Experiments

Making chemically competent cells (for heat-shock transformation):

  • Requirements
    • Sterile LB
    • Overnight liquid culture containing desired cells
    • 10% glycerol
    • 25 ml, 10 ml, 5 ml pipettes and PIPETBOY
    • Ice and dry ice
    • 1ml cuvettes
    • Sterile Eppendorf tubes
    • Sterile conical flasks
    • Spectrophotometer
    • 4°C centrifuge
    • Gloves
  • Make a 1/10 dilution of the overnight culture and measure the OD600 (100 µl liquid culture in 900 µl sterile LB in 1 ml cuvette)
  • In two sterile conical flasks, combine LB and overnight culture to get a 50 ml solution with an OD600 of roughly 0.15 (using OD600 previously obtained)
  • Shaking incubate the two flasks at 37°C and regularly check OD600 until it reaches 0.4 and then put on ice OD600 of 0.4, transfer the 50 ml cultures into two sterile 50 ml falcon tubes
  • Centrifuge the two balanced tubes at 4000 rpm 4°C for 10 mins. Discard the supernatant and resuspend in 50 ml of 10% glycerol
  • Centrifuge again at 4000 rpm 4°C for 10 mins. Discard the supernatant and resuspend in 50 ml of 10% glycerol.
  • Centrifuge again at 4000 rpm 4°C for 10 mins. Discard the supernatant and resuspend in 25 ml of 10% glycerol.
  • Centrifuge again at 4000 rpm 4°C for 10 mins. Discard the supernatant and resuspend in 5 ml of 10% glycerol.
  • Centrifuge again at 4000 rpm 4°C for 10 mins. Discard the supernatant and resuspend in 1 ml of 10% glycerol.
  • Prepare 50 µl aliquots into sterile Eppendorf tubes on dry ice and transfer into a -80°C freezer

Making electro-competent cells (for transformation via electroporation):

  • Requirements
    • Sterile LB
    • Overnight liquid culture containing desired cells
    • 10% glycerol
    • 25 ml, 10 ml, 5 ml pipettes and PIPETBOY
    • Ice and dry ice
    • 1ml cuvettes
    • Sterile Eppendorf tubes
    • Sterile conical flasks
    • Spectrophotometer
    • 4°C centrifuge
    • Gloves
  • Make a 1/10 dilution of the overnight culture and measure the OD600 (100 µl liquid culture in 900 µl sterile LB in 1 ml cuvette)
  • In two sterile conical flasks, combine LB and overnight culture to get a 50 ml solution with an OD600 of roughly 0.15 (using OD600 previously obtained)
  • Shaking incubate the two flasks at 37°C and regularly check OD600 until it reaches 0.4 and then put on ice OD600 of 0.4, transfer the 50 ml cultures into two sterile 50 ml falcon tubes
  • Centrifuge the two balanced tubes at 4000 rpm 4°C for 10 mins. Discard the supernatant and resuspend in 50 ml of 10% glycerol
  • Centrifuge again at 4000 rpm 4°C for 10 mins. Discard the supernatant and resuspend in 50 ml of 10% glycerol.
  • Centrifuge again at 4000 rpm 4°C for 10 mins. Discard the supernatant and resuspend in 25 ml of 10% glycerol.
  • Centrifuge again at 4000 rpm 4°C for 10 mins. Discard the supernatant and resuspend in 5 ml of 10% glycerol.
  • Centrifuge again at 4000 rpm 4°C for 10 mins. Discard the supernatant and resuspend in 1 ml of 10% glycerol.
  • Prepare 50 µl aliquots into sterile Eppendorf tubes on dry ice and transfer into a -80°C freezer

Gel Electrophoresis:

  • Requirements
    • Agarose
    • 1% TAE/TBE
    • Gel tray and comb
    • DNA ladder (1kb plus)
    • 6X loading dye
    • GelRed® stain
    • Microwavable flask
    • Precise weight scales
    • Microwave
    • Electrophoresis tank and power pack
    • Gel imager
    • Gloves
  • Mix 0.5 g of agarose with 50 ml of TAE in a microwavable flask
  • Microwave for 1-2 minutes until the agarose powder is dissolved
  • Let it cool before adding 3 µl of GelRed and pour into the gel tray containing the comb. Leave to set at room temp for 20-30 minutes.
  • Add 6X purple loading dye to samples and 3 µl of DNA ladder to the first well (e.g. 2 µl 6X purple loading dye to a 10 µl sample)
  • Run at 100V for 20 minutes or until the bands are as desired

