Team:XMU-China/Experiments

Team:XMU-China/Experiments - 2018.igem.org

Model
Project
Model
Molecular Cloning Hardbook
Competition experiment
Aptamer binding protein

● SYL3C-FAM (Sangong)
● EpCAM (ACROBiosystems)
● Binding Buffer (see Appendices)
● Random Sequence-FAM
● 30K ultrafiltration tube (Amicon)
● Black 96-well ELISA plate (Corning)
● Microplate reader (Tecan Infinite M200 Pro)

1. Make distributions in 1.5 mL centrifuge tubes:
1) Tube 1(Negative Control): 7.7 μL 13 μM(100 pmol) RS-FAM +192.3 μL binding buffer.
2) Tube 2(Random Sequence): 7.7 μL 13 μM(100 pmol) RS-FAM +172.3 μL binding buffer.
3) Tube 3(Aptamer Group): 1 μL 100 μM(100 pmol) SYL3C-FAM +179μL binding buffer.
2. Sample processing:
The above 3 tubes are simultaneously subjected to the following steps:
1) Denature at 95℃ for 10 min.
2) Remove the tubes, and then put them on ice for 10 min at 0°C.
3) Put the tubes at room temperature for 10 min.
4) Add 10 μL EpCAM protein to each tube.
5) Incubate all tubes in shaker for 40 min.(37℃, 200 rpm) Note: Avoiding light in whole process is necessary.
3. Ultrafiltration:
The above 3 tubes are simultaneously subjected to the following steps:
1) Add 300 μL binding buffer to each tube so that the volume is 500 μL.
2) Pipette 500 μL solution and add it to 0.5 mL ultrafiltration tube.
3) 14, 000×g centrifuge for 10 min.
4) Collect the filtrate and transfer it to the ELISA plate.
5) Add 400 μL binding buffer to each ultrafiltration tube.
6) Repeat steps 3) to 5) once.
7) Add 200 μL binding buffer to the inner tube of the ultrafiltration tube and pipette up and down to resuspend.
8) Transfer the resuspended solution from the inner tube of the ultrafiltration tube to the ELISA plate.
Note: Avoiding light in whole process is necessary.
4. Measure fluorescence intensity by microplate reader:
1) Set temperature: 30℃, choose plate type: Black.
2) Set excitation wavelength: 495 nm and emission wavelength: 525 nm.
3) Choose mode: Optimal.
4) Place the ELISA plate properly, then start to measure fluorescence intensity.

● Binding Buffer: 1×PBS buffer with 5 mM MgCl2

Forming double strands

● SYL3C-biotin (borui)
● C3-FITC (borui)
● C4-FITC (borui)
● Binding Buffer (see Appendices)
● 50×TAE concentrate Solution (Solarbio)
● Agarose (Biowest)
● DNA dye (TransGen GelStain)
● Distilled water
● Microwave oven
● 10×Loading buffer (Takara)
● Electrophoresis instrument

1. Make distributions in 1.5 mL centrifuge tubes:
1) Tube 1 (Aptamer Group): 1 μL 100 μM (100 pmol) SYL3C-biotin + 19 μL binding buffer.
2) Tube 2 (C4 Group): 1 μL 100 μM (100 pmol) C4-FITC + 19 μL binding buffer.
3) Tube 3 (C3 Group): 1 μL 100 μM (100 pmol) C3-FITC + 19 μL binding buffer.
4) Tube 4 (C4&Aptamer Group): 1 μL 100 μM (100 pmol) SYL3C-biotin +1 μL 100 μM (100 pmol) C4-FITC + 18 μL binding buffer.
5) Tube 5 (C3&Aptamer Group): 1 μL 100 μM (100 pmol) SYL3C-biotin +1 μL 100 μM (100 pmol) C3-FITC + 18 μL binding buffer.
Note: The Tube 2, 3, 4 and 5 should be coated by tin foil properly after distributing.
2. Sample processing:
The above 5 tubes are simultaneously subjected to the following steps:
1) Denature at 95℃ for 10 min.
2) Anneal to room temperature for 20 min.
3. Prepare 3% agarose gel:
1) Weigh 0.9 g agarose in a 100 mL Erlenmeyer flask.
2) Add 30 mL 1×TAE buffer into the flask from 1).
3) Make agarose melt by microwave oven (medium-high heat, about 3 min).
4) Add 3 μL GelStain, mix by shocking.
5) Assemble gel pouring apparatus by inserting gate into slots.
6) Pour agarose gel into the gel tray.
7) Cool for 40 min to solidify the DNA agarose gel.
8) Remove the pouring apparatus, put the gel into an electrophoresis instrument.
4. Electrophoresis:
1) Add 2 μL 10×Loading buffer to each tube, then pipette up and down to evenly mix.
2) Pipette all samples which have been mixed with loading buffer into the slots.
3) Turn on the electrophoresis instrument, set the working electric current at 150 mA and the working electric voltage at 110 V.
4) Electrophoresis for 50 min.
5. Gel imaging:
 Turn off the instrument, take the gel into the gel formatter to take and save photos.

