Team:Calgary/Experiments



General Wet Lab

  1. Mix 100ml of 1X TAE buffer with 1g of regular or LMP agarose (for a 1% gel). For a small gel, mix 30ml of 1X TAE with 0.3g agarose.
  2. Microwave covered for about 1.5 minutes or until agarose is dissolved and pour into a balanced casting tray. LMP gels should be cast in the fridge.
  3. Add a volume of Safe-Red dye to be 1/6th the total volume of the sample.
  4. Run samples at 100V (regular gel) or 80V (LMP gel) until sample is ¾ of the way down the gel.

This protocol was not used by the team yet, however we are providing this protocol from NEB International and is to be used with the NEB Gibson Assembly Kit.

  1. Set up the following reaction on ice:

 

Recommended Amount of Fragments Used for Assembly

2-3 Fragment Assembly

4-6 Fragment Assembly

Positive Control**

Total Amount of Fragments

0.02–0.5 pmols*
X μl

0.2–1 pmols*
X μl

10 μl

Gibson Assembly Master Mix (2X)

10 μl

10 μl

10 μl

Deionized H2O

10-X μl

10-X μl

0

Total Volume

20 μl***

20 μl***

20 μL

* Optimized cloning efficiency is 50-100 ng of vector with a 2-fold molar excess of each insert. Use 5 times more of inserts if size is less than 200 bps. Total volume of unpurified PCR fragments in Gibson Assembly reaction should not exceed 20%.


** Control reagents are provided for 5 experiments.


*** If greater numbers of fragments are assembled, additional Gibson Assembly Master Mix may be required.

  1. 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).

  2. Transform competent coli cells with 2 μl of the assembly reaction

  1. To a microcentrifuge tube, add:
    • Digested vector DNA (in appropriate ratio)
    • Digested insert DNA (in appropriate ratio)
    • 1/10 of total final volume of aliquoted 10X T4 DNA ligase buffer
    • 1μl of T4 DNA ligase (1U/μl)
    • ddH2O to final volume
  2. Incubate at room temperature for 2 hours
  3. Transform chemically competent cells with the ligation product.
  4. Leave remaining ligation product at room temperature overnight, and transform again the following day.
  5. Store remaining product at -20°C

  1. Add 10μl of ddH2O to the desired well of the distribution kit plate
  2. .Pipette up and down 3-5 times (until solution becomes red)
  3. Incubate at room temperature for 10 minutes
  4. Transform cells with 1μl of rehydrated DNA as per transformation protocol

  1. Combine in a 0.2ml microcentrifuge tube:
    • 5μl of NEB 10X standard Taq buffer (final concentration = 1X)
    • 0.25μl of NEB Taq (final concentration = 1.25U/50μl)
    • 1μl of 10μM forward primer (final concentration = 0.2μM)
    • 1μl of 10μM reverse primer (final concentration = 0.2μM)
    • 1pg to 1ng of template plasmid DNA (final concentration < 1000ng/μl)
    • 1μl of 10mM Kapa dNTPs (final concentration = 200μM)
    • ddH2O to 50μl
  2. Vortex 2 to 3 seconds to mix, then centrifuge briefly to settle
  3. Place in thermocycler and select or set up the appropriate program as follows:
    • Initial denaturation → 95°C for 5 minutes
    • Repeat 30x:
      • Denaturation → 95°C for 30 seconds
      • Annealing → Tm - 5°C (45 to 68°C) for 15 to 60 seconds
      • Extension → 68°C for 1 minute per kilobase
      • Final extension → 68°C for 5 minutes
    • Final extension → 68°C for 5 minutes
    • Hold at 4°C

  1. Add 10μl of ddH2O to the desired well of the distribution kit plate
  2. .Pipette up and down 3-5 times (until solution becomes red)
  3. Incubate at room temperature for 10 minutes
  4. Transform cells with 1μl of rehydrated DNA as per transformation protocol

  1. To a microcentrifuge tube, add:
    • Required amount of DNA to be digested (can use NEBioCalculator to determine amount if performing a ligation with digested DNA)
    • 1/10 final total volume of appropriate 10X buffer
    • 1μl of each restriction enzyme (diluted)
    • ddH2O to final volume
  2. Incubate at 37°C (or other optimal temperature for enzyme activity) for 30 minutes to 3 hours (be careful of star activity)
  3. Heat inactivate restriction enzymes at 82°C for 20 minutes
  4. For digest confirmations, run on regular agarose gel. For subsequent ligation, run on LMP gel and excise or gel-extract. Otherwise, store at -20°C

