM CAPS, 20% methanol) using Wet/Tank Blotting Systems and transfer the proteins from the gel to a PVDF membrane at 150 mA for 3h at 4℃.
2. Treat the membrane with 10 mL blocking buffer (TBST containing 5% Easy Blocker) in a plastic container at room temperature with gentle agitation for 1 h.
3.Treat the membrane with 10 mL blocking buffer containing 1:1,000 anti His tag IgG in a plastic container at room temperature with gentle agitation for 1 h.
4.After washing with 20 mL TBST for three times of 5 min agitation, treat the membrane with 10 mL blocking buffer containing 1:2,000 anti-mouse IgG conjugated with alkaline phosphatase in a plastic container at room temperature with gentle agitation for 1 h.
5. After washing with 20 mL TBST for three times of 5 min agitation, treat the membrane with a staining buffer without agitation for 5 min.
6.After bands appear, dip the membrane in TE buffer supplemented with 10 mM EDTA to stop overstaining and then dry the membrane on paper towel.
7.Measure the band intensity by densitometry scanning.

SDS – PAGE

I. Equipment and Instrument:

1. Eppendorf tubes
2. Pipette and pipette tips
3. Centrifuge
4. Vortex
5. lce bucket
6. Empty box
7. Dry Thermounit
8. Glass plates
9. 10-well comb
10. Spacer
11. Clamp
12. Casting stand
13. Buffer tank
14. Voltage source
15. Shaker

II. Reagent:

1. H20
2.30% acrylamide
3.1.5M tris pH 8.8
4.1M tris pH 6.8
5.10% SDS
6. 10% APS
7. TEMED
8. Prestained Protein Marker - Bioman
9. Protein dye
10. Overnight culture (sample)
11. Destaining buffer
12. Tank buffer (1×)
13. lso-propanol

III. Protocol:

1. Configure SDS - PAGE gel.

2. Sample preparation.
3. Assemble electrophoresis device.
4. Spotting.
5. Electrophoresis at 80V.
6. After the blue front of the sample enters the separation gel, adjust voltage to 130 V.
7.After the blue front of the sample reaches the bottom of the gel, turn the power supply off.
8. Dyeing.

Electrotransformation

1. Thaw electrocompetent cells on ice, keep on ice once thawed
2. Because quickness is important in this procedure, gather the following for each sample:
- one 15 ml culture tube with 1 ml SOC（All tubes should be equilibrated and kept in ~37℃ H2O in an ice bucket.）
- one sterile pasteur pipette
- one sterile electroporation cuvette on ice; either 0.1 or 0.2 cm gap cuvettes may be used
3. Mix DNA ( < 5 ng with low salt content to prevent arcing) with 100 µl cells in an eppendorf tube, keep on ice or alternatively, add DNA to the electro-cuvette (pre-chilled) and then add the electrocompetent cells, making sure that the DNA is mixed well with the cells and skip step 4.
4. Transfer the cell/DNA mix into an electroporation cuvette.
Note: the gene pulser should already be set properly:

- time constant = 4.5 - 5.0 ms
- resistance = 200 W
- capacitance = 25 mFD
（For 0.1 cm gap cuvettes, set the volts to 1.7 kV; for 0.2 cm gap cuvettes, set the volts to 2.5 kV）

5. Pulse the cells once; the voltage displays blinks and the gene pulser beeps.
6. Quickly transfer 37℃ SOC to the cuvette, mix by gently pipetting up and down, and transfer SOC/cells back to the culture tube; replace in 37℃ H2O bath. -At this time you may choose to record the actual volts sent to the cuvette as well as the time constant (these values are determined by pressing the appropriate softkeys on the gene pulser)
7. Incubate for 60 min at 37℃, followed by 120 min at 37℃/shaking
8. Plate cells* on appropriate antibiotic

BCA quantification

1. Standard solution preparation: Take a microplate, add reagents as follow.
2. Sample solution dilution: Estimate the sample concentration, prepare several diluted solutions ensuring that the concentration is in the range of the standard curve.
3. Working solution preparation: Prepare proper volume of BCA working solution with BCA Reagent A : BCA Reagent B = 50 : 1. Mix fully and add 200µl working solution per well.
4. Shake gently for 20s, incubate at 37℃ or 60℃ for 30min and measure A562 using Microplate Reader.
5. According to the reads, draw a standard curve and calculate the function and R2.
6. Calculate sample concentration using the function of the standard curve.