● Binding Buffer: 1×PBS buffer with 5 mM MgCl2

Competition

● SYL3C-biotin (Borui)
● C3-FITC (Borui)
● EpCAM (ACROBiosystems)
● Magnetic Beads (Invitrogen)
● Binding & Washing Buffer (see Appendices)
● Incubation Buffer (see Appendices)
● Vortex (IKA lab dancer)
● Fluorescence Spectrometer (Shimadzu RF-6000)

1. Make distributions in 1.5 mL centrifuge tubes:
1) Tube 1 (Blank Control): 100 μL incubation buffer.
2) Tube 2 (Negative Control): 25 μL diluted SYL3C-biotin + 25 μL 1×B&W buffer.
3) Tube 3 (Competition Group): 25 μL diluted SYL3C-biotin + 25 μL diluted C3-FITC.
4) Tube 4 (Competition Group without EpCAM): 25 μL diluted SYL3C-biotin + 25 μL diluted C3-FITC.
5) Tube 5 (Positive Control): 0.6 μL 100 μM C3-FITC + 100 μL incubation buffer. Note: The Tube 3, 4 and 5 should be coated by tin foil properly after distributing.
2. Denature, anneal and renature:
The Tube 2, 3, and 4 are denatured at 95°C for 5 min, then anneal to room temperature.

3. Wash magnetic beads:
1) Resuspend the beads and vortex for 10 seconds to make them even.
2) Transfer 75 μL 10 mg / mL beads to 3 200 μL centrifuge tubes (ie 25 μL per tube).
3) Add 25 μL 2×B&W buffer to each tube.
4) Vortex for 5 seconds to resuspend.
5) Place the tubes in a magnetic field for 1 minute, then aspirate the supernatant.
6) Remove the tubes from the magnetic field and add 50 μL 1×B&W buffer to each tube.
7) Repeat steps 4) to 5).
8) Add 50 μL 1×B&W buffer to each tube.
4. Bind to magnetic beads
1) Add 50 μL solution of Tube 2,3 and 4 from step 2 to 50 μL washed beads, respectively.
2) Vortex for 5 seconds to resuspend.
3) Incubate at room temperature for 30 min in the dark.
4) Vortex for 5 seconds to resuspend.
5) Place the tubes in a magnetic field for 2 minute, then aspirate the supernatant.
6) Remove the tubes from the magnetic field and add 100 μL 1×B&W buffer to each tube.
7) Repeat steps 4) to 5).
8) Add 100 μL incubation buffer to Tube2, 3 and 4, respectively.
5. Compete:
1) Add 17 μL diluted EpCAM to Tube 2 and 3.
2) Incubate at room temperature for 40 min in the dark.
3) Place the tubes in a magnetic field for 2~3 min, and then pipette 100 μL of the supernatant into a new 200 μL centrifuge tube and mark it.
4) Measure the fluorescence intensity of Tube 1, 5 and the supernatant from 3).
Note: Before measuring, the solution of each tube will be further diluted by adding 3, 000 μL incubation buffer into it.

