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Experimental Protocols

Preparation and Analysis of pET-14b-EGFP Vectors and Toehold Switches

Maxiprep pET-14b-EGFP

Materials

EndoFree Maxi Plasmid Kit, TIANGEN (Cat.no. GDP117), centrifuge (all centrifuge steps should be carried out at 8,000 RPM)

Procedures

  1. Harvest 100 ml overnight cultured bacterial cells by centrifuge for 3 min at room temperature (15-30°C), and then remove all traces of supernatant.
  2. Resuspend pellet bacterial cells in 8 ml Buffer P1 (Ensure that RNase A has been added). The bacteria should be resuspended completely by vortex or pipetting up and down until no cell clumps remain.
  3. Add 8 ml Buffer P2 and mix thoroughly by inverting the tube 6-8 times, then incubate at room temperature for 5 min. Mix by inverting the tube. Do not vortex.
  4. Add 8 ml Buffer P4, and mix immediately and thoroughly by gently inverting 8 times, until the whole solution becomes cloudy. Incubate at room temperature for 10 min.
  5. Centrifuge for 10 min, the white material should be in the bottom of the centrifuge tube (prolong centrifugation time properly). Transfer the supernatant into a Filtration CS1.
  6. Add 0.3× volume isopropanol to the cleared lysate (too much isopropanol will lead to RNA contamination), mix completely by reverting upside down and then transfer all solution to the Spin Column CP6 (put Spin Column CP6 into 50 ml Collection Tube).
  7. Centrifuge for 2 min. Discard the flow-through and place the Spin Column CP6 back into the same Collection Tube. Note: Centrifuge for two times under the above condition.
  8. Add 10 ml Buffer PW (Ensure ethanol has been added before use) to the Spin Column CP6 and centrifuge for 2 min.
  9. Discard the flow-through and place the Spin Column CP6 back into the same Collection Tube. Repeat this step.
  10. Add 3 ml 100% ethanol to the Spin Column CP6 (put the CP6 in a Collection Tube). Centrifuge for 2 min, discard the flow-through.
  11. Put Spin Column CP6 back to the Collection Tube, centrifuge for 5 min for removing residual ethanol.
  12. To elute DNA, place the Spin Column CP6 in a clean 50 ml Collection Tube (supplied in the kit) and add 1-2 ml Buffer TB to the center of the membrane and incubate 5 min at room temperature, centrifuge for 2 min.
  13. Transfer the eluate from 50 ml centrifuge tube to a clean 1.5 ml centrifuge tube.
  14. Perform the DNA sequencing and nanodrop concentration testing.

Restriction Digestion of pET-14b-EGFP

Materials

20,000 units/ml NcoI-HF (NEB, R3193S), 20,000 units/ml NdeI (NEB, R0111S), 10X rCutSmart buffer (NEB, B6004S), Nuclease-free water, 300ng/ul pET-14b-EGFP vectors

Procedures

Set up reaction as follows:

Component 50 ul Reaction
pET-14b-EGFP 7 ul (2 ug)
10X rCutSmart buffer 5 ul (1X)
NcoI-HF 1 ul (20 units)
NdeI 1 ul (20 units)
Nuclease-free water 36 ul

Place the samples in a 37℃ incubator for 2 hours. Stop the reaction by adding 10 µL of purple loading dye (6X), and run gel electrophoresis to isolate cut vector fragments.

Gel Electrophoresis

Materials

Agarose powder, 1x TAE buffer, gel electrophoresis system, 100-5000 bp DNA ladder, 10,000X SYBR®Safe DNA Stain (Invitrogen, S33102)

Procedures

Prepare 1% agarose gels using a 1x TAE buffer. Melt agarose by heating, then cool it to 60℃. Add SYBR®Safe DNA Stain at a 1:10,000 dilution.

Run gel electrophoresis of the digested DNA plasmid using 120 volts for around 30 mins.

Gel Purification of Digested pET-14b-EGFP

Materials

Monarch® Spin DNA Gel Extraction Kit (NEB, T1120S), Blue light transilluminator, Scalpel, Microcentrifuge (all centrifugation steps should be carried out at 13,000 RPM)

Procedures

  1. The gel fragments (DNA bands at 5 kb) can be excised using a scalpel under a blue light transilluminator and stored at -20°C if time is limited. Take care to trim any excess agarose.
  2. Transfer the gel slice to a 1.5 ml microfuge tube and weigh it. Calculate the amounts of Monarch buffer BY and WZ according to the ratio: 400 µl of buffer BY and 200 µl of buffer WZ per 100 mg of agarose.
  3. Add buffer BY to the gel slice. Incubate the sample at 50 ℃ without vortexing. The gel slice will typically dissolve completely within 5–10 minutes.
  4. Insert the Monarch Spin Column into the collection tube and load the sample onto the column. Spin for 1 minute, then discard the flow-through.
  5. Re-insert the column into the collection tube. Wash by adding buffer WZ, wait for 2 minutes, and spin for 1 minute. Then discard the flow-through.
  6. Repeat wash (step 5).
  7. Transfer the column to a clean 1.5 ml tube. Take care to ensure that the tip of the column does not touch the filter.
  8. Add 20 ul of buffer EY to the center of the matrix to elute the DNA. Wait for 2 minutes, and spin for 1 minute.

