PROTOCOLS AND FORMULATIONS

PROTOCOL FOR BACTERIAL TRANSFORMATION AND COMPETENT CELL HANDLING

Developed by: Pedro Ribeiro Faria & Fernando Segato

Tested by: Pedro Ribeiro Faria & Manuela Bernardes de Oliveira

Handling Competent Cells

Transformation Procedure

  1. Sanitize materials with 70% ethanol and place in the laminar flow hood.
  2. Thaw competent cells on ice.
  3. Add DNA (1 µL for minipreps or full volume for Gibson) to the cells and mix. Incubate on ice for 30 minutes.
  4. Heat shock at 42°C for 30 seconds, then place on ice for 3 minutes.
  5. Add 1 mL SOC broth, mix, and incubate at 37°C for 1 hour in a shaker.

Plate Preparation

  1. Melt LB agar in the microwave, cool until holdable.
  2. Add antibiotic (1:1000 ratio) to LB agar, mix, and pour 15 mL per plate. Label plates.

Plating

  1. Centrifuge cells at 9500 rcf for 7 minutes. Remove 900 µL of supernatant, resuspend in 100 µL.
  2. Plate 50 µL onto each plate and spread evenly.
  3. Incubate plates at 37°C for 18-24 hours.

GIBSON ASSEMBLY CLONING

Objective

This protocol describes all stages needed for cloning genes into plasmids via the Gibson Assembly method, using a homemade Gibson Assembly master mix.

Methodology

  1. Amplify your gene, fragments, biobricks and/or plasmids.
  2. Linearizing vectors with a restriction enzyme facilitates PCR-amplifying vectors. You can design your primers around an enzyme cut site on the vector’s MCS.
  3. Make sure to include overlaps of at least 15bp on your PCR primers – They are needed in order to make the Gibson Assembly cloning work!
  4. Run an agarose gel electrophoresis to check your PCR products.
  5. Purify your PCR products. You might want to run a gel with your purified products to double check them. If your template was a plasmid, or if you’re amplifying a plasmid, it might be useful to treat the PCR product with DpnI for 1h at 37°C before purifying. Just add 0.5μL of DpnI straight to your PCR reaction after it comes out of the thermocycler!

Cloning

  1. In a PCR tube, add 1,5μL of your purified gene fragment and 3,5μL of your purified PCR-amplified plasmid. If more than one fragment is being cloned into a plasmid, use 0.75μL or less of each purified fragment, totaling 1,5μL. Try not to exceed this volume, but if needed, adjust the plasmid volume accordingly, and make sure you use 3 times the total DNA volume in 1.33X homemade Gibson Assembly Master Mix. (More exact quantities can be calculated from the proportions of gene/plasmid fragment on the NEBuilder calculator website (NEBuilder Calculator)).
  2. Add 15μL (or 3x the total volume of fragments) of the 1.33X homemade Gibson Assembly Master Mix.
  3. Incubate at 50°C for 60 to 120 minutes.

Transformation:

We proceed with E. coli transformation as described before, using the entire reaction volume as the transformation DNA.

AGAROSE GEL PREPARATION PROTOCOL

For a thin, 0.8 to 1% agarose gel:

  1. Weigh 0.27-0.3 g of agarose powder using an analytical balance.
  2. Measure 30 mL of freshly prepared 1X TAE buffer.
  3. Combine the 0.3 g of agarose and 30 mL of 1X TAE buffer in an Erlenmeyer flask. Heat the mixture in a microwave, pausing every 30 seconds to swirl gently until the agarose is completely dissolved. Ensure there are no visible particles. Allow the agarose solution to cool for a few minutes until it is warm but not hot to the touch (~50°C). Be careful to avoid forming bubbles in the gel.
  4. Carefully pour the agarose solution into a prepared gel tray for electrophoresis. Place the comb at the appropriate position to create wells. Let the gel solidify at room temperature for approximately 20-30 minutes.