E. coli Protocols

  1. Add ≤ 5μl (≤ 1/10 of the cell aliquot amount) of the DNA sample to a chemically competent cell aliquot. Mix by pipetting gently, then incubate on ice for 30 to 45 minutes.
  2. Heat shock at 42°C for 1 minute
  3. Incubate on ice for 5 minutes
  4. Add 250μl of plain LB or SOC media aseptically, then incubate for 30 to 90 minutes at 37°C, shaking. *If resistance is Kan, must incubate for at least 1 hour
  5. Plate 100μl (if big plates) or 50μl (if small or half plates) aseptically
  6. Incubate plate at 37°C overnight or until growth is observed

*If transformation fails:

  • Spin down transformed cells for 5 minutes to pellet
  • Resuspend pellet in 100μl of media
  • Plate and incubate

  1. Culture overnight culture (O/N) in 2ml LB at 28°C, shaking
  2. Subculture (1:50) by adding 1ml O/N culture to 50ml LB with 10mM MgSO4 (500μl of 1M MgSO4) and 1mM KCl (50μl of 1M KCl)
  3. Shake at 28°C to OD600 = 0.3 to 0.4
  4. Chill on ice for at least 10 minutes
  5. Put into 50ml pre-chilled Falcon tube and centrifuge at 2500g for 8 minutes at 4°C (3450rpm in Allegra X-12 centrifuge)
  6. Resuspend in 10ml ice-cold 100mM CaCl2, gently mix on ice, then ice for at least 10 minutes
  7. Centrifuge at 2500g for 8 minutes at 4°C
  8. Resuspend in 500μl 100mM CaCl2 with 10% glycerol on ice, then incubate on ice for 10 minutes
  9. Aliquot 50μl into pre-chilled 1.5ml microcentrifuge tubes on ice. Store at -80°C
  10. Perform a test transformation with a reporter gene (i.e RFP)

  1. Create cPCR mastermix as follows:
    • 20μL 10X Taq Buffer
    • 4μL 10μM VF2 primer (or other forward primer)
    • 4μL 10μM VR primer (or other reverse primer)
    • 4μL 10mM dNTPs
    • 1μL Taq Polymerase
    • 127μL ddH2O

    *Each mastermix aliquot is enough to run 10 cPCR reactions/screen 10 colonies

  2. Add 4μL of ddH2O to each PCR tube or well (of a 96-well plate)
  3. Using pipette tips or sterile toothpicks, touch an individual labelled colony, swirl in the ddH2O, and then streak on masterplate. Repeat for as many colonies as desired.
  4. Add 16μL of cPCR mastermix to each tube/well
  5. Place in thermocycler and select or set up the appropriate program as follows:
    • Initial denaturation → 95°C for 5 minutes
    • Repeat 30x:
      • Denaturation → 95°C for 30 seconds
      • Annealing → Tm - 5°C (For VF2/VR primers, 53°C) for 30 seconds
      • Extension → 68°C for 1 minute per kilobase
      • Final extension → 68°C for 5 minutes
    • Final extension → 68°C for 5 minutes
    • Hold at 4°C

  1. Add 500μl of 50% glycerol to a 1.5ml tube aseptically
  2. Add 500μl of 50% of overnight culture to the tube aseptically. Mix gently
  3. Store at -80°C

  1. Mix into 1L of dH2O:
    • 10g of Tryptone (1%)
    • 5g of yeast extract (0.5%)
    • 10g NaCl (1%)
    • 15g Agar (1.5%)
  2. Autoclave and cool
  3. Add necessary antibiotics and mix thoroughly
    • 1ml/L Ampicillin (stock = 100mg/ml, final = 100μg/ml)
    • 1ml/L Kanamycin (stock = 50 mg/ml, final = 50μg/ml)
    • 0.5ml/L Chloramphenicol (stock = 50 mg/mlEtOH, final = 25μg/ml)
  4. Pour into plates aseptically, swirl to remove bubbles, and let set. Store in fridge upside-down.

  1. Mix into 1L of dH2O:
    • 10g of Tryptone (1%)
    • 5g of yeast extract (0.5%)
    • 10g NaCl (1%)
  2. Autoclave and cool
  3. Add necessary antibiotics and mix thoroughly
    • 1ml/L Ampicillin (stock = 100mg/ml, final = 100μg/ml)
    • 1ml/L Kanamycin (stock = 50 mg/ml, final = 50μg/ml)
    • 0.5ml/L Chloramphenicol (stock = 50 mg/mlEtOH, final = 25μg/ml)
  4. Pour into plates aseptically, swirl to remove bubbles, and let set. Store in fridge upside-down.
  5. Aliquot as necessary

