Team:Queens Canada/Notebook

Protocols

Protocol for expression and purification of 3-24 ScFv for OspA biosensor

Griffin Watson-Boehnisch, Aidan Booker, Kathryn Jalink, Kody Klupt

Queen’s University at Kingston Department of Biomedical and Molecular Sciences


A Queen’s Genetic Engineered Machine Team project for the iGEM 2021 Competition

Additional project information can be found at queensigem.ca

Background

The goal of this year’s project is to develop a novel antibody-based test to detect Borrelia Burdorferi through outer surface protein A (OspA). To develop this test, we will design a single chain variable fragment (ScFv) to express in an E. coli system based on previous research by Ghosh and Huber (1). This ScFv will be fused, through glycine linkers, to the reporter enzyme Alkaline Phosphatase (phoA) to identify successful binding to low concentrations of ospA with fluorescence visible to the naked eye (2). Initially, however, a green fluorescence protein (GFP) will be fused to the ScFv in order to monitor successful protein folding within the E. coli system and binding affinity before testing is conducted using phoA. Finally, a His-tag will be sequence on the C-terminal to aid in the purification of the recombinant protein.

The intent of this test is to aid in the early detection of Lyme Disease caused by spirochete bacteria Borrelia Burdorferi. Lyme disease is a bacterial disease with no known cure which can only be treated properly if caught very early. In the province of Ontario, where our team is located, Lyme diseases infection rates are higher than anywhere else in our country and are continuing to grow rapidly. Therefore, as the spread of Lyme disease increases our team aims to produce a rapid field test that can aid in the diagnostic process of Lyme Disease.

This protocol will focus on the expression and purification of the fluorescent protein constructs used in our biosensor apparatus. Topics will include creating cells that will translate the synthetic DNA into our proteins, plasmid ligation, protein purification techniques, and assembly techniques. This protocol provides instructions for the building of our modular fusion protein.

A critical assumption being made in this protocol is that the entire fluorescent binding protein can be synthesized and ordered as one long DNA strand. This is not always the case for larger proteins. If this is the case, a non-modular or scarless assembly method must be considered. The proteins used in this protocol are quite small and should not be limited by the ordering limitations.


Creating Competent T7 E.Coli Cells

The protocol we use for creating competent T7 Express E. coli cells is based on the protocol used by iGEM, found in the Registry of Standard Biological Parts (3). The bacteria that we grew is the E. coli, T7 strain. This bacterium is primarily used for safety and environmental reasons. It is not able to aggregate in the environment and is also non-toxic, should an accident occur, it will likely not have a dangerous effect (4).

Our E. coli K-12 will be stored in the -80C freezer for future work. To do this we need to grow our cells on an SOB plate and have them colonize. SOB stands for sugar optimal broth, otherwise known as Hanahan’s Broth. This provides nutrition and the chance for our bacteria to grow.

Materials
  • Petri plates with SOP agar
  • Sterile loop
  • Glycerol
  • Cryot ubes
Methods (1)
  1. Add cells onto a SOB plate and grow (room temperature).
    1. Protocol lists about 16 hours as the time for growth.
  2. Remove single colonies and place into 2ml of SOB.
    1. Shake overnight using an incubator for about 14-16 hours at room temperature.
  3. Add glycerol to about 15%.
  4. Add 1 ml samples into Nunc cryotubes.
  5. Add tubes to a sealed bag and place bag into a dry ice/ethanol bag for 5 minutes.
  6. Store in the -80C freezer until needed for use.

When growing competent cells, it is important to 1) ensure the quality of the cells and 2) ensure a sterile environment, which can be done by treating the lab area with ethanol. This production protocol uses SOB as previously discussed and a CCMB80 buffer.

CCMB80 buffer contains:

Item Amount
KOAc 10 mM @ pH 7.0 10 ml of a 1M stock/L
CaCl2 x 2 H2O 80 mM @ 11.8 g/L
MnCl2 x 4H2O 10 mM @ 4.0 g/L
MgCl2 X 6H2O 10 mM @ 2.0 g/L
Glycerol 10 % @ 100 ml/L

**It is important to make sure the pH is 6.4. To do this you may have to titrate down using HCl. Store the buffer at 4C in the fridge. Now to grow the competent cells.

Materials
  • SOB
  • CCMB80 buffer
Methods (2)

Add 250 mL of SOB to a 1 mL vial of cell stock, grow at 20C to an optical density of 0.3 at 600 nm. Inoculate for 16hrs.

