Experiments

Plasmids and DNA fragments ordered from synthesis companies often come in powder form in small tubes. They need to be resuspended before experimental use

Materials

• Dry DNA
• Tris-HCl ph 8.0 buffer

Procedure

1. First, given the weight of DNA received, n, use n = cv to calculate the required volume of liquid, v, to add to in order to achieve the desired final working concentration, c
   • This is usually 10ng/μL, but vary based on the use case.
2. Briefly centrifuge the tube to ensure that all of the dry DNA is at the bottom.
3. Add the required volume v of Tris-HCl ph 8.0 buffer to the tube.
   • It is possible to resuspend DNA in ddH₂O but this is not recommended for long term storage.
4. Gently mix by pipetting up and down a few times to ensure all the DNA has been dissolved.
5. Store the resuspended DNA solution at -20°C.

Competence is the ability for a cell to take up exogenous DNA. E. coli have a low degree of natural competence but can be treated with CaCl₂ to make their cell walls more permeable and increase their competence.

Materials

• Liquid ON culture
• SOB medium
• Ice-cold 0.1M CaCl₂
• Ice-cold 0.1M CaCl₂/20% glycerol

Procedure

1. Take a 500μL aliquot from an overnight culture and add it into 49.5 mL of SOB medium (1:100 dilution).
2. Grow the culture at 37°C with shaking until it reaches an OD₆₀₀ of 0.4.
3. Place the culture on ice for ~15 minutes, swirling occasionally.
4. Pour the culture into a 50mL Falcon™ tube.
5. Centrifuge at 3500 rpm for 5 minutes at 4°C.
6. Carefully remove as much supernatant as possible without disturbing the pellet.
7. Resuspend the pellet in 100μL ice-cold 0.1M CaCl₂ using a sterilized loop.
8. Add 15mL of ice-cold 0.1M CaCl₂ and mix gently by pipetting up and down.
9. Incubate the cells on ice for 30 minutes.
10. Centrifuge again at 3500 rpm for 5 minutes at 4°C.
11. Resuspend the pellet in 2mL of ice-cold 0.1M CaCl₂/20% glycerol solution.
12. Incubate for 45 minutes on ice.
13. Pipette 50μL aliquots into chilled 1.5mL tubes.
    • Mix gently during aliqouting as cells can settle.
14. Snap freeze any aliquots not required for immediate use in liquid nitrogen and store at -80°C.
• Cells cannot be refrozen once thawed, as this kills them.

Digestion

Materials

• DNA: Plasmid (vector) or gene of interest, variable ng (see formula below for desired molar amount).

copies = (ng × (6.022 × 10²³)) / (length × 10⁹ × 650)

• Restriction buffer (10×): 5 µL (50 µL reactions) or 2.5 µL (25 µL reactions).
• Compatible restriction buffer: e.g., Smart Buffer (NEB) or FastDigest Buffer (Thermo Fisher Scientific).
• Restriction enzymes: 1 µL each (double digest) or 2 µL (single digest).
  • Use the appropriate restriction enzymes for your cloning strategy.
• ddH₂O: adjusted to the final volume.
• Total reaction volume: 50 µL (or 25 µL for half-volume reactions).

General note: Perform all steps on ice unless otherwise stated.

DNA Preparation

1. Add the required amount of DNA to reach the desired ng (see formula above).
2. Adjust volume with ddH₂O, accounting for the addition of 1 µL of each enzyme:
   • 50 µL reactions → 43 µL ddH₂O.
   • 25 µL reactions → 20.5 µL ddH₂O.
3. Add 10× reaction buffer (any compatible restriction buffer may be used):
   • 50 µL reactions → 5 µL buffer.
   • 25 µL reactions → 2.5 µL buffer.
4. Do not reduce enzyme volume for smaller reactions.
5. For a double digest: Add 1 µL of each restriction enzyme for both 50 µL and 25 µL reactions (2 µL total).
6. For a single digest: Add 2 µL of restriction enzyme to both 50 µL and 25 µL reactions.
7. Gently tap or flick tubes to mix.
8. Incubate at 37 °C for 30 min.
9. Heat-inactivate enzymes at 80 °C for 20 min.
10. Store the samples at −20 °C.

