Team:IISER TVM/wetlab engineering

IGEM-IISERTVM

Wetlab


Engineering

Research:

The prospect of tackling real-life problems of both local and global relevance by leveraging the powers of synthetic biology was the primary motivating factor behind our iGEM project. We were inspired by the project of iGEM Ruia Mumbai 2018, Catechewing coli, an eco-friendly enzyme to remove paan stains from walls. In this regard, we explored many different issues, mainly focusing on environmental issues.



Concept:

Our team noticed black patches on walls both inside and outside our campus and was motivated to explore the potential reasons behind this. A detailed literature survey revealed that these patches are fungal molds that grow due to high atmospheric humidity. The spores from these colonies accumulate on building surfaces and can be inhaled by people inhabiting these spaces, leading to a plethora of respiratory illnesses. The year 2021 saw astronomical rises in fungal infections like mucormycosis, aspergillosis, and invasive candidiasis caused by Mucorales, Aspergillus, Candida, respectively in India, in conjunction with the COVID-19 pandemic. We also discovered that the fungal pathogens that cause these infections are ‘opportunistic’ in nature, meaning that they primarily affect immunocompromised patients, such as AIDS patients, people receiving organ transplants, COVID-19 patients, etc. Patients with severe COVID-19 or those recovering from such conditions have been reported to develop severe illness accompanied by a high mortality rate due to fungal infections.


Design:

We decided to develop Moldemort, a chimeric chitinase enzyme, as a solution to the problem we wanted to address. We hypothesized the enzyme would synergistically combine the activity of their parent molecules and would have higher chitinolytic activity than the wild-type chitinases. Our design takes inspiration from the domain architecture of class 18 and 19 endochitinase enzymes which degrade glycosidic bonds at non-specific internal sites of the chitin polymer, thereby disrupting the structural integrity of fungal cell walls. Our enzyme design combines functional domains from diverse chitinases, essentially combining the most favorable characteristics of each enzyme into a single enzyme. We also propose the possibility of nano-encapsulation mediated drug delivery as a potential mode for therapeutic intervention. We are also working on the potential applicability of this idea in diverse settings like agriculture and hospital settings where fungi are invasive and harmful.


Fig. Recombinant Chitinase Combo Gene inserted in pET28a Expression Vector

Why did we choose chitinase?

Chitin (a long-chain polymer of N-acetylglucosamine) is an underlying and conserved polysaccharide found in the cell wall of all fungal species. Chitinases are enzymes naturally produced by plants, bacteria, and other multicellular organisms to defend themselves from fungal infestation. Chitinase is used as an antifungal agent in combination with antifungal specifics therapeutic for various fungal infections.[1] Chitinase can also be used as a possible biomarker in the prognosis and verdict of several antifungal illnesses and allergies.[2]


The chitin biosynthesis pathway is rather complex and involves the orchestrated action of several enzymes in a well-regulated fashion. Hence, we hypothesize that it is unlikely for fungi to be able to alter this pathway to generate different chitin structures which our enzymes would be unable to lyse. In essence, since chitin is an essential molecule for fungi and its biosynthesis pathway is complex, we think that the probability of the emergence of drug-resistant fungi is low.


Therefore we develop new recombinant chitinases that combine functional domains from diverse chitinases, essentially integrating the best attributes of each enzyme into one. However, it'll contribute to a new class of antifungals, If successful. The design of unprecedented chitinase supports may act as vital and rational scaffolds in designing novel remedial agents in the treatment of various inflammatory diseases. Over the past decades, only five classes of antifungals have been discovered of which Olorofim is still under study. Amphotericin B (AmB), belonging to the class of polyenes, is shown to be effective against severe systemic fungal infections. The mechanism of action of AmB is based on the binding of the AmB molecule to the ergosterol that is present in the fungal cell membrane. It produces an aggregate that creates a transmembrane channel, allowing the cytoplasmic contents to leak out and leading to cell death[3]. Clinical manifestations of AmB nephrotoxicity include renal insufficiency, hypokalemia, hypomagnesemia, metabolic academia, and polyuria due to nephrogenic diabetes insipidus.[3] Their hefty cost also makes them less accessible to the poorer sections of our society. Olorofim antifungal agents called the orotomides work by targeting the enzyme dihydroorotate dehydrogenase (DHODH) in the de novo pyrimidine biosynthesis pathway. They've been designed to be administered orally and intravenously.[4] [5] Ultimately alarmingly, rampant use of these antifungals over the stretches has led to the emergence of various resistant populations of fungi, which necessitates the development of further robust remedial agents.