  1. Pre chill flat bottom centrifuge bottles in an ice bucket.
  2. Transfer the culture made into the centrifuge bottle.
    1. Weigh and balance bottles.
  3. Centrifuge at 3000g at 4C for 10 minutes.
  4. Decant the supernatant from the centrifuge bottles into waste and keep pellet.
  5. Resuspend the pellet in 80 ml of ice chilled CCMB80 buffer.
  6. Incubate on the ice bucket for 20 minutes.
  7. Re-centrifuge at 3000g at 4C and decant the supernatant into waste.
  8. Again, resuspend cell pellet in 10 ml of ice cold CCMB80 buffer.
  9. Measure the OD of the mixture of 200 µL SOC (growth medium) and 50 µL of the resuspended cells.
    1. The 200 μl SOC and 50 μl CCMB80 buffer is the blank.
  10. Add chilled CCMB80 to get a final OD of about 1.0-1.5.
  11. Incubate on ice for 20 minutes and prep for aliquoting.
    1. Prep dry ice
  12. Aliquot into chilled 2 mL microcentrifuge tubes 13. Store at -80C

It is expected according to the iGEM protocol that cells should yield about 100-40 colonies. To calculate the competent cell efficiency, refer to our supplementary tests section in this document.


PCR Amplification of desired DNA Sequence

Materials
  • Taq or Phusion PCR Kit
  • DNA of interest
  • Forward and Reverse Primers
  • Pipette and pipette tips
  • Thermal Cycler
  • PCR Tubes
  • Betaine
  • ddH20
Methods (3)
  1. Reaction mixture is created which includes 10ng of ScFv DNA, 0.5µL of Taq or Phusion polymerase, 5µL of 10x PCR Buffer, 1 µL of primer 1), 1 µL of primer 2), 1 µL of 10mM dNTPs per reaction (deoxynuclotide mix).
  2. Obtain distilled water to achieve a 50 µL final volume per reaction. Thus, 23.5 µL of water per reaction should be available to bring the total volume up to 50 µl.
  3. Place a 96 well plate into the ice bucket as a holder for the 0.2 mL thin-walled PCR tubes.
  4. Pipette the following reagents into PCR tube in the following order: distilled water, 10X PCR buffer, dNTPs, both primers, DNA, and polymerase.
  5. Add PCR tube to thermal cycler and set cycle parameters based on required annealing temperature and type of polymerase used.
  6. Repeat this process for 30 cycles, leading to 230 amplified DNA segments.
  7. Following PCR conduct PCR cleanup to purify the sample.

*Annealing Tm based on individual primers and type of polymerase used.


Restriction Digestion

This step is conducted to allow for the insertion of the DNA into the plasmid backbone. Our DNA is optimized to be pET28A compatible, so we adopt the restriction digest protocol used by iGEM in the Registry of Standard Biological Parts (5).

Materials
  • BamHI
  • Xhol1
  • CutSmart Buffer
  • ddH2O
  • DNA/Plasmid to be digested
Methods (4)

Note: Keep all components on ice.

  1. Add 250 ng of DNA/Plasmid to be digested.
    1. Add water to reach a total volume of 16 µL.
  2. Add 2.5 µL of NEBuffer2 (for optimal color activity).
  3. Add 0.5 µL of BSA.
  4. Add 0.5 µL of both restriction digests Xhol1 and BamHI.
    1. Mix and spin the tube.
  5. Incubate at 37C for 30 min, followed by heat incubation at 80C for 20 min to ensure the restriction digests enzymes are denatured.
  6. Run 8 µL of the mixture on a gel (To test for DNA/plasmid length).
    1. Based on the size of the band you can ensure the part length and plasmid are both accurate, it is a good idea to have a standard MW band.

DNA Ligation into Plasmid

The DNA ordered for our modular protein components must be inserted into a plasmid backbone. We use restriction digest enzymes recommended by iGEM. Our plasmid used is the pET28A vector. This ligation protocol is taken from the iGEM Registry of Standard Biological Parts (6).

