Loading menubar.....

Team:ICS BKK/Experiments

Team:ICS BKK/Human Practices - 2020.igem.org

EXPERIMENTS


COMPUTATIONAL WORK OVERVIEW


  The purpose of the computational portion of this lab is to be able to identify specific properties of mutated Phage P2 that are able to bind to S. pyogenes. The overview of the method is as follows:

  1. Calculate binding energy of Phage P2 with S. pyogenes (Control)

  2. Create 450 Phage P2 mutants by permuting residues crucial to bacteria receptor binding

  3. Compute binding energy of the 450 candidates with S. pyogenes

  4. Compare the binding energy of the best candidate with the control

  5. Calculate binding energy of native phage (Phage A25) with S. pyogenes and compare with the binding energy of the best candidate


  We utilized three different softwares: The Scripps Research Insitute’s Autodock Vina [1], BIOVIA Discovery Studio [2], and MODELLER [3]. Autodock Vina is the main tool we used to predict the binding affinity between the phage and the bacteria.



WET LAB SUMMARY


  In order to acquire the gene sequence responsible for the binding mechanisms of bacteriophages to Streptococcus pyogenes, we used biopanning, enzyme-linked immunosorbent assay (ELISA), and gene sequencing. During biopanning, KM13 phage clones were exposed to S. pyogenes antigens, and bound phage clones were selected and isolated. By identification, isolation, and gene sequencing of the phages bound to ​S. pyogenes​, we can gain insight to the genetic mechanisms that allow phages to bind to S. pyogenes, and engineer them to inhibit ​S. pyogenes ​colonies in the future.


  However, due to COVID-19, we were unable to acquire S. pyogenes cultures and blood agar in a timely manner. Therefore, for this year’s project, we decided to use our limited time in the lab to validate the process of biopanning, ELISA, and gene sequencing by binding phage M13 to Staphylococcus aureus, a bacteria already accessible in our lab, instead of Streptococcus pyogenes. Since both S. pyogenes and S. aureus are gram-positive bacteria, we expect for the results of biopanning on S. aureus to be informative for the future tests on S. pyogenes. In future projects, we hope to complete our goal of sequencing the mutated Phage M13 that binds to S. pyogenes best.


  KM13 phage was determined to be a suitable phage candidate because it is lysogenic, in which the viral genetic material is integrated into the bacterium’s genome, and a recombinant protein is produced on the surface of the phage.



METHODOLOGY OVERVIEW


  The steps of the wet lab process are as follows: display, panning, enrichment, amplification, and ELISA (enzyme-linked immunosorbent assay).

(1) Display: Filamentous phage displaying variants of protein antibodies were displayed on the surface of the KM13 bacteriophages.

(2) Panning: The variant KM13 phage library is then exposed and incubated to immobilized S. aureus antigen. The excess of KM13 phages in 1000-fold allows for stability of the binding and minimal non-specific binding between KM13 and S. aureus.

(3) Enrichment: Non-binding phages were washed off, and bound phages were eluted by incubation with trypsin protease. This caused disruptions in the antigen-antibody interactions, leading to enrichment of the specific bound phages.

(4) Amplification: The eluted phages were then amplified overnight via infection of E. coli, resulting in an enriched phage population produced in a 96-well microtiter plate that displayed antibodies that bound to the S. aureus target.

(5) ELISA: To test for affinity of the phage clones after biopanning, ELISA, also known as enzyme-linked immunosorbent assay, was used to determine whether antigen binding had occurred. The dilutions of the selected phages were added to both the ELISA plates and a control protein. After incubation for 2 hours at room temperature, it was washed six times with TBST. The final activity was detected by the absorbances with an ELISA microplate reader. The absorbances were used to determine the affinity found among the selected M13 phages.




  Dr. Kitsricharoenchai and Dr. Hunsuwan both asserted that forming regular brushing habits is a more sustainable and healthy solution than engineering a product that patients need to depend on, even if the product is very effective.



  Therefore, we determined that our project would not be useful if dental cavities can already be simply prevented by tooth brushing. However, we already designed our project and determined our protocol. So, we shifted our interest to targeting S. pyogenes to treat strep throat and tonsillitis instead.



WET LAB PROTOCOL


Catalase test
   The catalase test is a biochemical test that detects if the organism produces the catalase enzyme. We tested it on S. aureus which is a catalase-positive organism, meaning that when we dropped in hydrogen peroxide, catalase would hydrolyze the hydrogen peroxide into water and gaseous oxygen, resulting in a reaction of gas bubbles. This test ensures if the organism that we are working with is purely that organism and not contaminated with any other species.