Sample Loading for Gel Electrophoresis

In order to be visible on the gel, load a minimum of approximately 125 ng of DNA in each well on a 1% agarose gel.

Fragment Recovery

• Excise and save the plasmid (vector) and gene of interest fragment for gel purification (see Gel Extraction Purification protocol).
• Freeze samples at −20 °C.

Ligation

Materials

• DNA of interest.
• T4 DNA ligase.
• 10× T4 DNA ligase buffer.
• ddH₂O.

Procedure

1. Label all tubes clearly to distinguish digested samples and controls.
2. Determine plasmid-to-insert ratios based on molarity. For the desired number of copies of plasmid and gene of interest (GOI) added to each reaction, see the formula in the Digestion protocol based on DNA amounts in ng and the number of base pairs. The desired DNA volume for each reaction can be determined using the formula:
   volume = n(mol) / c(mol/dm³)
3. The plasmid should be pre-digested with appropriate restriction enzymes leaving restriction cuts compatible with the GOI sequence.
4. Prepare corresponding control tubes: one without DNA and one without ligase. Substitute the missing component with ddH₂O.
5. For a 20 µL reaction, add 2 µL of 10× ligase reaction buffer (T4 DNA ligase buffer) to each appropriate tube.
6. Add 1 µL of T4 DNA ligase to each tube.
7. Adjust with ddH₂O to reach a final reaction volume of 20 µL.
8. Gently mix by tapping or flicking.
9. Incubate at 22 °C for 30 minutes.
10. Heat-inactivate ligase at 80 °C for 20 minutes.
11. Store samples at −20 °C until further analysis.
12. Run samples on a 1% agarose gel for visualization and comparison of ligation and control reactions (if needed), and see the Transformation protocol.

Transformation is the process of introducing exogenous DNA to a cell. Transformed E. coli are an effective way to replicate and store DNA

Materials

• Competent cells
• Plasmid of choice
• SOB medium

Procedure

1. Incubate 50μL competent cells on ice for 15 minutes
2. Add the ~5μL of plasmid to competent cells
   • This may vary slightly depending on the concentration of plasmid DNA
3. Incubate on ice for a further 30 minutes
4. Heat shock the cells at 42°C for 45 seconds
5. Incubate on ice again for 5 minutes
6. Add 950μL of SOB media to the cells
7. Incubate at 37°C for 90 minutes
8. Centrifuge cells for 5 minutes at 4000rpm to pellet
9. Remove 900μL of supernatant, taking care not to disturb the pellet
10. Resuspend the pellet in the remaining 100μL and plate

Re-streaking

1. Take one colony from a plate using an inoculation loop and smear it out on one edge of a new plate.
2. Draw one line vertically through the first lines to the centre of the plate.
3. Sterilize the loop and then go back and forth through the vertical line all the way to the other edge of the plate.

Overnight Cultures

Materials

• LB media
• Antibiotic of choice (stock solution)
• Plate with grown colonies

Procedure

1. Prepare growth media by mixing 5 ml of LB media with adequate amount of antibiotics to obtain a 1× antibiotic concentration.
2. Take one colony from a plate and resuspend it in the growth medium.
3. Incubate at 37 °C, 110 rpm for at least 8 hours (overnight).

Gel electrophoresis is a technique used to separate DNA fragments by size. An electric field is applied accross the gel, which causes the negatively charged DNA molecules to move toward the positive electrode. The agarose matrix slows the movement of the DNA through the gel; smaller molecules move faster, while larger molecules move slower.