Materials
  • T4 DNA Ligase
  • Buffer
  • BSA
Methods (5)
  1. Add 2 µL of BamHI – XhoI digested plasmid backbone, about 25 ng.
    1. Use the pET28A backbone.
  2. Add under 3 µL of BamHI—XhoI digested fragment.
    1. Should be a 300:50 ratio of backbone to insert.
  3. A ligation control should also be made with all materials except insert to confirm successful ligation.
  4. Add 1 µL of the T4 DNA ligase buffer.
  5. Add 0.5 µL of DNA ligase.
  6. Add water so that the final volume of the tube is 10 µL.
  7. Set on the benchtop for 1 hour or overnight at 16 oC.
  8. 1-2 µL of plasmid product will be used for transformation.
Figure 1 - pET28A plasmid map with ligated DNA of ScFV + GFP

Transformation of DNA into Cells

Transformation of DNA is required to enter our competent cells in order for those cells to grow our proteins of interest. Colonies can then be selected and purified. This protocol is from the iGEM Registry of Standard Biological Parts (7). Our group used the single tube transformation protocol since the number of samples we must transform is limited. This protocol requires a transformation control like an empty plasmid made during ligation, SOC media (for growth), and an antibiotic which will aid in colony selection (7).

Materials
  • Resuspended DNA to be transformed
  • 10pg/µl Positive transformation control DNA
  • Competent Cells (50µl per sample)
  • 1.5mL Microtubes
  • SOC Media (950µL per sample)
  • Petri plates w/ LB agar and antibiotic (2 per sample)
  • Glass beads
  • Pipette and Pipette tips
Methods (6)
  1. Add resuspended DNA to selected wells with 10 µL of distilled water.
    1. Sit for several minutes (should be red colour).
  2. Label the 1.5 mL tubes and pre-chill tubes on ice.
    1. Should be one tube per transformation.
  3. The competent cells created should be thawed over ice.
  4. Add 50 µL of competent cells via pipette to the 1.5 mL tubes followed by adding 1 µL of resuspended DNA to the 1.5mL tubes.
  5. Add 1 µL of the control DNA via pipette into a 2 ml tube.
  6. Incubate the 1.5 mL tubes on ice for 30 min.
  7. Heat tubes for 45 seconds at 42C – water bath.
  8. Return the tubes to ice and incubate on ice bucket for 5 min.
  9. Pipette 950 µL of the SOC media into each transformation tube.
  10. Incubate the tubes at 37C for 1 hour, shaking at about 200-300 rpm.
  11. Add 100 µL of the transformation via pipette onto petri plates.
    1. It is important to spread the plates with spreader or glass beads so single colonies can be selected.
  12. Spin down at 6800g for 3 mins, discarding 800 µL of the supernatant after centrifugation.
    1. Suspend the cells in the 100 µL of remaining supernatant and pipette onto the petri plates.
  13. Incubate overnight for about 14-18 hours at 37C (agar side up).
  14. Pick single colonies from the transformations, you can do a colony PCR to verify the part size, or make stocks, or grow up the cell cultures.

Plasmid Miniprep

Miniprep is used to identify the cloned DNA in the competent cell. This is a quality control technique for our application. The DNA elution’s from the cell may be run on an electrophoretic gel and the bands will provide insight regarding if our transformation was successful. iGEM recommends the use of a Qiagen Miniprep Kit – after a brief internet search, this seems to be a popular choice (8). This miniprep method adopts the protocol described by iGEM in the Registry of Standard Biological Parts. Note that ‘TE’ used is a buffer solution that includes Tris, a pH buffer, and EDTA.

Materials
  • Qiagen Miniprep Kit
  • Cell Culture
  • 1.6 ml Microcentrifuge tubes (2 per a miniprep)
  • TE (1:10)
  • Centrifuge
  • Pipette and Pipette tips
Methods (7)
  1. Centrifuge cell culture to pellet cells – supernatant should be discarded.
  2. Suspend the pellet in 250 µL Buffer P1 and transfer to microcentrifuge tube (fully dissolve).
    1. Note Buffer P1 has RNase A and is from the Qiagen Miniprep Kit.
  3. Add 250 µL Buffer P2, invert tube several times until viscous and clear.
    1. Limit reaction to 5 mins and do not vortex.
  4. Add 350 µL Buffer N3, invert tube several times – the solution should be cloudy.
  5. Centrifuge at 13,000 rpm for 10 mins.
  6. Collect supernatant from centrifugation and pipette it into the QIAprep spin column.
  7. Centrifuge for 30 – 60 seconds, discarding the flow-through.
  8. (Optional step) Add 0.5 mL of Buffer PB to the spin column and wash it by centrifuging for 30 – 60 seconds, discarding the flow-through. This will remove nuclease activity.
  9. Wash the QIAprep spin column adding 0.75 mL Buffer PE, centrifuge for 30 – 60 seconds.
  10. Discard flow-through, centrifuge for 1 min to remove wash buffer residue.
  11. Place QIAprep column in new 1.5 mL microcentrifuge tube.
    1. Elute the DNA by adding 50 µL of Buffer EB (10 mM Tris Cl, pH 8.5) or water to the center of the QIAprep.
      • Stand for 1 min, then centrifuge for 1 min.