Restriction Digest

  • Requirements
    • CutSmart buffer
    • Relevant restriction enzyme
    • Relevant plasmid
    • Water
    • Gel electrophoresis equipment
    • Gel imager
    • Gloves
  • Add 1 µl of CutSmart buffer, 0.5 µl total of restriction enzyme (or 0.25 µl of each enzyme), and 7.5 µl of water to a 1.5 ml tube
  • Add 1 µl of plasmid and mix well throughout
  • Incubate at 37°C for 1 hour in a static incubator
  • Afterward, add 2 µl of 6X loading dye to each sample and mix well
  • Load onto gel with DNA ladder and run gel electrophoresis

Heatshock

  • Requirements:
    • 2/3 µl desired plasmid/ligation mix
    • 50 µL of chemically competent cells
    • Sterile SOC
    • 42°C water bath
    • Sterile Eppendorf tubes
    • Selective media plate (with relevant antibiotics)
    • Bunsen burner
    • Spreaders
    • Gloves
  • Procedure:
  • Take out competent cells from -80°C freezer and thaw on ice for 15 minutes
  • After thawed, add 2 µl of plasmid (or 3 µl of ligation mix) into 50 µl of chemically competent cells and mix by flicking the tube
  • Incubate on ice for 30 minutes
  • Heat shock in 42°C water bath for 45 seconds
  • Place the cells back on ice for 2 minutes
  • Add 500 µl of SOC and shake incubate at 37°C for 45 minutes
  • Plate 50 µl of mixture onto one plate and 300 µl onto another (don’t forget to use plates that have the desired antibiotic)
  • Incubate plates overnight at 37°C

Electroporation:

  • Requirements:
    • 1/2 µl desired plasmid/ligation mix
    • 50 µL of electro-competent cells
    • Sterile SOC
    • 2mm blue electroporation cuvettes
    • Electroporation machine
    • Sterile Eppendorf tubes
    • Selective media plate (with relevant antibiotics)
    • Bunsen burner
    • Spreaders
    • Gloves
  • Procedure:
  • Take out competent cells from -80°C freezer and thaw on ice for 15 minutes
  • After thawed, add 2 µl of plasmid (or 3 µl of ligation mix) into 50 µl of electro-competent cells and mix by flicking the tube (keep on ice)
  • Transfer all of the mix into a 2mm blue electroporation cuvette, on ice
  • Dry the slides so no moisture is on the metal plates and ensure there are no bubbles present in the cuvette
  • Shock at 25 µF and 2.5 volts, hold down buttons until it beeps
  • Immediately add 500 µl of 37°C SOC and incubate at 37°C for 1 hour
  • Plate 300 µl of mixture onto one plate and 50 µl onto another
  • Incubate plates overnight at 37°C

Gel Extraction

  • Requirements:
    • 96% ethanol
    • Gel containing desired bands
    • Gel imager
    • QIAquick®reg; Gel Extraction Kit
    • 50°C water bath
    • 13,000rpm tabletop centrifuge
    • Gloves
  • Procedure:
  • First ensure that buffer PE contains the added ethanol
  • After looking at the gel bands, using a clean scalpel, cut out the bands as precisely as possible (keep checking on the machine)
  • Weigh the gel slice in a colorless 1.5 ml Eppendorf tube and add 100 µl of QG buffer for every 100 mg of gel slice.
  • Incubate at 50°C for 10 min and vortex occasionally (or until the gel slice has completely dissolved).
  • Transfer the solution into a QIAquick spin column and centrifuge for 1 minute at 13,000 rpm, discard the flow-through
  • Add 500 µl buffer QG, centrifuge for 1 minute, discard flow-through
  • Add 750 µl buffer PE, centrifuge for 1 minute, discard flow-through.
  • Centrifuge for a further minute to remove residual wash buffer
  • Place the QIAquick column into a clean 1.5 ml microcentrifuge tube and elute by adding buffer EB (20-50 µl depending on desired concentration). To increase concentration, incubate it for 4 mins before spinning.