1. Bacterial culture preparation

  a. Finding the growth curve:

  - Sampling S.Aureus broth

    - We used a liquid solution of S.Aureus and broth (food for S.Aureus). We put that solution into a shaking incubator and took it out every 30 minutes to measure its OD600 value in a spectrophotometer. After 10 rounds (300 minutes) , we took the data for time and OD600, then plotted it on a scatter plot. Time is on the x-axis and OD600 value on the y-axis.

  - Scatterplot analysis:

    - Analyzing the growth curve, we looked for the biggest increase in the OD600 value, aka the mid log phase, which is when S.aureus is most productive. We determined the mid log phase by checking the greatest difference between two intervals.


  b. Growing S. aureus

  - Culture S. aureus (goal : OD600 = 0.4)

  - Put 30 microliters of S. aureus into new broth then put into shaking incubator and wait for one hour

  - Put broth into a cuvette and put into the spectrophotometer, then zero it to set a standard. Set wavelength to 600nm because OD600

  - Wash the cuvette then put solution into the cuvette. Got around 0.23, so put the solution back into the shaking incubator for 30 minutes. When measured again, we got 0.8.. so we diluted the solution, but the most we got was 0.6, using 350 microliters broth and 500 microL of S. aureus.

  - Dry the plates that came out from the fridge because there is water vapor that might contaminate the bacteria by putting them into the heating box for around an hour


  c. Pick single colony for bacterial growth curve plotting and colony forming unit assay

  - Spread plate

  - Use serial dilution to find the colony forming unit (CFU). Dilute by 10 times every time. Start by putting 900microL of LB into 5 out of 6 tubes. Put all of the solution into the first tube. Label as undiluted. Take out 100 microL of the undiluted solution into the next small tube, containing 900 microL of LB, and mix it using the vortex. Label as 10^-1. Take out 100 microL of the 10^-1 solution, put it into the next tube and mix it again. Label as 10^-2. Continue for the rest (Undiluted, 10^-1, 10^-2, 10^-3,10^ -4, 10^-5)

    - The reason for 100 and 900 is because the total volume is 1000 microL and we want the ratio of S. aureus and whole volume to be 1:10.

  - Put 100 microliters of each solution into separate plates, 6 in total. Use a metal rod to spread the solution. If not the colonies are going to be clumped together and cannot be used for biopanning. Be sure to dip the rod into alcohol and heat it up two times after using.

  - Put finished plates into incubator overnight

2. Biopanning

  a. Coating plate

  b. Put 50 microliters of S. aureus and BSA into two separate wells in the 96-well plate. Leave it for a day.

  c. Identify S. aureus or check for contamination through using hydrogen peroxide.

  - Hydrogen peroxide will catalyze gram positive bacteria (S. aureus) and form H2.

  d. Prepare buffers for Bio Panning

  - PBST + 0.1% Tween

  - PBST + 0.05% Tween

  - 5% BSA in PBS

  - Trypsin in TBSC buffers

  e. Bio Panning

  - 1) Wash the well with BSA and S. aureus with 200 microliter of PBST+0.05% Tween.

  - 2) Add 200 microliter of 5% BSA in PBS in the two wells.

  - 3) Incubate at 37 degrees celsius for 1 hour.

  - 4) Wash BSA well three times by using 200 microliter of 0.05% PBST

  - 5) Add the phage library

  - 6) incubate at 37 degrees celsius for 1 hour

  - 7) take the phage that didn’t bind with S. aureus well out

  - 8) Wash the S. aureus well 10 times with 200 microliter of 0.1% PBST

  - 9) Add 1xtrypsin dissolved in TBSC buffer

  - 10) Incubate at 37 degrees celsius for 1 hour

  - 11) take 1xtrypsin dissolved in TBSC buffer out

  - 12) Add in 200 microliter of buffer elution buffer

  - 13) Incubate at room temperature for 15 minutes

  - 14)Add in 100 microliter of elution buffers : 0.2 molar glycine of 2.2 pH and then take it out

  - 15) Add 15 microliter of neutralization buffer, 1 molar His - hcl (ph 9.14) to the solution that was taken out in step 14

  - 16) Take elute phages to infect phage TG.1

Round 2:
Same as above except changing from 10 μl of solution with 200 μl of TG, tris-glycine buffer, to 100 μl of solution with 200 μl of TG for the undiluted In case there are some bacteria left in the well, we also put 200 μl of TG - not sure) into it and incubate at 37C for 1 hour. Then do the same, using serial dilution to make 10^-1, 10^-2 solutions.