Introduction of Protein Production via IPTG-Induction

To induce protein expression IPTG is used to remove the lac operon repressor allowing for overexpression of the recombinant protein sequence in large quantities (9).

Materials
  • LB Medium
  • IPTG
Methods (8)
  1. From a relatively fresh plate pick a colony and grow overnight at 37°C and 225 rpm in 10 mL LB+KAN.
  2. Inoculate 3 mL into 30 mL LB+CAM and incubate at 37°C and 225 rpm.
  3. When the cells reach an OD600 between 0.4 and 0.8, induce the protein expression with 0.2 mM IPTG (IPTG concentration can vary from 0.1 to 1M).
  4. Incubate at 37°C and 225 rpm for 5 hours (Hours and temperature can vary based on protein).

Lysis of Cells

To isolate the cellular contents and remove the cell membranes of our bacterium – eventually leading to a purified protein, the cell must undergo lysis. Our preferred method is sonication. Our lab has a Branson sonicator to conduct this, and the method described in this document is what a PhD candidate advisor of ours (Nolan Neville) typically employs.

Materials
  • Lysis Buffer
  • Sonicator
Methods (9)
  1. Cells will be resuspended first in lysis buffer. To know the appropriate buffer pH we have the solution at either +/- 1 pH unit from the proteins theoretical pI. TRIS and HEPES buffers will be used for a pI less than or greater than 7.5, respectively. Also, add 150 mM NaCl and a reducing agent like DTT to keep cysteines reduced.
    Protein pI Buffer Choice
    Phosphate binding with FRET 5.47 TRIS @ pH ~ 6.47
    Potassium binding with FRET 6.16 TRIS @ pH ~ 7.16
    Glucose binding with FRET 5.83 TRIS @ pH ~ 6.83
    PTH receptor with mNeonGreen 6.46 TRIS @ pH ~ 7.46
    PTH with mCherry 6.65 TRIS @ pH ~ 7.65
    Alpha klotho with mNeonGreen 7.12 TRIS @ pH ~ 8.12
    FGF23 with mCherry 6.66 TRIS @ pH ~ 7.66
    E coil with TEV cut side and GFP 5.18 TRIS @ pH ~ 6.18
    K coil with TEV cut side and GFP 6.22 TRIS @ pH ~ 7.22
  2. Pre-treat the cells with 0.2 mg/ml lysozyme.
    1. Lysozyme is used to weaken the cellular membranes in our cells.
  3. Sonicate the sample with the Branson Sonicator Probe for 5 mins.
    1. Alternate, 5 seconds sonicating, 15 seconds off. The sonicator generates lots of heat. Since proteins can denature at high temperatures it is important to rest the sample on the ice during the ’15 seconds off’.

Protein Purification Steps

For our larger protein construct, adding a 6-residue histidine tag that may bind to nickel – using principles of affinity chromatography – will allow us to capture our proteins quickly and without disturbing native function (9). Histidine is an amino acid that will most readily associate with nickel, which is a transition metal. Nickel is immobilized on the chromatography column. The protocol used here is adopted from Bornhorst et al. in the journal Methods of Enzymology (10).

Materials
  • NaCl
  • Imidazole
  • Tris base
  • Ni Column Kit
Methods (10)
  1. Centrifuge the cell lysate at 30,000 g for 30 mins at 4C.
  2. Discard the pellet.
  3. Add QIAgen 50% Ni2+-NTA (nickel-nitrilotriacetic acid) which should be mixed with ice-chilled loading buffer, this will bind the protein that is His-tagged.
    1. Add about 5-10 mg/ml of the 50% Ni2+-NTA.
    2. Stir for 1 hour at 4C.
  4. Load this resin into a column, washing with loading buffer at 4C (loading buffer volume should be 20 times the column volume).
  5. Wash the resin now with wash buffer, 20 times the column volume.
    1. This is the same as the loading buffer, however, it contains 10 mM imidazole, pH set to 8.0 (1 unit from physiological pH which our proteins operate at)
  6. Elute the protein with 20 column volumes of 10 to 250 mM imidazole in loading buffer.
    1. Collect 1 ml fractions of protein.