Plasmid Purification:

  • Requirements:
    • RNase
    • 96% ethanol
    • Overnight culture of cells containing plasmid
    • QIAprep®reg; Spin Miniprep Kit
    • 13,000rpm tabletop centrifuge
    • Gloves
  • Procedure:
  • Start by adding RNase to buffer P1 and ethanol to buffer PE
  • Add 1.5 ml of overnight liquid culture to an Eppendorf tube and centrifuge it at 13,000 rpm for 3 mins at room temperature (15-25°C).
  • Remove the supernatant and leave just the pellet.
  • Resuspend the pellet in 250 µl of buffer P1
  • Add 250 µl buffer P2 and mix by inverting, the reaction will turn the solution blue. Don’t let the reaction occur for longer than 5 mins
  • Add 350 µl N3 and mix by inverting, the solution will turn colorless
  • Centrifuge for 10 mins at 13,000 rpm
  • Apply 800 µl of supernatant to a QIAprep 2.0 spin column and centrifuge at 13,000 rpm for 60 seconds, discard the flow-through
  • Wash the spin column with 750 µl of buffer PE and centrifuge for 60 seconds, discard flow-through. Centrifuge for another minute to remove the residual wash buffer.
  • Place the QIAprep 2.0 spin column into a 1.5 ml Eppendorf tube and add buffer EB (20-50 µl), let stand for 1 minute then centrifuge.

Overlap Extension PCR:

  • Materials:
    • All relevant 10 µM primers (1-4)
    • 5X Q5 reaction buffer
    • Q5 polymerase
    • 2 mM dNTPs
    • Purified template DNA
    • Nuclease-free water
    • Nanodrop device
    • Phosphatase
    • T4 ligase
    • T4 Ligase buffer
    • Gel electrophoresis materials
    • Gel extraction materials
    • Restriction enzymes
    • Thermocycler
    • PCR tubes
    • Selective media plate (with relevant antibiotics)
    • Heatshock/electroporation materials
    • Bunsen burner
    • Spreaders
    • Gloves
  • Procedure:
  • Using four pre-designed primers (primers 1-4), determine the annealing temperature and extension time for each.
  • Prepare a reaction mixture on ice with 10 µl of 5X Q5 reaction buffer, 5 µl of 2 mM dNTPs, 2.5 µl of 10 µM forward primer, 2.5 µl of 10 µM reverse primer, 1 µl of purified template DNA, and 0.5 µl of Q5 polymerase. Make up to 50 µl with nuclease-free water (28.5 µl).
  • Repeat the above step for primer pairs 1:3 (2.3 kb) and 2:4 (0.4 kb).
  • Put the reaction mixtures in a thermocycler for the following conditions:
    • Initial denaturation: 98°C for 30 seconds
    • Denaturation: 98°C for 10 seconds
    • Annealing: 67°C for 20 seconds
    • Extension: 72°C for 85 seconds
    • Final extension: 72°C for 120 seconds
    • Hold: 4-10°C for 10 mins
  • Run the PCR products on a gel and cut out the bands containing the amplified region.
  • Use a QIAquick gel extraction kit to purify DNA, resulting in fragment A (from 1:3 primer amplification) and fragment B (from 2:4).
  • Prepare another reaction mix on ice with 10 µl of 5X Q5 reaction buffer, 5 µl of 2 mM dNTPs, 2.5 µl of 10 µM forward primer 1, 2.5 µl of 10 µM reverse primer 4, 5 µl of fragment A, 5 µl of fragment B, and 0.5 µl of Q5 polymerase. Make up to 50 µl with nuclease-free water (23.5 µl).
  • Put the reaction mixture in a thermocycler for the following conditions:
    • Initial denaturation: 98°C for 30 seconds
    • Denaturation: 98°C for 10 seconds
    • Annealing: 50-72°C for 20 seconds
    • Extension: 72°C for 25 sec/kb
    • Final extension: 72°C for 120 seconds
    • Hold: 4-10°C for 10 mins
  • Run the PCR products on a gel and extract the 2.7 kb band with the joined AB fragment.
  • Nanodrop the fragment to determine the DNA concentration (ng/µl).
  • Using 1000 ng of AB fragment, add 1 µl of each restriction enzyme (EcoRI and SacI), 10 µl of CutSmart buffer, and make up to 100 µl with water.
  • Meanwhile, make up another solution containing 5 µl CutSmart buffer, 2 µl of enzyme (or 1 µl of both), 1000 ng of original plasmid DNA, and make up to 50 µl with water.
  • Incubate both solutions for 1 hour at 37°C, then add phosphatase and incubate for a further 5 minutes.
  • Run both solutions on a gel and extract the digested AB fragment (2.7 kb) and the digested plasmid (12.5 kb) with sticky ends made by enzymes.
  • Prepare a solution of 1 µl T4 ligase, 2 µl T4 ligase buffer, 12 µl of cut plasmid, and the calculated volume of fragment (in 1:3 ratio). Make up to 20 µl with water. Do a control without any fragments.
  • Incubate the ligation mix at room temperature for 1 hour and then heatshock the formed plasmid into cells and plate onto an antibiotic plate.