Materials

• Agarose powder
• 1x Tris/Borate/EDTA (TBE) buffer
• 1x SYBR™ Safe DNA Gel Stain
• DNA sample of choice
• 6x DNA gel loading dye

Preparation of 1% Agarose Gel (150mL)

1. Add 1.5g of agarose powder to an Erlenmeyer flask
2. Add 150ml of 1x Tris/Borate/EDTA (TBE) buffer to the flask and swirl to mix
3. Microwave until the agarose dissolves
   • Take care when handling the flask as it will be hot; agar dissolves at 85-90°C
4. Let the flask cool until it can be safely touched
5. Add 1x SYBR™ Safe DNA Gel Stain to the solution and swirl to ensure it is fully mixed
6. Insert the comb into the casting tray
7. Pour the gel solution into the casting tray
8. Gently tap the tray on the bench a few times or use a clean pipette tip to carefully pop any air bubbles
9. Once the gel has solidified, it is ready for use

Electrophoresis

1. Ensure that the gel is oriented correctly in the chamber, with the sample wells nearest to the cathode (negative electrode)
2. Pour in 1x TBE buffer until the gel is fully covered
3. Prepare the sample mixes in separate Eppendorf tubes or on a clean piece of Parafilm
   • Mix 5μL of sample DNA with 1μL of 6x DNA gel loading dye
4. Pipette the 6μL sample mixes into separate wells on the gel
5. Place the lid and power supply leads onto the electrophosis unit
6. Turn on the power supply and run the gel
   • The gels can be run anywhere in the range of 20-150 volts
   • Higher voltages give shorter run times but may carry the risk of melting the gel

Purify DNA fragments from agarose gels using the Thermo Scientific GeneJET™ Gel Extraction Kit^[4]. Optional: Add 100% isopropanol to improve recovery for small or large fragments.

Materials

• Agarose gel slice containing DNA fragment of interest (≤ ~1 g)
• Binding Buffer (provided)
• Wash Buffer (provided; supplemented with ethanol)
• Elution Buffer (provided; 10 mM Tris-HCl, pH 8.5)
• GeneJET purification column + collection tube
• Microcentrifuge tubes (1.5 mL)
• Pipettes and tips
• 96–100% ethanol (molecular biology grade)
• (Optional) 3 M sodium acetate, pH 5.2
• Microcentrifuge capable of >12,000 × g

Procedure

1. Excise the gel slice containing DNA using a clean scalpel or razor blade.
2. Place gel slice in a pre-weighed tube and add an equal volume of Binding Buffer (1:1 weight-to-volume).
3. Incubate at 50–60°C for 10 min until gel is completely dissolved, mixing occasionally.
4. (Optional) For DNA ≤500 bp or >10 kb, add equal volume of 100% isopropanol and mix.
5. Transfer up to 800 μL of solution to the purification column and centrifuge at >12,000 × g for 1 min. Discard flow-through.
6. Wash column with 700 μL of Wash Buffer and centrifuge at >12,000 × g for 1 min. Discard flow-through.
7. Centrifuge column empty at >12,000 × g for 1 min to remove residual buffer.
8. Transfer column to a clean tube. Add 50 μL Elution Buffer to center of membrane and centrifuge at >12,000 × g for 1 min.
9. Discard column and store purified DNA at −20°C or use immediately in downstream applications.

Luria-Bertani (LB) Media, also known as Lysogeny Broth is a nutritionaly rich solution that can be used to grow bacterial cultures. LB Media typically contains peptides, carbohydrates, vitamins, and minerals which are needed by bacteria. LB can be prepared by mixing its constituent components, or from commerically available powder mixes.

Materials

• NaCl
• Bacto tryptone
• Yeast extract
• NaOH
• ddH₂O

Procedure

1. In a 1L bottle, add NaCl (0.17M), Bacto tryptone (1% w/v), yeast extract (0.5% w/v), ddH₂O to 600mL, 100µL of 5M NaOH.
2. Autoclave for 20 min within 2h, store in the fridge.

LB agar is LB media with added agar. The agar sets the LB into a gel, which provides a solid media for bacterial colonies to grow on. Antibiotics can be added to the gel before pouring onto plates, which can then be used to screen bacteria for antibiotic resistance. LB Agar can be made by adding agar powder while preparing LB media or by from commercially available powder mixes.