Ligation of pET-14b-EGFP and Toehold Switch Oligos

Toehold Switches 1 and 2

Materials

4.5 ng/uL Oligos of Toehold Switches 1 and 2 (insert DNA), T4 DNA Ligase Buffer (10X), 12.5 ng/uL digested pET-14b-EGFP (vector DNA), uncut plasmid, Nuclease-free water, 40,000 unit/ml T4 DNA Ligase

Procedures

Prepare tubes of a 4.5 ng/uL oligos solution (Toehold Switches 1 and 2) that are in powder form.

Set up the following reaction in a microcentrifuge tube on ice, T4 DNA ligase should be added last (1:10 insert:vector).

Component Negative Control Toehold Switch 1 Toehold Switch 2 Positive Control
Vector DNA (ul) 8 8 8 /
Toehold Switch 1 oligo (ul) / 4.5 / /
Toehold Switch 2 oligo (ul) / / 4.5 /
Ligase buffer (ul) 2 2 2 /
T4 DNA ligase (ul) 1 1 1 /
Uncut plasmid (ul) / / / 1
Water (ul) 9 4.5 4.5 19

Overnight ligation (2 hours) at room temperature.

Toehold Switches 7, 8, and 9

Materials

4.5 ng/uL Oligos of Toehold Switches 7, 8, and 9 (insert DNA), T4 DNA Ligase Buffer (10X), 12.5 ng/uL digested pET-14b-EGFP (vector DNA), uncut plasmid, Nuclease-free water, 40,000 unit/ml T4 DNA Ligase

Procedures

  1. Prepare tubes of a 4.5 ng/uL oligos solution (Toehold Switches 7, 8, and 9) that are in powder form.
  2. Set up the following reaction in a microcentrifuge tube on ice, T4 DNA ligase should be added last (1:20 vector:insert).
Component Negative Control Toehold Switch 7 Toehold Switch 8 Toehold Switch 9 Positive Control
Vector DNA (ul) 8 8 8 8 /
Toehold Switch 7 oligo (ul) / 9 / / /
Toehold Switch 8 oligo (ul) / / 9 / /
Toehold Switch 9 oligo (ul) / / / 9 /
Ligase buffer (ul) 2 2 2 2 /
T4 DNA ligase (ul) 1 1 1 1 /
Uncut plasmid (ul) / / / / 1
Water (ul) 9 / / / 19

Overnight ligation (16 hours) at room temperature.

Transformation of E.coli with Plasmids

Materials

E.coli DH5α Competent cells, water bath, ice bath, LB broth medium, Ampicillin agar plate

Procedures

  1. Thaw E.coli DH5α Competent cells in an ice bath just before use.
  2. Gently mix cells and aliquot 50 ul into a 1.5 ml tube. It is important not to use a vortex to mix cells.
  3. Add 20 µL of ligation DNA samples and mix gently but thoroughly.
  4. Keep in an ice bath for 30 minutes.
  5. Incubate cells for 45 seconds at 42 °C.
  6. Return to the ice bath for 2 minutes.
  7. Add LB broth medium (pre-incubated at 37 °C) up to a final volume of 1 ml.
  8. Perform a recovery step by shaking (160-225 rpm) for 2 hours at 37 °C.
  9. Spin down bacteria and remove 900 ul LB broth medium. Resuspend the pellet and plate 100 ul of culture on an Ampicillin agar plate.
  10. Incubate overnight at 37 °C.

Isolation of Single Colony and Inoculation

Materials

Bunsen burner, ampicillin (100mg/ml) LB agar plate, disposable inoculation loop, LB medium with Ampicillin (100mg/ml), 75% alcohol

Procedures

  1. Sterilize the workbench with 75% alcohol. Turn on a Bunsen burner to create an aseptic area. Use a disposable inoculation loop to pick up a single colony and culture it in LB broth medium containing ampicillin. Incubate by shaking (160-225 rpm) at 37 °C for 16 to 18 hours.
  2. Use a disposable inoculation loop to streak the culture on an LB agar plate containing ampicillin. Incubate at 37 °C for 16 to 18 hours.
  3. Use a disposable inoculation loop to pick up 3 individual colonies, and add each to 5 ml LB broth medium containing ampicillin. Incubate by shaking (160-225 rpm) at 37 °C for 16 to 18 hours.