P1 (Resuspension buffer) at 4°C on ice
  • 50mM Tris•HCl (pH = 8), 10mM EDTA, 100μg/ml RNase A
P2 (Lysis buffer) at room temperature
  • 200mM NaOH, 1% SDS
P3 (Precipitation buffer) at room temperature
  • 3M potassium acetate (pH = 5.5)
  1. Grow 2 to 6ml culture O/N
  2. Transfer 2ml (at a time) to 2ml microcentrifuge tube(s) and pellet at 14000rpm for 5 minutes. Discard supernatant and repeat as necessary
  3. Resuspend in 300μl ice-cold P1
  4. Add 300μl P2, gently invert 3x. Quickly add 300μl P3 and invert 3x
  5. Spin at 14000rpm for 10 minutes at room temperature
  6. Retain supernatant in 1.5ml microcentrifuge tube
  7. Add 650μl of 100% isopropanol, gently invert, and incubate for 10 minutes at room temperature
  8. Spin at 14000rpm for 10 minutes, then discard supernatant
  9. Wash pellet with 500μl of cold 70% ethanol
  10. Spin at 14000rpm for 5 minutes
  11. Discard supernatant. Carefully tap tube to remove remaining ethanol. Dry pellet in vacufuge
  12. Resuspend pellet in 20μl of sterile ddH2O or TE buffer. Nanodrop to determine concentration. Store at -20°C

Yeast Protocols


We would like to thank Dr. Vanina Zaremberg, Maria Laura Sosa Ponce, and Dr. Rodrigo Ledesma Amaro for graciously providing us with these protocols for Saccharomyces cerevisiae and Yarrowia lipolytica .


Reagents:

  • 6.7g Bactoyeast Nitrogen base without amino acids, with ammonium sulphate
  • 20g sugar (typically glucose)
  • 2.0mL 1% L-tryptophan
  • 2.0mL 1% L-methionine
  • 2.0mL 1% L-arginine
  • 2.0mL 1% L-leucine
  • 2.0mL 1% L-lysin
  • 10mL 0.2% Adenine sulfate
  • 10mL 0.2% Uracil

  1. In 500mL of ddH2O dissolve:
    • Bactoyeast nitrogen base (TNB, final concentration= 0.67% w/v)
    • Sugar (final concetration= 2% w/v)
  2. Under aseptic conditions add:
    • Amino acids from AUTOCLAVED 1% solutions
    • Bases from AUTOCLAVED 0.2% solutions

    *Add the amino acids and bases in the oder listed above to fill the desired auxotrophies. For example, to make SD-Ura (media lacking uracil), add all the amino acids and adenine, but not uracil.

  3. Bring volume to 1000mL with ddH2O, accounting for the volume(s) of amino acid and base solutions added.
  4. FOR PLATES: Add agar to final concentration of 2% (i.e 20g in 1L of media)

  1. Dissolve the following reagents in 1L of distilled water
    • 10g Yeast extract
    • 20g Peptones
    • 20g Dextrose
    • 20g Agar powder (FOR PLATES ONLY)
  2. Autoclave and allow media to cool slightly
  3. Add antibiotic/selection marker
  4. Store broth in the fridge OR aseptically pour into plates, swirl to remove bubbles, let set, and then store in the fridge upside-down.
NOTES:
  • Yeast extracts = contain vitamins, growth factors, minerals, or digested nucleic acids needed for growth.
  • Peptones = protein degradation productions (source of amino acids and nitrogen)
  • Dextrose = biochemically identical to glucose

*For other sugar media, REPLACE Dextrose with 20g of your desired sugar

You can either:

  • Resuspend cells from a fresh plate in 1mL sterile 15% glycerol
  • Add 600uL of log phase culture (overnight culture) to 400uL of sterile 50% glycerol- vortex the culture first.

Work under aseptic conditions and store the tubes in the -80ºC freezer

  1. Vortex the starter tubes which were grown the day before.
  2. Prepare cuvettes in a 1:10 dilution and measure OD for each starter. The linear range of the measurement device is 0.5-1. lf other results are received then dilute your sample further. How to use the OD device? Press "L" so the reference stand (the blue stand) will move to the scanning place -> insert all cuvettes, starting with the reference one -> press "F2" -> press OK (the reference has been set) -> continue pressing OK for measuring.
  3. Make new starters that will reach mid-log (at this phase, the yeast cells have a thin cell wall, so it's easier to transform them with foreign DNA) according to the following principles: Cell cycle in YPD is 90 minutes and in SD is 120 minutes. You should calculate for a total of 0.4 OD after the waiting- at least 6h in SD and 4.5h in YPD. lt takes 2 divisions in order to be in mid log with no matter what is the measured OD. When the cells reach 1 OD they finish up all the sugar in the medium and go through diauxic shift. Since it takes one cell cycle for them to start dividing (in the first 90 minutes in YPD or L2O minutes in SD their OD remains exactly the same after back dilution) then at LEAST 3 cell cycles are needed to reach mid log.