● 2×Binding & Washing Buffer (pH 7.5) contains: 10 mM Tris 1 mM EDTA 2 M NaCl
● Incubation Buffer (pH 7.5) contains: 20 mM Tris 140 mM NaCl 5 mM KCl 1 mM MgCl2

The reaction rate equation of competition

● SYL3C-biotin (Borui)
● C3-FITC (Borui)
● EpCAM (ACROBiosystems)
● Magnetic Beads (Invitrogen)
● Binding & Washing Buffer (see Appendices)
● Incubation Buffer (see Appendices)
● Vortex (IKA lab dancer)
● Fluorescence Spectrometer (Shimadzu RF-6000)

1. Make distributions in 1.5 mL centrifuge tubes according to the following:
1) Tube 1 (0 min): 25 μL diluted SYL3C-biotin + 25 μL diluted C3-FITC.
2) Tube 2 (1 min): 25 μL diluted SYL3C-biotin + 25 μL diluted C3-FITC.
3) Tube 3 (4 min): 25 μL diluted SYL3C-biotin + 25 μL diluted C3-FITC.
4) Tube 4 (10 min): 25 μL diluted SYL3C-biotin + 25 μL diluted C3-FITC.
5) Tube 5 (40 min): 25 μL diluted SYL3C-biotin + 25 μL diluted C3-FITC.
Note: All tubes should be coated by tin foil properly after distributing.
2. Denature, anneal and renature:
All tubes are denatured at 95°C for 5 min, then anneal to room temperature. 3. Wash magnetic beads:
1) Resuspend the beads and vortex for 10 seconds to make them even.
2) Transfer 125 μL 10 mg / mL beads to 5 200 μL centrifuge tubes (ie 25 μL per tube).
3) Add 25 μL 2×B&W buffer to each tube.
4) Vortex for 5 seconds to resuspend.
5) Place the tubes in a magnetic field for 1 minute, then aspirate the supernatant.
6) Remove the tubes from the magnetic field and add 50 μL 1×B&W buffer to each tube.
7) Repeat steps 4) to 5).
8) Add 50 μL 1×B&W buffer to each tube.
4. Bind to magnetic beads
1) Add 50 μL solution of each tube from step 2 to 50 μL washed beads, respectively.
2) Vortex for 5 seconds to resuspend.
3) Incubate at room temperature for 30 min in the dark.
4) Vortex for 5 seconds to resuspend.
5) Place the tubes in a magnetic field for 2 min, then aspirate the supernatant.
6) Remove the tubes from the magnetic field and add 100 μL 1×B&W buffer to each tube.
7) Repeat steps 4) to 5).
8) Add 100 μL incubation buffer to each tube.
5. Compete:
1) Add 17 μL diluted EpCAM to each tube.
2) Incubate at room temperature for 0, 1, 4, 10, 40 min in the dark, respectively.
3) Place the tubes in a magnetic field, then pipette 100 μL of the supernatant into a new 200 μL centrifuge tube and mark it.
4) Measure the fluorescence intensity of the supernatant from 3).
Note: Before measuring, the solution of each tube will be further diluted by adding 3, 000 μL incubation buffer into it.

● 2×Binding & Washing Buffer (pH 7.5) contains: 10 mM Tris 1 mM EDTA 2 M NaCl
● Incubation Buffer (pH 7.5) contains: 20 mM Tris 140 mM NaCl 5 mM KCl 1 mM MgCl2

Characterization

● LbCpf1 (NEB)
● crRNA (Sangong)
● 1×Binding Buffer (see Appendices)
● DNaseAlert Kit (IDT)
● Supernatant from competition
● Fluorescence Spectrometer (Shimadzu RF-6000)

1. Pretreatment of LbCpf1-crRNA
1) Incubate LbCpf1 with crRNA at 37℃ for 30 min to form the LbCpf1-crRNA complex.
2) Dilute the complex by 1×Binding Buffer.
2. Pretreatment of Substrate:
1) Add 5 μL of Nuclease-Free Water to a DNaseAlert Substrate single-use tube for each assay.
2) Add 5 μL of 10×DNaseAlert Buffer to each tube.
3. Make ditributions
1) Sample Group: LbCas12a-crRNA + Supernatant from competition + DNaseAlert Substrate from step 2.
2) Negative Control: LbCas12a-crRNA + DNaseAlert Substrate from step 2.
3) Positive Control: DNase I + Supernatant from competition + DNaseAlert Substrate from step 2.
4. Measure
Incubate the reaction system at 37℃ for 2 hours, and measure fluorescence intensity once every 30 seconds.