Overlap extension PCR

1. Prepare separate PCR reactions of each part needed to be assembled.
2. Gel-extraction to purify the fragment of correct size.
3. Set up PCR mixture without primers. Use a molar ratio of ~1:1 of fragments as template.
4. The total volume of template should be around 1/2 to 3/4. Example using iV6 HiFi polymerase:
5. Run PCR at around 15 cycles, with annealing temperature of the homologous regions.
6. Proceed a second PCR. Add two primers flanking the outer parts as in normal PCR.
7. Rerun PCR at 30 cycles, with annealing temperature of the flanking primers.
8. Gel-extraction to separate the desired products.

MEGAWHOP (The Megaprimer PCR of Whole Plasmids)

I. MEGAWHOP Reaction Mix (50uL total volume):

II. MEGAWHOP PCR amplification program:

III. DpnI Digest:

Add 20U (1ul) of DpnI (NEB) directly into the PCR reaction. Mix well. Incubate at 37°C for 2-3h. IV. Transforming chemically competent cell:
1. Add 2ul of the DpnI-treated mixture to 50ul competent cells.
2. Ice for 30min.
3. Heat shock at 42°C for 30sec.
4. Ice for 2min.
5. Add 900ul warmed culture medium.
6. Incubate at 37°C with shaking for 1h.
7. Plate 200ul on warmed LB plate containing appropriate antibiotic (option: centrifuge at 4000rpm for 3 min and extract 800ul).

Digestion with restriction enzymes

I. Equipment and Instrument:

1.Thermostatic Water Bath
2.Eppendorf tubes
3.Vortex
4.Pipette and pipette tip
5.Ice bucket
6.Centrifuge

II. Reagent:

1.Restriction enzyme
2.ddH2O
3.10× digestion buffer
4.Plasmid or segment needed

III. Protocol:

1.Thew substrate (plasmids or segments) on ice.
2.Add 18ul ddH2O into a 1.5mL Eppendorf tube.
3.Add 25ul (5ug) substrate into the Eppendorf tube.
4.Add 5ul 10× digestion buffer.
5.Add 1ul restriction enzyme-1 and 1ul restriction enzyme-2 (total volume: 50ul)
6.Vortex the tube and centrifuge shortly.
7.Incubate the mixture in the thermostatic water bath at 37℃ for 40 minutes.

Electrophoresis after digestion

I. Equipment and Instrument:

1.Electrophoresis apparatus
2.Electronic scale
3.Electrophoresis gel sheet
4.Electrophoresis gel tank
5.Comb
6.Conical flask
7.Graduated cylinder
8.Microwave oven

II. Reagent:

1.Agarose
2.TAE buffer
3.DNA marker
4.6× loading buffer
5.Gel-red
6.Enzyme-digested product

III. Protocol:

1.Weigh 20 grams of agarose on a scale.
2.Measure 20ml TAE buffer with graduated cylinder and mix agarose with buffer.
3.Heat the mixture in microwave oven until it boils.
4.Add 2ul gel-red into gel.
5.Put gel-sheet onto gel-tank and place comb; pour gel onto gel-sheet.
6.Allow 30 minutes for the gel to cool.
7.Mix 5ul digested product with 1ul 6× loading buffer.
8.Load 6ul prepared sample and 5ul DNA marker.
9.Start electrophoresis at 150 voltage for 30 minutes.

Ligation

I. Equipment and Instrument:

1.Temperature incubator
2.Eppendorf tube
3.Pipette and pipette tip
4.Vortex
5.Centrifuge
6.Nanodrop

II. Reagent:

1.T4 ligase
2.Gel-extraction product
3.ddH2O
4.10× ligation buffer

III. Protocol:

1. Use nanodrop to measure concentration of gel-extraction products.
2.Add 100ng vector and segment whose amount is three times as vector into an EP tube.
3.Add 2ul T4 ligase and 2ul ligase buffer into the EP tube.
4.Add ddH2O until the total volume is 20ul.
5.Vortex the tube and then centrifuge shortly.
6.Place the tube in the temperature incubator at 16℃ overnight.

Microscopic examination

1. Inoculate the strains of both E. coli and Bacillus subtilis in 5ml of liquid medium and cultivate overnight.
2. 1% transfer and cultivate for 1.5-2h to OD600 0.2-0.4.
3.10%transfer, add inducer(1%IPTG), and then culture to OD600 0.2-0.4.
4. Add 150 ul H2O onto the slide, add 2ul induced bacteria solution, and then cover with the cover slide.
5. Fluorescence microscope observation.