  4. Place the new starters at 30oC until they reach logarithmic phase according to your plan. Measure the OD again (this time no need to dilute, just place 750 pl from the starters to the cuvettes.

    Important: steps 3+4 are only performed when you transform genomic DNA and want it to be integrated into the genome. Homologous recombination is obligatory in order to achieve this goal and it only takes place during cell cycle. lf you transform a plasmid then it is possible to use stationary phase yeast because the plasmid only needs to insert the nucleus and homologous recombination is not required.

  5. Make up sufficient Transformation Mix for the planned number of transformations plus one extra. It is not necessary to keep the transformation mix in ice. Once the Salmon Sperm's DNA has been boiled and cooled quickly it no longer has the energy to find its complementary strand. In addition, once you dilute it nearly 10 fold in the transformation mix, the chances of it complementing are really small so no problem leaving it outside the ice. Also the PEG is so viscous that there is not a lot of movement of the DNA (unlike in water).
    6. Reagents 1 5 (6X) 10 (11X)
    PEG 3500 50% w/v 240 uL 1440 uL 2640 uL
    Lithium Acetate 1.0 M 36 uL 216 uL 396 uL
    Boiled Salmon Sperm-carrier of DNA 50 uL 300 uL 550 uL
    Plasmid/Genomic DNA plus Water 34 uL 204 uL 374 uL
    Total 360 uL 2160 uL 3960 uL

    Notes:

    • *SS carrier should be boiled at 100 C for 5 minutes and then transferred straight to ice prior to its usage.
    • *PEG is highly sensitive to changes in concentration. If the transformation isn't efficient--change the PEG.
    • *If you use genomic DNA then run 5uL of the PCR product in gel to verify the reaction was successful. The PCR band must be brighter (ie. more DNA) than the corresponding ladder band. The rest (20uL) of it is inserted to the Transformation Mix. If you use purified plasmid then you need to have 1ug of plasmids.
    • *Remember to prepare one mix without any DNA as a control. This will allow the comparison between background growth and actual transformants.
  6. For each starter tube:
    • If the cells were raised in SD medium they might not be centrifuged well. Add 100uL of YPD to solve this.
    • If you are transforming a stationary culture:
      1. Take 500uL into a new eppendorf tube.
      2. Centrifuge at 3000g for 3 minutes.
      3. Vacuum the supernatant.
    • If you are transforming a mid-log culture:
      1. Take a 15 mL conical tube or an eppendorf tube.
      2. Add 3 OD of cells into the conical tube.
      3. Centrifuge at 3000g for 3 minutes.
      4. Vacuum the supernatant.
      5. Move the cells to a new eppendorf tube.
    • Wash: add 1 mL of DDW to the eppendorf tube.
    • Vortex.
    • Centrifuge at 3000g for 3 minutes.
    • Vacuum the supernatant.
    • Add the DNA to the pellet and suspend it.
      1. In the case of genomic transformation, add the 20uL of the PCR product.
      2. In the case of plasmid transformation, add the adjusted volume for 50 ng of DNA.
    • Add the Transformation Mix to each transformation tube and immediately re-suspend the cells by vortex. Make sure there are no clumps of yeast in the mix and that it is completely homogenous.
  7. Incubate all the tubes in a 42 C water bath for 40 minutes.
  8. Centrifuge at 3000g for 3 minutes and remove the Transformation Mix with a micropipette.
  9. If the selection marker is an amino acid then plate the transformants:
    • Pipette 100uL of sterile water into each tube.
    • Make sure the mix is homogenous, and then quickly dispense onto the appropriate plate with beads.
  10. If the selection is carried out via antibiotics:
    • Add 1 mL of YPD for each tube and allow the yeast to grow for 1 hour (if the selection is Kan) or 3 hours (if the selection is Nat) at 30 C.
    • Centrifuge at 3000g for 3 minutes.
    • Aspirate 800uL of YPD so 200uL are left.
    • Vortex and make sure the mix is homogenous. Then, quickly dispense onto the appropriate plate with beads.
  11. After the plating, incubate the plates at 30 C for 2 days.
  12. Choose several transformants from each plate and streak them into a new plate.
  13. If you transformed a genomic DNA then check PCR should be done in order to verify that the homologous recombination took plate on the right place. If you transformed a plasmid then there is no need to perform check PCR.
  14. If there is a lot of background growth after 2 days, perform a replicate plating protocol. The background growth might be due to cells that received the template plasmid (that cannot be replicated inside the yeast). It is possible to replicate each plate 3 times only. Also each velvet sheet can be used 3 times (with different plates). Only the true transformants should grow.