● Binding Buffer (pH 7.3~7.5)contains:
1×PBS buffer with 5 mM MgCl2
● DNaseAlert Kit contains:
1) DNaseAlert Substrate (25 single-use tubes (50 pmol per tube) or 2 tubes bulk substrate (2 nmol per tube))
2) DNaseAlert Buffer (250 μL)
3) Nuclease-Free Water (2 mL)
4) DNase I Enzyme (25 μL)
5) Nuclease Decontamination Solution (50 mL)

OMVs
Preparation of OMVs-free culture medium

● 0.45 m syringe filters
● LB broth culture
● Optima XE-90 Ultracentrifuge (Beckman Coulter)
● SW41 Ti Swinging-Bucket Rotor

1. Prepare 100 mL 10×LB broth medium.
2. Pass the culture through a 0.45 m syringe filter.
3. Ultracentrifuge each tube for 18 hours at 100,000 g in a Swinging-Bucket rotor to precipitate the biomass.
4. Recover the supernatant of each tube and dilute them to 1×LB broth culture.
5. Sterilize the 1×LB broth culture.

OMVs Extraction for HSFCM[1] analysis and electron microscope analysis

● PBS
● 0.45 m syringe filters
● OMVs-free LB broth culture
● OptimaTM Max-XP Ultracentrifuge (Beckman Coulter)
● TLA120.2 Rotor

a. Bacteria Growth
1. Inoculate each strain for analysis to 10 mL OMVs-free culture with appropriate antibiotic, and place them on a shaking incubator at 37 °C, 200 rpm.
2. After about 5 hours of incubation (when the OD600 of each culture was 0.6-0.8), add any necessary inducing agents (IPTG at 0.5 mM or Arabinose at a 0.2% final concentration) into the cultures, and then leave them on the shaking incubator at 37 °C, 200 rpm overnight.
b. Cell Extraction
1. Measure the OD600 of each culture before OMVs harvest.
2. Centrifuge each culture for 10 min at 1,000 g and then 10 min at 2,000 g in a fixed angle rotor to pellet the biomass.
3. Recover the supernatants and then pass them through a 0.45 m syringe filter.
c. OMVs Purification
1. Load 10 mL syringe-filtered supernatant into ultracentrifugal tubes, and then ultracentrifuge them at 100,000 g in a TLA120.2 rotor for 17 min at 4 °C。
2. Discard the supernatant, add enough PBS buffer and repeat the step 1 above, and then discard the supernatant again.
3. Add 100 L PBS into the tubes and then pipet up and down for about 200 times.
4. OMVs are stored at 4 °C until analysis.

[1] https://pubs.acs.org/doi/10.1021/nn505162u

OMVs Extraction for SDS-PAGE analysis

● PBS
● 0.45 m syringe filters
● OMVs-free LB broth culture
● Optima XE-90 Ultracentrifuge (Beckman Coulter)
● SW41 Ti Swinging-Bucket Rotor

a. Bacterial Growth
1. Inoculate each strain for analysis into 100 mL OMVs-free culture with appropriate antibiotic, and then place them on a shaking incubator at 37 °C, 200 rpm.
2. After about 5 hours of incubation (when the OD600 of each culture was 0.6-0.8), add any necessary inducing agents (IPTG at 0.5 mM or Arabinose at a 0.2% final concentration) into the cultures, and leave them on the shaking incubator at 37 °C overnight.
b. Cell Extraction
1. Measure the OD600 of each culture before OMVs harvest.
2. Centrifuge each culture for 10 min at 1,000 g and then 10 min at 2,000 g in a fixed angle rotor to pellet the biomass.
3. Recover the supernatants and then pass them through a 0.45 m syringe filter.
c. OMVs Purification
1. Load 100 mL syringe-filtered supernatant into ultracentrifugal tubes, and then ultracentrifuge them at 100,000 g in a Swinging-Bucket rotor for 2 hours at 4 °C。 2. Recover the supernatant, repeat the step 1 above, and then discard the supernatant.
3. Add 100 L PBS into the tube and then pipet up and down for about 200 times.
4. OMVs are stored at 4 °C until analysis.
d. SDS-PAGE
1. Prepare the 1×Tris-Glycine electrophoresis buffer (2 L)
2. Add 5×Protein loading buffer (V/V) to the OMVs pellet, and then pipet up and down to mix the sample.
3. Put the tube in 100 °C water bath for 10 min.
4. Make the gel for SDS-PAGE according to the manufacture’ s instructions.
5. Add 10 L protein marker to the first well and add about 25 L samples to other wells.
*Tips: You must keep enough sample loaded in every well. You can add 25 L firstly and after the sample pass through the stacking gel, stop the instrument immediately and add 25 L sample again.
6. Start the electrophoresis apparatus, the parameters are showed below:
7. Turn off the instrument, take the gel into a clean plate with deionized water, wash it gently and then discard the water.
8. Add the Coomassie blue stain into the plate and then heat it to about 50 °C.
9. Incubate for 30 min on a shaking bed.
10. Discard the stain and add the destaining solution (acetic acid: ethyl alcohol: deionized water=10: 50: 40, V/V/V), then heat it to about 50 °C.
11. Incubate for 30 min on a shaking bed.
12. Repeat step10-11 for 3 times.
13. Take the gel into the gel formatter to take and save photos.