Materials

• LB media
• Agar powder
• Antibiotic of choice (1000x)
• 30 petri dishes

Procedure

1. In a 1L bottle, add 600mL of LB media and 9g of agar and shake.
2. Autoclave for 20 min within 2h.
3. Let it cool until you can touch the bottle without burning yourself and add 600 µL of 1000x antibiotics of choice.
4. Gently mix, pour into petri dishes (20mL per dish) and let it solidify for an hour at room temperature. Once solid, turn upside down the dishes at room temperature for a few hours, then store in the fridge.

Super Optimal Broth (SOB) media is a nutritionaly rich solution that is optomised for the preparation and transformation of competent cells. SOB is an adjusted version of LB media. SOB can be prepared by mixing its constituent components, or from commerically available powder mixes.

Materials

• Yeast extract
• Bacto tryptone
• NaCl
• KCl
• NaOH
• ddH₂O

Procedure

1. In a 1L bottle, add yeast extract (0.5% w/v), Bacto tryptone (2% w/v), NaCl (10mM), KCl (2.5mM), 600mL of ddH₂O, 120µL 5M NaOH.
2. Autoclave for 20 min within 2h, store in the fridge.

To extract the produced protein from the cell, we rupture the cells with lysozyme.

Materials

• 1 M Tris-HCl, pH 8.0
• 0.5 M EDTA, pH 8.0
• NaCl
• Triton X-100
• Lysozyme stock solution (10 mg/mL in 10 mM Tris-HCl, pH 8.0)
• BL21 Z17-induced samples

Procedure

1. Prepare the lysis buffer with the following composition:
   • 10 mM Tris-HCl, pH 8.0
   • 1 mM EDTA
   • 100 mM NaCl
   • 0.5% (v/v) Triton X-100
2. Resuspend the cell pellet in 300 µL of lysis buffer.
3. Add 25 µL of lysozyme stock solution.
4. Mix thoroughly by vortexing for a few seconds.
5. Incubate the samples at 37 °C for 30 minutes.
6. Centrifuge the samples at maximum speed for 3 minutes.
7. Carefully collect the supernatant, which contains the soluble protein fraction.
8. To obtain data about the protein content of the cell perform SDS-page gel

Since the proteins expressed were insoluble, and were not present in a supernatant after cell lysis, we have performed Urea denaturation to solubilize and purify the protein

Materials

• Urea powder (for preparing 4 M, 6 M, and 8 M solutions)
• Deionized or distilled water
• Sample pellets (from 2 mL bacterial culture)

Procedure

1. Prepare urea solutions of 4 M, 6 M, and 8 M concentrations.
2. Add 200 µL of the appropriate urea solution to each sample.
   Note: Since the samples contain only pellets from 2 mL of culture, 200 µL was used instead of the original Costa Rica protocol volume.
3. Incubate the samples for 60 minutes at room temperature.
   Observation: The first incubation of 35 minutes was insufficient; 60 minutes gave better results.
4. Centrifuge the samples at 5000 × g for 5 minutes.
5. Carefully collect and store the supernatant. Proceed to the next urea concentration.
6. Repeat steps 2–5 until the samples have been treated with the highest (8 M) urea concentration.
   Note: When urea concentraiton at which Pretein denatures is known, just perform one step at given urea concentration
7. Run an SDS-PAGE gel using 10 µL of each sample.
8. Electrophorese the gel at 100 V for 45 minutes.

The protocol has been recomended by Cytiva _[2]

Materials

• Urea powder (for preparing 8 M, 6 M, 5 M, 4 M, 3 M, 2 M, 1 M, and 0 M buffers)
• Imidazole (prepare 5 mM solution; 0.023828 g per 20 mL)
• Binding buffer (base solution, modified by adding urea and imidazole)
• Elution buffer (Cytiva HisTrap HP protocol base, with added urea)
• ddH₂O (filtered, non-pyrogenic “Steril-R”)
• 20% ethanol (for column storage)
• HisTrap HP column (1 mL)
• Protein sample (urea-extracted)
• Microcentrifuge tubes (1.5 mL and 2 mL)
• 15 mL Falcon tubes
• Pipettes, tips, and syringes (for buffer loading and washing)