SACCHAROMYCES CEREVISIAE TRANSFORMATION PROTOCOL

Preparation of Competent Yeast Cells

  1. For the pre-inoculum, a single colony of the SC9721 strain was transferred to an Erlenmeyer flask containing 10 mL of YPD medium (2.0% (w/v) peptone, 1.0% (w/v) yeast extract, 2.0% (w/v) glucose) and incubated at 200 rpm for 16 hours at 30°C.
    1. After growth, 5 mL of the culture was inoculated into 200 mL of YPD medium and incubated under the same conditions for 4 hours.
      1. After this period, the yeast cells were centrifuged at 5,000 g for 5 minutes, the pellet was resuspended in 20 mL of sterile MilliQ water, and centrifuged again.
        1. Next, 1 mL of TE/LiAc solution (10 mmol/L Tris-HCl, 1 mmol/L EDTA, pH 8.0, and 100 mmol/L lithium acetate) was added to the cells.
  1. Add 100 µL of the competent cell suspension to each transformation reaction.

Preparation of Mixtures

Mixture 1:

  1. Use 10-20% of the total volume of competent cells
  2. 5-10 µg of linear plasmid (pre-assembled plasmid)

Mixture 2:

  1. Prepare 250 mL of TE buffer (10 mmol/L Tris-HCl, 1 mmol/L EDTA, pH 8.0)
  2. Separate a portion and mix with:

- 600 µL of LiAc (100 mmol/L)

- PEG

Transformation Protocol:

  1. Combine the mixtures and add them to the competent cell suspension.
  2. Incubate the mixture at 200 rpm for 30 minutes at 30°C (the Falcon tube can be left on the shaker).
  3. After incubation, add 70 µL of DMSO to the mixture.
  4. Homogenize the mixture by inversion and incubate at 42°C for 15 minutes.
  5. Cool the mixture on ice for 2 minutes.
  6. Add 700 µL of MilliQ water to the mixture and centrifuge at 13,000 rpm for 30 seconds.
  7. Discard approximately 800 µL of the supernatant.
  8. Plate the transformed cells on the appropriate selective medium.

Media Preparation

Buffered Minimal Glucose Medium (BMGH)

1. Prepare 1 liter of Buffered Minimal Glucose medium with the following components:

- 100 mM potassium phosphate, pH 6.0

- 1.34% YNB (filter sterilized, do not autoclave)

- 4 x 10⁻⁵% biotin (filter sterilized, do not autoclave)

- 1% glucose

2. Autoclave 780 mL of water and 100 mL of 1 M potassium phosphate solution, pH 6.0

3. After cooling to room temperature, add:

- 100 mL of 10X YNB (filter sterilized, not autoclaved)

- 10 mL of solubilized biotin (filter sterilized, not autoclaved)

- 10 mL of sterile 1g/ml glucose (can be added directly to the Erlenmeyer flask)

4. Add amino acids to a final concentration of 0.004%, including:

- Leucine

- Lysine

- Tryptophan

5. Adjust the water volume in the medium before autoclaving, considering the addition of amino acids and other components.

6. Aminoacids added can change depending on auxotrophic marks on different yeast strains.

Note: Sterilize 50 mL of MilliQ water to use in the experiment.

BIOREACTOR PROTOCOL

Nutritional Medium

The S. cerevisiae SC9721 strain will be grown in YPD medium (1% yeast extract, 2% peptone, 2% glucose, Leucine 0.1g/L, Lysine 0.1g/L Tryptophan 0.1 g/L, Histidine 0.05 g/L, Uracil 0.1g/L, Uridine 0.1 g/L) at 30°C for 8-10 hours.

Yeast activation

  1. Prepare two 80 mm Petri dishes containing 15 ml of YPD medium.
  2. After the medium has solidified, prime both plates with the yeast stock solution.
  3. Keep the plates inverted in an oven at 30°C for two days.