Evaluate the efficiency of the C/Dbox

● PBS
● 0.45 m syringe filters
● LB broth culture
● OptimaTM Max-XP Ultracentrifuge (Beckman Coulter)
● TLA120.2 Rotor

a. Bacterial Growth
1. Inoculate each strain for analysis into 100 mL OMVs-free culture with appropriate antibiotic, and then place them on a shaking incubator at 37 °C, 200 rpm.
2. After about 5 hours of incubation (when the OD600 of each culture was 0.6-0.8), add arabinose at a 0.2% final concentration to initiate the production of siRNA-C/Dbox.
After an additional 3 h incubation period, add IPTG to a final concentration of 0.5 mM to initiate the production of L7Ae-SpyCatcher. Incubate the culture at 37 °C and 200 rpm for an additional 18 h.
b. Cell Extraction
1. Measure the OD600 of each culture before OMVs harvest.
2. Centrifuge each culture for 10 min at 1,000 g and then 10 min at 2,000 g in a fixed angle rotor to pellet the biomass.
3. Recover the supernatants and then pass them through a 0.45 m syringe filter.
c. OMVs Purification
1. Load 100 mL syringe-filtered supernatant into ultracentrifugal tubes, and then ultracentrifuge them at 100,000 g in a Swinging-Bucket rotor for 2 hours at 4 °C.
2. Discard the supernatant, add enough PBS buffer and repeat the step one above, then discard the supernatant again.
3. Add 100 L PBS into the tube and then pipet up and down for about 200 times.
4. OMVs are stored at 4 °C until analysis.
d. RNASelect dye
The experiment below is designed according to the manufacture’s instruction.[2]
1. Prepare a 1 mM DMSO stock solution of the SYTOTM RNASelectTM.
2. Add 1 L of the dye stock solution to the 100 L OMVs sample and mix them to obtain a final dye concentration of 10 M. Incubate it at 37 °C for 20 min (protect from light).
3. Remove excess unincorporated dye from the labeled OMVs with ultracentrifugation at 100, 000 g, 17 min, 4 °C. (Twice)
4. Analyze the efficiency of OMVs labeling using HSFCM.

[2] https://www.thermofisher.com/order/catalog/product/S32703

Addenda
Motor speed testing

● PBS solution with protein EpCam (imitative plasma)
● Microfluidic chips

1. Pipette appropriate imitative plasma solution into the sample chamber;
2. Gradually increase the motor speed by software and find the critical speed for the second chamber in which the competition reaction happens.
3. Find a second critical speed for the third chamber-incubation chamber

Combine the aptamer with ssDNA

● ssDNA (10 nt)
● Biotinylated aptamers
● PBS contained Mg2+ (5.0 mmol)
● 100× SYBR Green Ⅰ (exited by 488 nm)
● Microplate Reader (Bio ?)