3. Colony counting

  a. For the 10^-2 on plate, it was 1.99 x 10^5 cfu/ml. For the 10^-2 elute, 5 x 10^2 cfu/ml. We picked 15 colonies from each plate and swirled it into each well in a 96-well plate as well as streaking it onto a plate of agar, also called masterplating.

  b. 

4. Superinfection with helper phage KM13

  a. Added 60 microliter of each well into each tube of LB agar with 100 microgram/ml ampicillin. Put tubes in the incubator shaker at 37C and 180 rpm and measure the OD until it is at 2 McFarland. After that, we put in the KM13 helper phage at a concentration of 10^8 pfu/ml. Incubate at 37C for one hour with no shaking. Add 50 microgram/ml of kanamycin since the helper phage has a kanamycin resistant gene. Add IPTG as an inducer that overexpresses the gene function that targets S. aureus. Let the tubes incubate at 30C overnight with shaking.

5. Phage precipitation

  a. We took the 30 tubes and centrifuged them at 8000 rpm for 5 minutes. After the centrifuge, we collected 1 ml of the supernatant. Then we added PEG6000 and 250 l of 2.5M NaCl into each of the tubes, and incubated them on ice for 1 hour. After the incubation, we centrifuged it again for 30 minutes at 3200g. After the centrifuge, we collected the pellet, and resuspended the pellet by 1 ml of PBS buffer. Repeating a step, we added PEG6000 and 250 l of 2.5M NaCl then incubated the tube with ice for 30 minutes. Following the ice incubation, we collected the pellet once again, resuspend by 1ml of PBS buffer and centrifuged it at 3200g for 10 mins. Lastly, we measured dsDNA by using a nanodrop spectrophotometer.

ELISA
After Biopanning (Prepare for ELISA) and biopanning

  1. Coat plate: coat antigen (S. aureus)

   - Culture S. aureus to achieve OD value of 0.65, which is mid-log phase

   - Calculate concentration of bacteria in ___ CFU/mL

   - Add 50 microliters of culture S. aureus to 96-well plate

   - Incubate to evaporate water for only bacteria

   - Wash 3 times by PBST to get only the bacteria that was immobilized

   - Blocking with 5% BSA 100 microliters because BSA is a protein which will block non-specific surface, surfaces without antigen and will prevent non-specific binding

Put 50 microliters of culture S. aureus into each well in a 96-well plate. Next incubate the culture to remove all the water from the bacteria. After incubation, wash each well with PBST to only get the bacteria that was immobilized remaining. Wash each well 3 times, taking out the PBST after every wash. For the final wash, wait 5 minutes before taking the PBST out. Next, put 100 milliliters of 5% BSA, a protein that will prevent non-specific binding, into each well. Then, wait 40 minutes before repeating the step of washing each well 3 times with PBST.

  2. Add phage

   - Add individual phage clone into each plate (10^9 pfu/mL) incubate at 4 degrees C overnight

Put 10^9 pfu/mL of phage clones from the tubes into each well, with each tube’s phages being put into 4 of the wells. Incubate the plate overnight at 4 celsius.

   - 3 populations:

     1. Bound phage S. aureus

     2. Contamination from the lab that does not display the binder molecules (unbound phage)

     3. Display nanobodies but cannot bind with the bacteria

  3. Wash

   - Remove weak bound phage to S. aureus with PBST (PBS and 0.1% Tween; tween will remove the weak hydrophobic binding between phage and bacteria) by washing thrice

  4. Anti m13

   - Add anti-M13 antibody (1st antibody), which will bind with pVIII (coat protein) of M13 phage

     - Will bind to all M13 phages. Therefore you need to wash well in order to avoid false positives

   - Add Goat-anti mouse HRP conjugate (horseradish peroxidase, enzyme, 2nd antibody) which will bind to the 1st antibody

   - Incubate for 1hr at 4 degrees C to have them bind

   - Wash 3 times by PBST to remove unbound 2nd antibody

   - The amount of HRP will tell you about the amount of high affinity

  5. Add TMB substrate 50 microliters

   - When the TMB substrate has been changed via HRP conjugate (oxidation reaction), it will change color to blue

  6. Stop reaction

   - If the OD is more than 2.0, it will not be reliable

   - Have to stop reaction

   - Add hydrochloric acid to increase hydrogen ions, stop reaction, solution becomes yellow

REFERENCES


[1] Morris, G. M., Goodsell, D. S., Halliday, R.S., Huey, R., Hart, W. E., Belew, R. K. and Olson, A. J. (1998), Automated Docking Using a Lamarckian Genetic Algorithm and Empirical Binding Free Energy Function J. Computational Chemistry, 19: 1639-1662.