Solution Preparation:

  1. Lithium Acetate (LiAc) Solution: 0.1M, pH 6.0
    • 5.1g LiAc for 500mL
    • Adujst to pH 6.0 with 10% acetic acid
    • Autoclave and store in the fridge
  2. Polyethylene Glycol (PEG) Solution: 40% PEG 4000 in 0.1M LiAc, pH 6.0
    • Add 50mL 0.1M LiAc to 40g PEG 4000
    • Adjust to pH 6.0 with 10% acetic acid
    • Adjust volume to 100mL with LiAc solution
    • Store in the fridge
  3. DNA Carrier: 5mg/mL fish DNA (salmon sperm)
    • 50mg DNA sodium salt/fish DNA
    • Dissolve in 10mL sterile TE buffer
    • Aliquot samples and store in -20ºC freezer

Part I: Preparation of Competent Cells

  1. Spread the strain that you want to transform on a YPD plate (Grow at 28°C for 16-24 hours)
  2. Put a loop of cells into 1ml of TE in sterile Eppendorf tubes
    • Prep for 5 transformations- i.e. 5 loops in 5 ml of TE
  3. Centrifuge 1 min at 10 000 rpm and remove supernatant
  4. Resuspend cell gently in 600µL of 0.1M LiAc pH 6.0
    • Prep for 5 transformations- i.e. 3mL
  5. Place in 28°C water bath without agitation for 1 hour
  6. Centrifuge for 2 min at 3000 rpm and remove supernatant
  7. Gently resuspend cells in 60µL of 0.1M LiAc pH 6.0
    • Prep for 5 transformations- i.e. 300µL

Part II: Transformation

  1. In a new eppendorf tube, mix together the following with a pipette:
    • 3-5μl of carrier DNA
    • 2-5µL your desired DNA
    • 40µL competent cells
  2. Incubate for 15 min in 28°C water bath without agitation
  3. Add 350µL PEG solution
  4. Incubate for 1 h in 28°C water bath without agitation
  5. Thermal shock the cells for 10 min at 39°C
  6. Add 600µL 0.1M LiAc pH 6.0
  7. Spread 200μl (or more) per plate containing the adequate selection medium

  1. Make a 2mL overnight culture with YPD media and a wild-type Y. lipolytica colony and grow overnight at 30ºC
  2. Make a 1:50 dilution with YPD media with 1mL of the overnight culture. Grow at 30ºC until it is a mid-log culture (OD600 = 0.8-1.2)
  3. Set up 7 culture tubes with 10mL of YPD media diluted 10x with autoclaved water
    1. 2x control
    2. 3x 42ºC for 1 hour
    3. 3x 42ºC for 3 hours
    4. 3x 42ºC for 1 day
  4. Add 1mL of yeast culture to each tube
  5. Place the three “42ºC for 1 hour” tubes in a 42ºC water bath for 1 hour
  6. Place the three “42ºC for 3 hours” tubes in a 42ºC water bath for 3 hours
  7. Place the three “42ºC for 1 day” tubes in a 42ºC water bath overnight
  8. Place the cultures back into the shaking incubator overnight
  9. Measure the OD again the next day and after 3 more days

  1. Make a 2mL overnight culture with YPD media and a wild-type Y. lipolytica colony and grow overnight at 30ºC
  2. Make a 1:50 dilution with YPD media with 1mL of the overnight culture. Grow at 30ºC until it is a mid-log culture (OD600 = 0.8-1.2)
  3. Set up 8 culture tubes with 10mL of YPD media diluted 10x with autoclaved water
    1. 1x negative control
    2. 1x positive control
    3. 3x shake once/day
    4. 3x shake once/3 days
  4. Add 1mL of yeast culture to each tube
  5. Place positive control in shaking incubator at 22ºC
  6. Shake the three shake once/day tubes once a day and streak the culture
    1. Measure pH with a pH strip
  7. Shake the three shake once/3 days tubes every 3 days and streak the culture
    1. Measure pH with a pH strip
  8. Don't shake the negative control

Cellulase Integration

  1. Create a 2mL overnight culture of Y. lipolytica beta-carotene strain in YPD
  2. Create a 1:50 subculture from overnight and grow the subculture to OD 0.8-1.2
  3. Transfer to falcon tube, spin down cells, and resuspend in 50mL 1x PBS
  4. Boil 500mL of tap water and allow to cool back to room temperature (sterilization process) or pour cold water over to cool faster
  5. To 100mL of the boiled water add 2g of glucose/dextrose (=2% sugar solution)
  6. Do serial dilutions to create 0.002%, 0.02% and 0.2% sugar solutions
  7. Set up culture tubes with each of the following:
    1. 2mL appropriate glucose solution
    2. 1mL resuspended Y. lipolytica cells
  8. Take 200µL of each sample to measure the 0D600 in a microtiter plate everyday