1. Mix equivalent aptamer and ssDNA (dissolved in the PBS with Mg2+)
2. Keep the solution away from light at room temperature/ 37℃ water bath / PCR 95 ℃ for 10 min
3. Mix 100×SYBR Green Ⅰ with the mixed solution for 20 min at room temperature
4. Use Microplate Reader to detect the fluorescence of the solution (using the solution contained only aptamer or ssDNA as blank control)

Ternary affinity coating (TERACOAT) method

● Biotinylated BSA
● NeutrAvidin
● Combined aptamer-ssDNA(with the fluorophore)
● PBS-0.1% Tween 20

1. Pour and coat Biotinylated BSA on our device for 10 min;
2. Wash the remaining Biotinylated BSA with PBS-Tween for 5 min;
3. Pour and coat Neutravidin on our device for 10 min;
4. Wash the remaining Neutravidin with PBS-Tween for 5 min;
5. Pour and coat Combined aptamers-ssDNA on our device for 10 min;
6. Wash the remaining Neutravidin with PBS-Tween for 5 min.
7. Detect the fluorescence of washing solution from step 6 to find whether ssDNA is tightly bonded to aptamer.

[1] Piraino, Francescoet, et al, ACS nano 2016, 10, 1699-1710

Fluorescence detection for Epcam

● Coated microfuidic chips
● EpCam
● PBS-0.1% Tween 20
● Cas12a & crDNA systems

1. Pipette appropriate imitative plasma solution into the sample chamber.
2. Control the motor speed and make the solution flow into the competitive chamber, and then remain for 20 min at room temperature.
3. Increase the motor speed and make the solution flow into the detection chamber, and then remain for 10 min at room temperature.
4. Collect the solution from the last chamber and detect whether there is any fluorescence.
5. Use the camera of the hardware to find out whether there is any fluorescence and compare the data.

Supporting project

1. Prepare polyacrylamide gel according to standard protocol. The parameters are showed below:

Stage Voltage (V) Current (mA) Time (min)
Ⅰ 100 80 30
Ⅱ 150 100 40

2. At this point, the gel can either be transferred to a membrane or stained with Coomassie blue (see below).
3. Place gel in a plastic container, cover over with isopropanol fixing solution and shake at room temperature (RT). For 0.75 mm-thick gels, shake for 10 to 15 min; for 1.5 mm thick gels, shake for 30 to 60 min.
4. Pour off fixing solution, cover over with Coomassie blue staining solution and shake at RT for 2 h.
5. Pour off staining solution, wash the gel with 10% acetic acid to destain and shaking at RT overnight.

1. Transformation:
1) Transform E. coli BL21star (DE3) with pET28a + SAHS plasmids.
2) Inoculate each single colon with 10 mL of LB broth culture with kanamycin at 60 mg/mL.
3) Shake each culture at 200 rpm, 37 ˚C overnight
2. Induced expression:
1) After about 3 hours of incubation (when the OD600 reaches to 0.5), add IPTG at a final concentration for 1 mM to induce expression.
2) Incubate the cultures for 8 h and pellet the cells at 7,000g, 25 ˚C for 10 min.
3. Centrifugation and lysis:
1) Store the cells pellet at -20 ˚C until use.
2) Resuspend the pellets in 12.5 mL PBS.
3) Lyse the cells by Ultrasonic Cell Disruptor.
4) Cool the lysates at 4 ˚C for 30 min.
5) Remove insoluble components by centrifugation at 25,000g, 4 ˚C for 20 min.
6) Harvest the supernatants to purify the SAHS protein.
4. Chromatography:
1) Equilibrate the Ni-NTA Agarose with PBS and then adjust the A280 value to the neutral line.
2) Load appropriate amount of supernatant into the Ni-NTA Agarose column and wash with lysis buffer until the A280 value is below 0.01.
3) Clean the column with PBS until the A280 value is below 0.01.
4) Elute the SAHS protein with Elution buffer and collect all eluates according to its A280.
5. Heat treatment: Collect all equates and place them in water bath at 85 ˚C for 15min. Remove insoluble components by centrifugation at 13,000g for 20 min.