Colony PCR

  • Requirements:
    • 2X DreamTaq MasterMix
    • 10 µM forward and reverse primer
    • Thermocycler
    • PCR tubes
    • Gel electrophoresis materials
    • Gloves
  • Procedure:
  • Set up a MasterMix containing 12.5 µl 2X DreamTaq, 1 µl of forward primer, 1 µl of reverse primer, 0.5 µl of plasmid (or ½ of a bacterial colony), and make up to 25 µl with 10.5 µl of water.
  • Gently mix the samples with a pipette.
  • Put the samples in a thermocycler for the following conditions:
    • Initial denaturation: 95°C for 10 minutes
    • Denaturation: 95°C for 30 seconds
    • Annealing: Tm-5 (~50-55°C) for 30 seconds
    • Extension: 72°C for 60sec/kb
    • Final extension: 72°C for 5 minutes
    • Hold: 4-10°C for 10 mins
    • 35 cycles
  • After the PCR, run it on a gel, and if the insert is present, you should see an additional band.
  • At this point, make a liquid culture of the ½ colony or plate out the liquid culture.

Gisbon Assembly

  • Requirements:
    • CutSmart® Buffer
    • Relevant enzyme
    • Relevant plasmid
    • Phosphatase
    • Thermocycler (heat denaturation and 50°C incubation)
    • QIAprep® Spin Miniprep Kit (if not heat denaturation eligible)
    • Gibson Assembly (GA) 2X MasterMix
    • Heatshock/Electroporation materials
    • Bunsen burner
    • Spreaders
    • Gloves
  • Procedure:
  • Begin the digestion with 2 µl CutSmart buffer, 2 µl of enzyme, 10 µl of plasmid, and 6 µl of water up to 20 µl. Incubate the tube for 2 hours at 37°C.
  • After 2 hours, add 2 µl of phosphatase and incubate for a further hour.
  • If eligible, heat denature the enzymes. If not, then add 22 µl of QG buffer and continue with column purification into 20 µl of elution buffer.
  • Use nanodrop to measure the concentration of the purified plasmid.
  • With 100 ng of plasmid, calculate the amount of plasmid in pmols and triple it (0.03 pmols) to find how much of the 10 µM fragments to use.
  • Add 10 µl of GA 2x MasterMix to 100 ng of plasmid and all the fragments and make up to 20 µl with water.
  • Incubate the mixture at 50°C for 1 hour in a thermocycler.
  • Add 2 µl of the mixture to 50 µl of competent cells (thawed on ice).
  • Transform the Gibson sealed fragment into cells using electroporation or heatshock and add 500 µl of SOC.
  • Shaking incubate at 37°C 220 rpm for 1 hour and plate 300 µl of the mixture onto one plate and 50 µl onto another.

Creating Growth Media for Producing Perillyl Alcohol

  • Materials:
    • Sterile LB
    • 1% glucose stock
    • Riboflavin
    • Arabinose
    • IPTG
    • Antibiotics (Kanamycin and Ampicillin)
    • 10 ml Falcon tube
    • Bunsen burner
    • Gloves
  • Procedure:
  • Begin by adding 5.4 ml of LB to a Falcon tube.
  • Then add 600 µl of 10% glucose stock (resulting in a 1% final concentration).
  • Dilute 5000x riboflavin (0.1 g in 1000 µl of water) and then add 1.25 µl to the final 6 ml media (resulting in 1x concentration in the final solution).
  • Add 0.12 g of arabinose to the 6 ml stock as well (to get 0.2% concentration).
  • Dilute 1000 mM IPTG stock to 1 mM using water. Next, add 150 µl to the media to achieve a 25 µM final concentration.
  • Finally, add any antibiotics needed for plasmid survival.