PREPARING DNS REAGENT:

  1. Slowly dissolve 2.18 g of DNS (MW=228.1 g/mol) in 80 mL of 0.5 M NaOH by heating and stirring at 70º C.
  2. Add 30 g of sodium potassium tartrate (MW=282.2 g/mol) and stir until dissolved
  3. Cool to room temperature and bring to 100 mL with distilled water

PRODUCING A STANDARD CURVE WITH GLUCOSE:

  1. Dissolve 0.2g of glucose into 100mL of water (=2mg/mL solution)
  2. Do dilutions to get 0.4mg/mL, 0.8mg/mL, 1.2mg/mL, and 1.6mg/mL
    1. 2mL stock glucose solution + 8mL ddH2O= 0.4mg/mL
    2. 4mL stock glucose solution + 6mL ddH2O= 0.8mg/mL
    3. 6mL stock glucose solution + 4mL ddH2O= 1.2mg/mL
    4. 8mL stock glucose solution + 2mL ddH2O= 1.6mg/mL
  3. Mix 2mL of each glucose solution with 2mL DNS reagent
  4. Boil for 5min
  5. Cool to room temperature under cool water
  6. Measure the A540

WITH CELLULOSE and CELLULASE:

  1. Dissolve 1g of cellulose in 100mL of water (it likely won’t dissolve= 1% w/v)
  2. Dissolve 1g of enzyme in 100mL of water (10mg/mL)
    1. Make 5 serial dilutions of 10x:
      1. 1mL +9mL = 1mg/mL or 1000µgmL
      2. 1mL + 9mL= 100µg/mL
      3. 1mL + 9mL= 10µg/mL
      4. 1mL + 9mL= 1µg/mL or 1000ng/mL
      5. 1mL + 9mL= 100ng/mL
    2. Can do another range of 1mg/mL - 5mg/mL
  3. Mix 1mL cellulose solution and 1mL of each enzyme solution
  4. Incubate at 45º for 30min
  5. Add 2mL DNS reagent
  6. Boil for 5min 
  7. Cool to room temperature under cool water
  8. Measure A540

GROWING Y. lipolytica ON PURE CELLULOSE:

  1. Create a 2mL overnight culture of Y. lipolytica beta-carotene strain in YPD
  2. Create a 1:50 subculture from overnight and grow the subculture to OD 0.8-1.2
  3. Transfer culture to falcon tube, spin down, and resuspend in 50mL of 1x PBS
  4. Set up culture tubes with three different enzyme concentrations
    1. Mix 1mL of 1% w/v cellulose solution and 1mL of either:
      1. 1mg/mL cellulase
      2. 5mg/mL cellulase
      3. 10mg/mL cellulase
    2. Mix 1mL of boiled water with 0.003% leucine
  5. Add 1mL of resuspended Y. lipolyticacells
  6. Grow at 30ºC shaking and measure OD over 5 days

 

GROWING Y. lipolytica ON CRUDE CELLULOSE:

  1. Create a 2mL overnight culture of Y. lipolytica beta-carotene strain in YPD
  2. Create a 1:50 subculture from overnight and grow the subculture to OD 0.8-1.2
  3. Transfer culture to falcon tube, spin down, and resuspend in 50mL of 1x PBS
  4. Set up culture tubes for three different enzyme concentrations:
    1. Measure out 0.2g of crude cellulose for each tube and mash it with a mortar and pestle
    2. Mix cellulose with 1mL of boil-sterilized water with 0.003% leucine in the culture tube and boil the tubes in a water bath.
    3. Mix 1mL of either:
      1. 1mg/mL cellulase
      2. 5mg/mL cellulase
      3. 10mg/mL cellulase
  1. Add 1mL resuspended Y. lipolyticacells
  2. Grow at 30ºC shaking and measure OD over 5 days

This protocol has not been used yet by our team, but here is a sample ELISA protocol from University of Washington.

General ELISA protocol for testing sera or antibodies against proteins.

Solutions Needed: 0.1M Sodium Bicarbonate (pH 9.4-9.6), Blocking Solution (1X PBS+10% Non-Fat Milk+0.3% Tween20), Dilution Buffer (1X PBS+10% Non-Fat Milk+0.03% Tween20), 1N Sulfuric Acid, Ammonium Thiocyanate.

  1. Add 50ng of protein in 100ul sodium bicarbonate to each well of a high-binding 96 well plate. Incubate over night at room temperature.