Standard growth curve

  1. Preparing the crucibles
  2. Prepare five previously washed porcelain crucibles (50 ml) and leave in an oven at 105°C for 24 hours.
  3. Place the crucibles in the desiccator for 15 minutes and weigh them on an analytical balance.
  4. After weighing, place them back in the desiccator for 15 minutes and weigh again.
  5. Repeat this process until you obtain the same value on the balance in two weighings (the value must be the same to 4 decimal places on the balance).

    ATTENTION: the crucibles must be handled with care, hold them wrapped in paper to avoid errors when weighing

Cell preparation

  1. Prepare the culture in 4 250 ml erlenmeyer flasks with 50 ml YDA medium + inoculum; leave to grow for 12 hours at 30°C
  2. Place all the culture medium in a 50 ml falcon tube and centrifuge at 2200 xg for 20 min.
  3. Discard the supernatant and rinse in 200 ml distilled water.
  4. Repeat this process twice.
  5. At the end, resuspend the pellet obtained with a small amount of distilled water (50 ml).
  6. Measure the final volume in the beaker (mother solution).
  7. From the mother solution, make multiple suspensions (1:10; 1:25; 1:50; 1:100;1:200; 1:500; 1:1000; 1:2000).
  8. Measure the DO of these solutions in order to obtain at least 7 points below 1.

Finding the mass of cells

  1. Use a volumetric pipette (or other precision volume device) to measure 10 ml of the stock solution and add this volume to each crucible.
  2. Place the crucibles with the sample in an oven at 60°C and leave for 24 hours.
  3. The next day, leave for another 24 hours in the oven at 105°C
  4. Leave in the desiccator for 15 minutes and weigh on an analytical balance.
  5. Carry out this procedure until the same weight is obtained for a crucible twice in succession (the values must be the same up to the 4th decimal place).
  6. Take the difference in mass between the empty crucible and the crucible with the dry sample.
  7. By dividing this value by the initial volume withdrawn (10 ml) we obtain the cell concentration of the mother solution.

Obtaining the optical density of the dilutions

Read all the diluted samples on the spectrophotometer (640 nm).

It is necessary to obtain 7 to 8 reading points within the OD= 1 limit (where the curve remains linear).

If necessary, dilute the mother solution more or less to obtain OD points within 1.

Construct the graph of the standard growth curve by optical density.

Preparing the inoculum

The inoculum must have a concentration of 3g/L to be added to the bioreactor.

To determine this concentration: we will first grow the activated yeast in 5 ml of YPD medium at 30°C for 10h at 200 rpm, and measure the OD using a spectrophotometer to obtain the absorbance curve.

Then, from the curve obtained, we will determine the concentration of cells in this initial solution compared to the standard curve.

After obtaining this initial concentration, we perform this step again, but in a 250 ml flask containing 50 ml of culture medium plus an arbitrary volume of inoculum, which will be cultivated at 30°C for 10 hours at 200 rpm.

Remove 2 ml of this solution and microcentrifuge at 2200 xg for 20 minutes.

Discard the supernatant and resuspend the pellet in sterile 0.9% saline solution.

Repeat this process twice, resuspending in the same volume of solution.

Next, we will take the inoculum (saline solution with the cells) to the spectrophotometer to obtain the OD and determine its concentration through absorbance.

After finding the concentration of this solution, calculations will be made to determine the volume of inoculum needed to obtain 3g/L.

Bioreactor preparation

The process will be conducted in a batch system fed into a previously sterilized bioreactor.

The useful volume of the fermentation will be 1L and the pH will be maintained at 5 for 12 hours.

The pH will be maintained by solutions of HCL 4 molar and NAOH 4 molar.

3g/L of inoculum will be inoculated into 1L of saline solution with 0,1g/L glucose

Feed the reactor with YDA medium (0,1 g/L glucose) according to the feed volume determined by the calculations every 30 minutes .

Sterile air should be supplied at a constant rate of 0.3 vvm (0.3L of air/min) and the bioreactor should be stirred at 300 rpm.