Using HP-20 Resin to Isolate Terpenes

  • Requirements:
    • HP20 resin
    • Ethyl acetate (EtOAc)
    • Growth media
    • Clean autoclaved bottle
    • Funnel
    • 4°C centrifuge
    • Fume hood
    • Gloves
  • Procedure:
  • Cover 5 g of HP20 resin with 15 ml of EtOAc in a clean autoclaved bottle and shake for 10 minutes.
  • Filter the resin using a funnel and discard EtOAc in flammable waste.
  • Add HP20 resin back into the used bottle and cover it with 15 ml of EtOAc.
  • Add 2.5 g of 5% HP20 resin into 50 ml media and autoclave it.
  • Mix 2.5 ml of bacterial culture with the 50 ml of media (containing 5% HP20), shaking incubate for 1 hour.
  • Centrifuge at 4000 rpm at 4°C for 10 mins and discard the supernatant.
  • Freeze the cell pellet/HP20 resin overnight at -80°C and dry the pellet.
  • Transfer the dry pellet/resin into a clean flask and add 20 ml EtOAc, shake overnight at 100 rpm.
  • Add another 20 ml of filtered EtOAc and shake overnight again.
  • Evaporate EtOAc under nitrogen and record the extract weight.

Homogenising Liquid Cell Cultures for GCMS (Without HP20 Resin)

  • Requirements:
    • Liquid culture of cells to homogenize
    • Ethyl acetate (EtOAc) or dichloromethane (DCM)
    • Sodium sulfate (Na2SO4)
    • 13,000 rpm centrifuge
    • GCMS-grade vial
    • Sterile Eppendorf tube
    • Fume hood
    • Gloves
  • Procedure:
  • Extract the POH from the sample by adding EtOAc in a 1:1 ratio to the media in an Eppendorf tube under a fume hood.
  • Centrifuge to pellet down particles at 13,000 rpm for 1 minute.
  • Transfer the supernatant to another sterile Eppendorf tube.
  • Add Na2SO4 to extract extra water remaining from the EtOAc extract.
  • Filter the supernatant to remove the solids and transfer the liquid to another sterile Eppendorf tube.
  • Centrifuge the filtrate again for 10 minutes at 13,000 rpm.
  • Transfer the supernatant to a GC/MS vial.

Preparing Perillyl Alcohol Sample from HP-20 Resin for GC/MS

  • Requirements:
    • Liquid culture containing HP-20 resin to homogenize
    • Ethyl acetate (EtOAc) or dichloromethane (DCM)
    • Sodium sulfate (Na2SO4)
    • 13,000 rpm centrifuge
    • GCMS-grade vial
    • Sterile Falcon tube
    • Filter paper
    • Scintillation vial
    • Rotary evaporator
    • Fume hood
    • Gloves
  • Procedure:
  • Transfer all the liquid culture into a sterile Falcon tube.
  • Centrifuge for 10 minutes at 4 degrees and 5000 rpm.
  • Remove the Falcon tube from the centrifuge and discard the supernatant.
  • Transfer the pellet with HP20 resin into a sterile conical flask and add 20 ml EtOAc/DCM, shake overnight at 100 rpm for extraction.
  • After overnight shaking, add Na2SO4 to the sample to remove residual water and filter the solid out into a pre-weighted 20 ml scintillation vial with filter paper.
  • Attach it to a rotary evaporator without heating (depending on time and speed), remove the EtOAc in a vacuum, record the exact weight.
  • The sample is then reconstituted into 1 ml of EtOAc and further diluted in a 1:50 ratio (10 uL in 490 uL EtOAc) for quantitative accuracy and comparison with the media extract.
  • Transfer the supernatant to a GC/MS vial.

Variable IPTG Experiment

  • Requirements:
    • Overnight liquid culture
    • IPTG dilutions
    • Growth media
    • Sterile conical flasks
    • Sterile 96-well plate
    • 37°C shaking absorbance plate reader
    • Bunsen burner
    • Gloves
  • Procedure:
  • Grow overnight culture of all cell types that will be tested (including any additional antibiotics).
  • Dilute the culture 1/10 with LB and measure the OD600. Using these values, make 10 ml dilutions of the cultures with an OD600 of roughly 0.15 in a sterile conical flask.
  • Incubate these flasks at 37°C and check frequently until they reach OD600=0.6. At which point, keep it on ice to prevent further growth.
  • In a sterile environment, set up 10 µL IPTG dilutions in each well at different concentrations and add a 20 µL of ‘growth media’ (comprising of 10% glucose, riboflavin, and arabinose).
  • Next, add 60 µL of OD600=0.4 liquid culture for each cell type to individual wells (excluding controls) and immediately after adding the culture, place in a 37°C shaking incubating absorbance plate reader.

Interlab Experiment 1 PDF Here

Find where we used the protocols in results.