  2. Empty plates and then block by adding 150ul/well blocking solution, incubate 1 hour at 37oC.

  3. Wash plate, then serial dilute the antibody/sera in dilution buffer in the plate, incubate 1 hour at 37oC.

  4. Wash plate, add 100ul/well diluted HRP conjugated secondary antibody (1:3000 in dilution buffer), and incubate 1 hour at 37oC.

  5. Wash plate, add 50ul/well TMB substrate, and incubate 3 min at room temperature.

  6. Stop development with addition of 50ul/well 1N sulfuric acid.

  7. Immediately read plate (absorbance is measured at 450nm).


To determine the avidity: sera diluted to 1:500 is added to the blocked plate at step 3 and serially diluted ammonium thiocyanate (ranging from 10M to 1M) is then added to the diluted sera and incubated for 20 min at room temperature on a plate shaker


Thymol Production

  1. Create overnight culture of Y. lipolytica beta-carotene strain in YPD
  2. Remove cells from suspension culture and centrifuge for 10 min.
  3. In 200 mL of the supernatant, add 2% NaHCO3 to pH 7.8 and wash with 20mL diethyl ether twice.
  4. Evaporate the solvent in a water bath at 40°C
  5. Analyze sample with split ratio was 10:1 onto a 30mm×0.25mm HP-5 (cross-linked phenyl–methyl siloxane ) column with 0.25 mm film thickness toa mass spectrometer and sniff port. Set injector temperatures to 220°C

    6. Reference: https://doi.org/10.18517/ijhr.2.1.40-49.2020


Biocontainment

Characterize amino acid production by engineered strains

Part 1: Make standard curve of OD600 vs amino acid concentration

  1. Make series of media with different concentrations of leucine (0, 3, 6, 9, 12, 15 mg/L)
    1. To do this:
      1. Take SD-Leu media add in the appropriate volumes of autoclaved leucine solutions (you will have to do math since the leucine solution we have is 1%) to get the right concentration of leucine.
      2. Make enough so that we can do triplicates for each type of media (6*3 =18 samples) (Each sample is 200uL)
  2. Load 200 uL of media into each well in 96-well plate, and introduce W303 cells into each well.
  3. To ensure concentration of cells is consistent for all wells, use mid-log culture of W303 yeast cells grown in YPD media
    1. Determine OD600 for culture, ensure that the OD of the culture is above 0.500
    2. Take culture with OD >  0.500 and centrifuge at 3000g at 3 minutes
    3. Discard the supernatant
    4. Resuspend cells in SD-Leu- media, ensuring OD > 0.500. Volume of media used dependent on OD of culture
    5. Inoculate 900uL of SD media with 100uL of yeast. Do this for all different amino acid concentrations of media.
    6. Load 3 wells for each level of amino acid concentration, with 200uL in each well (This is a total of 18 wells, or 600uL for each concentration level).
  4. Also load 6 wells with the SD media but no yeast cells in them as blanks
    1. SD media with 0, 3, 6, 9, 12, 15 mg/L Leu (6 wells in total)
  5. Repeat the same thing but with Trp, using SD-Trp media!
  6. Make sure you draw out plate configuration somewhere after you load the wells, so we know what is in each well.
  7. Set plate reader with the following parameters:
  • Plate type: 96 well plate
  • Read type: absorbance endpoint
  • Wavelength: 600nm
  • Temperature: 30 C
  • Run Time: 24 hrs
  • Interval: 15:00
  • Shake: continuous

Part 2: Test concentration of amino acid released into media by engineered and control strains (example for ymm60)

  1. Grow overnights, mid-log, 24 hr, and 48 hr cultures of yMM60 in SD-Leu media. Do the same protocol for all the cultures.
  2. Check OD600 of overnights and mid-log using spec and record them in notebook so we know.
  3. Centrifuge cultures to pellet the cells. Then take the supernatant and pass it through a 0.2 µm nylon membrane to remove cells.
  4. To ensure concentration of cells is consistent for all wells, use mid-log culture of W303 yeast cells grown in YPD media
    1. Determine OD600 for culture, ensure that the OD of the culture is above 0.500
    2. Take culture with OD >  0.500 and centrifuge at 3000g at 3 minutes
    3. Discard the supernatant
    4. Resuspend cells in SD-Leu- media, ensuring OD > 0.500. Volume of media used dependent on OD of culture
    5. Inoculate 900uL of filtered culture supernatant with 100uL of yeast.
  5. Load wells with 200uL in each well of yeast/supernatant mixture.
  6. Also include wells with 200uL supernatant but no yeast cells as the blank (control).
  7. Draw out how you set up plate in notebook so we know when we are doing analysis!!
  8. When setting up plate reader do all the same settings as last time!!!!