Samples will be taken every 1 hour to measure the level of biomass and glucose concentration.

Measure glucose and biomass by DNS and absorbance curve, respectively.

To obtain the absorbance of the sample: take 1 ml of sample, centrifuge at 2200 xg for 20 min, remove the supernatant and set aside for the DNS test; Resuspend the pellet to 1 ml to read the absorbance.

DNS test

Remove 1 ml of sample and add to a test tube.

Add 1 ml of DNS reagent.

Shake and heat in a water bath at 100 degrees for 5 minutes.

Cool the tube on ice for 5 minutes.

Take the absorbance reading on a spectrophotometer at 540 nm (zero the device first with the blank).

Blank solution: distilled water (1ml) plus DNS (1ml).

FORMULATIONS

LB Medium (Luria-Bertani)

-1% tryptone;

-0,5% yeast extract;

-1% NaCl;

-2% agar for solid medium.

SOC Medium (Super Optimal broth with Catabolite repression)

For 100 ml:

-2% tryptone - 2 g;

-0,5% yeast extract - 0,5;

-10 mM NaCl - 58 mg;

-2,5 mM KCl - 18 mg;

-10 mM MgCl2 - 95 mg;

-10mM MgSO4 - 250 mg;

-20 mM glucose - 180 mg.

-The medium’s final pH must be between 6,8 and 7,0.

TAE 10x Buffer

-Tris(hydroxymethyl)aminomethane: 48 g/L

-Disodium EDTA: 3,68 g/L

-Glacial acetic acid: 11,4 mL (for 1L of the TAE 10x Buffer)

Procedure:

  1. Add the Tris and the EDTA to 850mL of nuclease-free water in a beaker and adjust the pH to 8,5 so that the EDTA solubilizes.
  2. Then, in a Laboratory-type hood, open the acetic acid bottle, measure the needed amount in a graduated cylinder, and add it in.
  3. Top the volume to 1L with nuclease-free water.
  4. Keep refrigerated.

Resuspension of synthetic DNA

How we did it:

  1. We spun it for 1 minute at top speed in a microcentrifuge;
  2. We added 100µL of Tris-HCl buffer, 100mM, pH 8,0. ;
  3. Vortexed for 3s;
  4. We incubated the resuspended synthetic DNA in a thermal block for 20 minutes at 50°C;
  5. Vortex again for 3s, then spun it in the centrifuge until at top speed for 1 minute.

DNA Loading

-50% glycerol (molecular purity);

-25 mM EDTA;

-0,25% Bromophenol Blue;

-0,25% Xylan cyanol;

-0,25% Orange Blue;

  1. At the end, add Gel Red.

YPD-9721 Medium (1L)

  1. 20 g peptone
  2. 10 g yeast extract
  3. 0.1 g leucine
  4. 0.1 g lysine
  5. 0.1 g tryptophan
  6. 0.05 g histidine
  7. 0.1 g uracil
  8. 0.1 g uridine
  9. 980 mL water

Weigh all components and mix in water.

Adjust pH to 6.5 using 1 M NaOH or 1 M HCl.

Prepare a glucose solution: Dissolve 20 g glucose in 20 mL distilled water.

Autoclave the medium and the glucose solution.

After autoclaving, add 2% glucose to the medium.

Divide the medium into two portions:

For liquid medium: Use as is.

For solid medium: Add 5 g agar.

Referências:

UNIVERSIDADE DE SÃO PAULO FACULDADE DE MEDICINA DE RIBEIRÃO PRETO DEPARTAMENTO DE BIOQUÍMICA E IMUNOLOGIA ALINNE COSTA SILVA Análise global da regulação transcricional do gene de cerato-platanina epl1 em Trichoderma reesei. (2021). https://www.teses.usp.br/teses/disponiveis/17/17131/tde-09052022-101527/publico/ALINNECOSTASILVA.pdf