1) Step 1. Preferably, select single colony of E. coli from fresh LB plate for inoculating a 10 mL 2XYT overnight (O/N) starter culture. Alternatively, streak out frozen glycerol stock of bacterial cells onto LB plate, grow plate O/N, and then select single colony for starter culture. Grow 10 mL starter culture O/N in 37 °C shaker (250 rpm).
2) Step 2. Inoculate 1L of 2XYT media and place culture in 37 °C shaker. Grow cells and measure OD600 every 45 min. When the OD600 equals 0.6-0.9 (log phase growth), remove the cells from the shaker and place on ice.
NOTE: It is very important to keep the cells at 4°C (or on ice) for the remainder of the procedure.
3) Step 3. Split the 1 L culture into four equal parts by pouring ~250 mL of culture into each chilled 250mL Corning pointed bottle.
4) Step 4. Spin (#1) in GPR centrifuge at 4000 rpm, 25 min at 4 °C.
(if you chose to use the J6/ JS-4.2 rotor (E. Davidson Lab), use 1L bottles , fill half full, spin 4000rpm, 20min, at 4°C.)
5) Step 5. Place bottles on ice. Remove supernatant immediately as cell pellet begins to lift off quickly. Gently resuspend each pellet in 200 mL ice-cold dH20.
6) Step 6. Spin (#2) in GPR centrifuge at 4000 rpm, 25 min at 4°C.
7) Step 7. Place bottles on ice. Remove supernate. Gently resuspend each pellet in 100ml of ice-cold dH20.
8) Step 8. Spin (#3) in GPR centrifuge at 4000rpm, 25min at 4°C.
9) Step 9. Place bottles on ice. Remove supernatant. Gently resuspend each pellet in 20mL ice-cold 10% glycerol. For each pair of 250 mL Corning bottles, transfer both 20 mL cell suspension into one chilled 50 mL conical tube- therefore you should end up with two 50 mL conical tubes on ice where each tube contains ~40 mL of cells in 10% glycerol.
10) Step 10. Spin (#4) in GPR centrifuge at 4000 rpm, 10 min at 4 °C.
11) Step 11. Place tubes on ice. Remove supernatant. Gently resuspend each cell pellet in 1 mL of ice-cold 10% glycerol. Final OD600 of resuspended cells » 200-250.
12) Step 12. With cell suspensions on ice, prepared 70l aliquots of cells in pre-chilled 1.5 mL eppendorf tubes. Snap freeze tubes containing cells in liquid N2. Store frozen cells at -80 °C.
NOTE: liquid N2 very hazardous- use caution and don't contact N2 directly!

1. Thaw electrocompetent cells on ice.
2. Transfer 50 mL of electrocompetent cells to a pre-chilled electroporation cuvette with 1 mm gap.
3. Add 1 mL of the assembly product to electrocompetent cells.
4. Mix gently by pipetting up and down.
5. Once DNA is added to the cells, electroporation can be carried out immediately. It is not necessary to incubate DNA with cells.
6. Add 950 mL of room-temperature SOC media to the cuvette immediately after electroporation.
7. Place the tube at 37 °C for 60 minutes. Shake vigorously (250 rpm) or rotate.
8. Warm selection plates to 37 °C.
9. Spread 100 mL of the cells onto the plates.
10. Incubate overnight at 37 °C.

1. Transfer the plasmid pKD46 into E.coli DH5α, and Incubate at 30° for 12h.
2. Make electro-competent cell with E.coli DH5α contains pKD46.
3. PCR amplification of CAHS fragments, and add two segments homologous arm.
4. DNA gel electrophoresis and gel extraction and purification.
5. Transfer the fragments into the electro-competent cell.
6. Add 100 mM L-Arabinose, then induced for 90min at 37°.
7. Activate for 2h in 37° shaking incubator.
8. Add 1 M IPTG to make the final concentration to be 1 mM.
9. Add 250 L of the bacteria solution to plate.
10. Incubate at 37°C.
11. That the colony’s color turning to be red is regarded as success.

1. Set up the following reaction on ice:
2. Incubate samples in a thermocycler at 50 °C for 15 minutes when 2 or 3 fragments are being assembled or 60 minutes when 4-6 fragments are being assembled. Following incubation, store samples on ice or at –20 °C for subsequent transformation.
Note: Extended incubation up to 60 minutes may help to improve assembly efficiency in some cases (for further details see FAQ section).
3. Transform NEB 5-alpha Competent E. coli cells (provided with the kit) with 2 L of the assembly reaction, following the transformation protocol.

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