 

***Follow the same protocol for measuring amino acid concentration for ymm65 culture, Leu2 transformed culture, and Trp1 transformed culture. For the cultures that are producing leucine grow in SD-Leu media; for the cultures that are producing tryptophan, grow in SD-Trp media.

This protocol was adopted for two auxotrophic complementary strains of Saccharomyces cerevisiae

  1. Grow 20ml overnights mono-cultures of each auxotrophic strain in YPD media.
  2. Dilute the cultures by a factor of 10 into new media and grow for 4h to prevent cells from being in stationary phase at the beginning of the experiment.
  3. Measure the OD 600 of mono-culture.
    1. Dilute/concentrate the cultures so that they roughly have an OD600 of 0.7.
  4. Mix equal volume of each mono-culture (depends on how many replicates are desired) together to form the co-culture.
    1. Each replicate would need 2ml of co-culture (1ml of each mono-culture)
    2. For example, if a total of 10 replicates are desired, mix 10 ml of each mono-culture to obtain a 20ml volume of co-culture.
  5. Make 2ml-aliquots of the co-culture for each replicate.
  6. Centrifuge the 2ml-aliquots and discard the supernatant.
  7. Resuspend the pellets in SD media lacking the subject amino acids, centrifuge again, and discard the supernatant.
  8. Resuspend the pellets in 2ml of the appropriate SD media, containing desire levels of amino acids.
  9. Measure initial OD600 and fluorescence of the auxotrophic strains.
    1. The wavelength of fluorescence measure depends on the reporter constructs used in each strain.
  10. Grow co-cultures at 30 degrees shaking.
  11. Measure OD600 and fluorescence of the auxotrophic strains every 24 hours.

We developed this protocol to test for river and soil samples. The soil samples were suspended in water then filtered, to remove any solid and make a liquid media. Cloudy environmental samples, such as soil water, may affect the results of this experiment as the results of yeast growth may not be obvious. Try to ensure your environmental samples are as clear as possible.

  1. Grow mid-log cultures of ymm60 and ymm65 cells separately in YPD Media.
  2. Check OD600 of cultures and write them down in a notebook.
  3. Centrifuge the cells at 3000g for 5 minutes to pellet. Remove and discard YPD supernatant.
  4. Resuspend cells in new media such that OD600 > 0.500. Ensure you resuspend to the same OD for all groups of cells to ensure consistency in results.
    1. The media you will be resuspending the cells in is the media that you are growing them in. For example, if we are testing whether environmental amino acid concentrations can sustain the leucine auxotrophic strain ymm65, we would resuspend Leu- cells in SD-Leu- media. Resuspend yMM60, the Trp- strain, in SD-Trp- media.
  5. Make 1mL of each solution mentioned below, using 450uL of media, and 450uL of the environmental sample
  6. For Trp auxotroph (ymm60):
    • SD-Trp- + autoclaved river water
    • SD-Trp- + non autoclaved river water
    • SD-Trp- + autoclaved soil water
    • SD-Trp- + non autoclaved soil water
    • YPD (positive control)
    • YPD + autoclaved river water
    • YPD + nonautoclaved river water
    • YPD + autoclaved soil water
    • YPD + nonautoclaved soil water
  7. For ymm65 (tryptophan overproducing and leucine auxotrophic strain), the treatments were:
    • SD-Leu- + autoclaved river water
    • SD-Leu- + non autoclaved river water
    • SD-Leu- + autoclaved soil water
    • SD-Leu- + non autoclaved soil water
    • YPD (positive control)
    • YPD + autoclaved river water
    • YPD + nonautoclaved river water
    • YPD + autoclaved soil water
    • YPD + nonautoclaved soil water
  8. In a 96-well plate, pipette 200uL of each sample. These will serve as the blanks.
  9. Add 70uL of yeast solution to their corresponding treatments. This results in a 1:10 ratio of yeast to total volume.
  10. Pipette 200uL of each sample into a 96-well plate. Do this in triplicate, so you will have one blank and three experimental samples for each treatment.
  11. Grow yeast for 48 hours in the plate reader machine. You only need to measure the endpoint of the growth.
  12. Set the plate reader requirements as follows:
    • Plate type: 96 well plate
    • Read type: absorbance endpoint
    • Wavelength: 600nm
    • Temperature: 30 C
    • Run Time: 24 hrs
    • Interval: 15:00
    • Shake: continuous
  13. Make sure to write down the plate configuration before placing it in the plate reader! This will ensure you don’t get your samples mixed up.
  14. After getting your results, subtract the blank from the yeast-replicate readings to account for background reading. You’ll now be able to analyze and compare your environmental results!