Miniprep Plasmids

Materials

Monarch Spin Plasmid Miniprep Kit (NEB, T1110S), centrifuge (all centrifuge steps should be carried out at 13,000 RPM)

Procedures

  1. Spin down 5 ml of culture and remove supernatant.
  2. Resuspend the pellet in 200 ul of Monarch buffer B1.
  3. Add 200 ul of buffer B2, gently invert the tube 5-6 times, and incubate at room temperature for 1 minute. Do not vortex.
  4. Add 400 ul of buffer B3, and gently invert the tube until neutralized. Do not vortex.
  5. Centrifuge the lysate for 5 minutes.
  6. Carefully transfer the supernatant to the column and centrifuge for 1 minute. Discard flow-through.
  7. Re-insert the column in the collection tube and add 200 ul of buffer BZ. Centrifuge for 1 minute.
  8. Wash by adding 400 ul of buffer WZ and centrifuge for 1 minute.
  9. Transfer the column to a clean 1.5 ml microfuge tube. Add 30 ul of buffer EY to the center of the matrix. Wait for 1 minute, then spin for 1 minute to elute DNA.
  10. Perform the DNA sequencing and nanodrop concentration testing.

Optimisation of Positive Control

Materials

In Vitro Expression kit (PUREexpress kit) (NEB, E6800S), 333 ng/uL Plasmid DNA from miniprep

Procedures

Assemble the reaction in 5 tubes on ice using the contents listed in the table below:

Component Tube A Tube B Tube C Tube D Tube E
PURExpress kit Reagent A (uL) 2 2 2 2 2
PURExpress kit Reagent B (uL) 1.5 1.5 1.5 1.5 1.5
Nuclease Free water (uL) / 0.6 / / /
pET-14b-EGFP (uL) 1.5 (333ng/uL) 0.9 (333ng/uL) 1.5 (187.5ng/uL) 1.5 (37.5ng/uL) 1.5 (7.5ng/uL)
Total (uL) 5.0 5.0 5.0 5.0 5.0
  1. Mix gently and pulse-spin in microfuge to collect mixture at the bottom of the tube.
  2. Incubate all the tubes at 37°C for 2 hours.
  3. Stop the reaction by placing the tubes on ice.
  4. Measure the fluorescence intensity of each tube with a plate reader (excitation: ~480 nm, emission: ~510 nm): The content of each of the tubes was transferred to a separate well on a black plate. PBS was added.

In Vitro Expression of Toehold Switch Plasmids

Materials

5 uM miRNA, In Vitro Expression kit (PUREexpress kit) (NEB, E6800S), 7.5 ng/uL Plasmid DNA from miniprep

Procedures

Assemble the reaction in 10 tubes on ice using the contents listed in the table below:

No miRNA

Component Tube 1 Tube 2 Tube 3 Tube 4 Tube 5
PURExpress kit Reagent A (uL) 4 4 4 4 4
PURExpress kit Reagent B (uL) 3 3 3 3 3
Toehold Switch 1 Plasmid (uL) / 2 / / /
Toehold Switch 2 Plasmid (uL) / / 2 / /
Toehold Switch 3 Plasmid (uL) / / / 2 /
pET-14b-EGFP (uL) / / / / 2
Nuclease-free water (uL) 4 2 2 2 2
Total (uL) 11 11 11 11 11

With miRNA

Component Tube 6 Tube 7 Tube 8 Tube 9 Tube 10
PURExpress kit Reagent A (uL) 4 4 4 4 4
PURExpress kit Reagent B (uL) 3 3 3 3 3
Toehold Switch 1 Plasmid (uL) / 2 / / /
Toehold Switch 2 Plasmid (uL) / / 2 / /
Toehold Switch 3 Plasmid (uL) / / / 2 /
pET-14b-EGFP (uL) / / / / 2
Nuclease-free water (uL) 2 / / / /
miRNA (uL) 2 2 2 2 2
Total (uL) 11 11 11 11 11
  1. Mix gently and pulse-spin in microfuge to collect mixture at the bottom of the tube.
  2. Incubate all the tubes at 37°C for 2 hours.
  3. Stop the reaction by placing the tubes on ice.
  4. Measure the fluorescence intensity of each tube with a plate reader (excitation: ~480 nm, emission: ~510 nm): The content of each of the tubes was transferred to a separate well on a black plate. PBS was added to each well to make up 100 uL. In addition, a well with only 100 uL PBS was added.