Experiments

Describe the research, experiments, and protocols you used in your iGEM project.

  1. Thaw all reagents completely on ice.
  2. Add 1 μl of ligation reaction to thawed competent cells (E. coli NEB5α) and gently mix by tapping tube.
  3. Incubate on ice for 30 min.
  4. Heatshock for 45s at 42°C in heat block.
  5. Incubate for 2 min on ice.
  6. Fill to 950 μl with LB medium.
  7. Incubate at 37°C and shake at 250 rpm for 1 h.
  8. EITHER transfer into more LB medium and let grow overnight at 37°C while shaking,
  9. OR spread on selection plate and incubate overnight at 37°C.

  1. Pellet 1–5 ml bacterial culture by centrifugation for 30 seconds. Discard supernatant.
  2. Resuspend pellet in 200 μl Plasmid Resuspension Buffer (B1). Ensure cells are completely resuspended.
  3. Add 200 μl Plasmid Lysis Buffer (B2), invert 5–6 times, incubate for 1 minute at room temperature.
  4. Add 400 μl Plasmid Neutralization Buffer (B3), invert gently, incubate for 2 minutes.
  5. Centrifuge lysate for 2–5 minutes. Transfer supernatant to spin column and centrifuge for 1 minute.
  6. Add 200 μl of Plasmid Wash Buffer 1, centrifuge for 1 minute. Discard flow-through.
  7. Add 400 μl of Plasmid Wash Buffer 2, centrifuge for 1 minute.
  8. Transfer column to clean microfuge tube, add ≥30 μl DNA Elution Buffer, incubate 1 minute, centrifuge for 1 minute.

  1. Prepare a liquid culture by incubating a bacterial sample in 5-10 ml LB medium overnight.
  2. Dilute glycerol with distilled water to create a 50% glycerol solution.
  3. Transfer 750 μl of the glycerol solution into a microfuge tube.
  4. Add 750 μl of bacterial culture to the microfuge tube.
  5. Close lid, mix gently, and freeze at –80°C.

1 L 50x TAE Buffer:
  1. Tris base 2M (242 g)
  2. Acetic acid 1M (57 ml)
  3. EDTA 0.05 M (100 ml 0.5 M)
  4. Fill up to 1 L with MilliQ H2O.
Dilute 50x TAE to 1x TAE for use.
  1. Cast 50 ml 1% agarose gel (1g agarose, fill up to 100 ml with 1x TAE buffer).
  2. Load gel, including at least one lane for a DNA ladder.
  3. Run gel at 120V for 45 min.

Gel extraction using QIAgen DNA Gel extraction kit, QIAGEN kit manual.

PCR Mix:
Component Volume Final Conc
5X Q5 Reaction Buffer (NEB)10 µl1X
dNTP Mix, 10 mM each1 µl0.2 mM each dNTP
Fwd primer2.5 µl0.5 µM
Rev primer2.5 µl0.5 µM
Q5® High-Fidelity DNA Polymerase0.5 µl0.02 u
DNA to be amplified1 µl< 1,000 ng
ddH2O32.5 µl–
Total50 µl–
PCR Program:
Step Temp/°C Duration/sec n repeats
Denaturation98301
Denaturation981030
AnnealingDepends on primers3030
Extension7212030
Final Extension721201
Cooling71201

Q5 reaction mix

ComponentVolumeFinal Conc
5X Q5 Reaction Buffer (NEB)10 µl1X
dNTP Mix, 10 mM each1 µl0.2 mM each dNTP
Fwd primer2.5 µl0.5 µM
Rev primer2.5 µl0.5 µM
Q5® High-Fidelity DNA Polymerase0.5 µl0.02 u
ddH2O33.5 µl–
Total50 µl–
  1. Mix reaction mix for each colony to be analyzed.
  2. Pick colony using disposable pipette tip.
  3. Stab pipette tip onto replica plate and note number.
  4. Mix pipette tip in PCR mix.
  5. Run PCR (see program above).
  6. Analyze PCR using agarose gel to check if the expected insert was amplified.
  7. Incubate replica plate overnight.

  1. Add 10ng/μl Tetracycline to LB medium.
  2. Add signal molecule to medium at the appropriate concentration to induce regulator activation.
  3. Incubate at 37°C for 2-6 hours.
  4. Image reporter gene assay using a microplate reader at the appropriate wavelength to induce reporter gene fluorescence (e.g., for GFP: 480 nm).