[2] BIOVIA, D. S. (2015). Discovery studio modeling environment. San Diego, Dassault Systemes, Release, 4.

[3] A. Šali and T. L. Blundell. Comparative protein modelling by satisfaction of spatial restraints. J. Mol. Biol. 234, 779-815, 1993.

More information about the methods implemented in MODELLER, their use, applications, and limitations can be found in the papers listed on our web site at https://salilab.org/publications/. Here is a subset of these publications:

  1. A. Šali and T. L. Blundell. Comparative protein modelling by satisfaction of spatial restraints. J. Mol. Biol. 234, 779-815, 1993.

  2..A. Šali and J. P. Overington. Derivation of rules for comparative protein modeling from a database of protein structure alignments. Protein Science 3, 1582-1596, 1994.

  3. R. Sánchez and A. Šali. Comparative protein structure modeling: Introduction and practical examples with MODELLER. In Protein Structure Prediction: Methods and Protocols, D.M. Webster, editor, 97-129. Humana Press, 2000.

  4. M. A. Martı-Renom, A. Stuart, A. Fiser, R. Sánchez, F. Melo and A. Šali. Comparative protein structure modeling of genes and genomes. Ann. Rev. Biophys. Biomolec. Struct. 29, 291-325, 2000.

  5. A. Fiser, R. K. G. Do and A. Šali. Modeling of loops in protein structures. Protein Science 9, 1753-1773, 2000.

  6. F. Melo, R. Sánchez, A. Šali. Statistical potentials for fold assessment. Protein Science 11, 430-448, 2002.

  7. M. A. Martı-Renom, B. Yerkovich, and A. Šali. Comparative protein structure prediction. John Wiley & Sons, Inc. Current Protocols in Protein Science 1, 2.9.1 - 2.9.22, 2002.

  8. U. Pieper, N. Eswar, A. C. Stuart, V. A. Ilyin and A. Šali. MODBASE, a database of annotated comparative protein structure models. Nucleic Acids Research 30, 255-259, 2002.

  9. A. Fiser and A. Šali. MODELLER: generation and refinement of homology-based protein structure models. In Methods in Enzymology, C.W. Carter and R.M. Sweet, eds. Academic Press, San Diego, 374, 463-493, 2003.

  10. N. Eswar, B. John, N. Mirkovic, A. Fiser, V. A. Ilyin, U. Pieper, A. C. Stuart, M. A. Martı-Renom, M. S. Madhusudhan, B. Yerkovich and A. Šali. Tools for comparative protein structure modeling and analysis. Nucleic Acids Research 31, 3375-3380, 2003.

[4] Fukunaga, K., & Taki, M. (2012). Practical Tips for Construction of Custom Peptide Libraries and Affinity Selection by Using Commercially Available Phage Display Cloning Systems. Journal of Nucleic Acids, 2012(295719), 1-9. https://doi.org/10.1155/2012/295719

[5] Lee, C., Iorno, N., Sierro, F. et al. (2017). Selection of human antibody fragments by phage display. Nat Protoc 2, 3001–3008 https://doi.org/10.1038/nprot.2007.448

[6] Nian, S., Wu, T., Ye, Y. et al. (2016). Development and identification of fully human scFv-Fcs against Staphylococcus aureus. BMC Immunol 17, 8. https://doi.org/10.1186/s12865-016-0146-z

[7] Gagic, D., Ciric, M., Wen, W. X. et al. (2016). Exploring the Secretomes of Microbes and Microbial Communities Using Filamentous Phage Display. Frontiers in Microbiology. 7. https://doi.org/10.3389/fmicb.2016.00429

[8] Harada, L. K., Silva, E. C., Campos, W. F., et al. (2018). Biotechnological applications of bacteriophages: State of the art. Microbiological Research, 212-213(7), 38-58. https://doi.org/10.1016/j.micres.2018.04.007.