Note: Fractions may be assayed using SDS-PAGE ensuring that only the protein of interest is present. SDS-PAGE should be run with an MW marker and at 300 volts. Imidazole is also used to compete with histidine-nickel binding, therefore, allowing the purified protein to elute in fractions.


TEV-Protease Digestion

TEV-protease targets a sequence most commonly ‘ENLYFQS’ with its catalytic triad. Expressing our modular protein with a His tagged GFP and a TEV sequence will allow us to isolate and then cleave the GFP before testing for binding to ospA. A rule of thumb regarding how much TEV-protease to use is 1 OD at 280 nm of protease per 100 OD at 280 nm of substrate protein (11).

Calculating Competent Cell Efficiency

This test is to ensure that the cells used in the ligation and transformation of the synthetic DNA are efficient and have been grown correctly. This is important – having efficient cells means that our synthetic DNA will be translated into protein at the highest possible rate. This test uses plasmid controls and SOC, a growth medium. This protocol is used by iGEM and is in the Registry of Standard Biological Parts (12). Ensure the area is sterile with ethanol.

Methods (10)
  1. Thaw the competent cells on an ice bath. Label a 1.5 mL microcentrifuge tube for each transformation and chill these tubes on ice. (Triplicates of each concentration has been recommended).
  2. Spin the DNA tubes with the controls to collect the DNA.
    1. Spin for 20-30 seconds between 8000-10000 rpm.
  3. Pipet 1 µL of DNA into each of the microcentrifuge tubes.
  4. Pipet 50 µL of compotent cells into each tube and flick.
  5. Shock the cells (heat-shock) and place them in the water bath for 45 seconds.
  6. Transfer tubes back on the ice for 5 minutes.
  7. Add 950 µL SOC into each tube.
    1. Incubate at 37C for 1 hour while shaking at about 200-300 rpm.
  8. Pipet 100 µL from each tube onto the plate, spreading the mix evenly.
    1. Incubate at 37C for about 16 hours, positioning the agar side at the top and lid at the bottom.
  9. Count the number of colonies on the plate.

Using this equation, you can calculate the competent cell efficiency (11):

A competent cell should have an efficiency between 1.5e8 and 6.0e8 cfu/µg of DNA.


References

1. Ghosh, S., and Huber, B. T. (2007) Clonal diversification in OspA-specific antibodies from peripheral circulation of a chronic Lyme arthritis patient. J. Immunol. Methods. 321, 121–134

2. Part:BBa K1216001 - parts.igem.org [online] http://parts.igem.org/Part:BBa_K1216001#References (Accessed June 8, 2021)

3. Help:Protocols/Competent Cells - parts.igem.org [online] https://parts.igem.org/Help:Protocols/Competent_Cells (Accessed October 3, 2021)

4. NEB (2012) Safety Data Sheet for T7 Express lysY/lq Competent E.coli (High Efficiency). Mater. Saf. Data Sheet. 4, 8–10

5. Help:Protocols/Restriction Digest - parts.igem.org [online] https://parts.igem.org/Help:Protocols/Restriction_Digest (Accessed October 3, 2021)

6. Help:Protocols/Ligation - parts.igem.org [online] https://parts.igem.org/Help:Protocols/Ligation (Accessed October 3, 2021)

7. Help:Protocols/Transformation - parts.igem.org [online] https://parts.igem.org/Help:Protocols/Transformation (Accessed October 3, 2021)

8. Help:Protocols/Miniprep - parts.igem.org [online] https://parts.igem.org/Help:Protocols/Miniprep (Accessed October 3, 2021)

9. Protein Induction with IPTG

10. JA, B., and JJ, F. (2000) Purification of proteins using polyhistidine affinity tags. Methods Enzymol. 326, 245–254

11. Khan, S., Norris-Roozmon, G., Klupt, K., Hunter, B., and Neville, N. Protocol for expression and purification of a fluorescent binding proteins for a metabolite biosensor



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