Procedure

1. Prepare the buffers according to the Cytiva protocol [2]
   and prepare the prepare 5ml each of running buffer at a decreasing urea concentration
2. Wash the HiTrap HP column with 15 mL ddH₂O (filtered, non-pyrogenic “Steril-R”) to remove ethanol.
3. Wash with 5 mL binding buffer.
4. Perform a blank run:
   • 5 mL ddH₂O
   • 5 mL elution buffer
   • 10 mL binding buffer
5. Prepare the protein sample by mixing 0.5 mL of protein solution with 1 mL binding buffer and load it into the column.
6. Wash with 10 mL binding buffer.
   NOTE: for refolding run the colum with 5ml of each runnign buffer at decreasing concentraiton of urea
7. Elute and collect with 5 mL elution buffer.
8. Clean the column with 5 mL ddH₂O followed by 5 mL binding buffer.
9. Run 5 mL of 20% ethanol through the column for storage.
10. Replace upper and lower stoppers.

To express a prtein BL21 strain of E. coli has been used. in this strain protein produciton is induced by IPTG.

Materials

• LB medium (Lysogeny Broth)
• Kanamycin (antibiotic stock solution, typically 50 mg/mL)
• IPTG (Isopropyl β-D-1-thiogalactopyranoside, 1 M stock)
• E. coli BL21 strain (containing required plasmid)

Procedure

The steps of this protocol are devided into 3 sections for better oritentation

Initiation

1. Prepare 50 mL LB medium in a Erlenmeyer flask. Add Kanamycin (50 µL of stock) and mix thoroughly.
2. Add 500 µL of an overnight culture (we used E. coli BL21). Alternatively use a single colony from a plate (overnight culture is preferred for faster induction timing).
3. Incubate the flask at 37 °C with shaking at 100 RPM.
4. Monitor growth by measuring OD₆₀₀. Grow until OD₆₀₀ is between 0.6 – 0.9. Take measurements hourly and increase frequency to every 30 minutes as you approach the target range.
5. When the culture reaches the target OD, divide the remaining culture into two flasks: one will be induced and the other left as a control.
6. Induce the experimental flask by adding IPTG to a final concentration of 1 mM. (Leave the control flask uninduced.)

Measurement

1. After addition of IPTG, the samples (IPTG induced and control/non-induced) were left for a total of 5h at 37 °C at 100 RPM (samples can be taken even at 3h after induction).

Sample collection

1. Spin the Eppendorf tubes in 6000rpm in 5 min and pour of the supernatant. If you cant get all of the supernatant out, spin it down again and carefully pipet it out . freeze at -20 °C.
2. For the remaining culture in the e-flasks, add to Eppendorf tubes (aliquot 2ml), centrifuge down (6000RPM at 5 min) and pour off supernatant (pipet if required), freeze at -20 °C.

o Evaluate the protein content of cells we prepare samples and load on denaturing SDS-page gel

Materials

• Frozen cell samples
• ddH₂O (deionized/distilled water)
• Tris-Glycine SDS sample buffer
• β-mercaptoethanol (BME)
• Protein ladder (molecular weight marker)
• SDS-PAGE gel (precast or hand-cast)
• Electrophoresis buffer (Tris-Glycine-SDS running buffer)

Procedure

1. Resuspend the samples in 50 µL of ddH₂O.
2. From each sample, take 10 µL and add:
   • 9.5 µL of Tris-Glycine SDS sample buffer
   • 0.5 µL of β-mercaptoethanol (BME)
3. Mix gently and boil the samples for 5 minutes at 95 °C.
4. Assemble the SDS-PAGE gel into the electrophoresis tank and fill with SDS buffer.
5. Load your prepared samples into the wells.
6. Load 5 µL of protein ladder (we used Broad Range Protein Molecular Weight Markers from promega) into one well as a molecular weight reference.
7. Run the gel at 100 V (the current reached ~50 mA) until the dye front has migrated slightly beyond ¾ of the gel length.