Build, Test, and Improve

  1. Surface area-based growth model

    Design:
    The experiment was designed to determine the fungal species’ growth rate and lag time using the best fit model.

    Build:
    Fungal samples were inoculated in potato dextrose agar plates, and images were taken and analyzed after 6hr intervals.

    Test:
    The fungus growing on plates had the following issues:
    • The growth of the fungus is confirmed by the surface area of the Petri plate itself. Once the fungus covers the plate, it grows volumetrically, making it challenging to analyze the growth.
    • Most of the fungus present on our campus are sporulating. The spread of spores within the plate causes several colonies to grow together, rendering the experiment useless, as different colonies superimpose on each other, and no proper radius is visible.
    • Sometimes, the fungal species may not grow radially but in a non-uniform shape, making the area calculation laborious and tedious.

    Fig. Radial growth based measurements for Aspergillus versicolor (top left), Trichderma virens (top right) and Aspergillus niger (bottom left and bottom right)


    Improvement:

    The OD-based growth model was considered owing to these drawbacks. Scattering of light by a replicating population over time, and hence the changing OD is a good proxy for the growth of fungus instead of measuring its changing radius.[6]

  2. OD based growth model

    Design:

    The experiment was designed to determine the fungal species’ growth rate and lag time using the best fit model. Moreover, this method would be applicable while calculating the IC50 of the chitinases.


    Build:

    For each well of a sample, 100μL 1X PDB and 100μL spore suspensions were added to check the changing OD over time.


    Test:

    While adding 100μL spore suspension, the concentration of PDB changed to 0.5X. Since all the fungi species had been cultured in 1X PDA/PDB, to maintain the normalization, the concentration of the PDB needed to be increased.


    Improvement:

    The concentration of PDB was changed to 2X so that upon dilution, the concentration of PDB in the well remains 1X. A significant difference in OD can be observed due to this change.


    Fig: Growth curve of A.versicolor in 0.5X PDB


    Fig2: Growth curve of A.versicolor in 1X PDB


  3. Protein Purification of Bacterial Chitinase Combo 2 (BC2)

    Design:

    The Ni-NTA Purification System was designed to purify BC2 chitinase, which is cloned in pET28a with C-terminal 6X His tag and is expressed in BL21 E. coli strain.


    Build:

    Cloned BC2 in BL21 was inoculated in 10 ml LB. Secondary inoculum (1 litre) induced with 1mM IPTG and incubated overnight at 16°C. After incubation the culture was pelleted down at 13,000 RPM for 15 mins. Cells were resuspended in 50 ml lysis buffer (50 mM sodium phosphate buffer (pH 7), 1 mM PMSF, 0.5 M NaCl, 0.05 % BME, 5 % Glycerol, 0.5 % Triton X-100, 1 protease inhibitor tablet) and sonicated in pulse mode (30 seconds ON and 30 seconds OFF for 15 cycles with 55% amplitude). The supernatant was collected and loaded onto Ni-NTA column pre-equilibrated with equilibration buffer (50 mM sodium phosphate buffer (pH 7), 1 mM PMSF, 0.5 M NaCl, 0.05 % BME, 5 % Glycerol, 0.5 % Triton X-100, 1 protease inhibitor tablet). It was then washed using 50 mL wash buffer (50 mM sodium phosphate buffer (pH 7), 0.5 mM NaCl, 1 mM EDTA) and eluted in 30 mL elution buffer (50 mM sodium phosphate buffer (pH 7), 0.5 mM NaCl, 1 mM EDTA, 250 mM Imidazole).


    Test:

    Collected samples were analyzed using SDS-PAGE (13% Resolving Gel, 6 % Stacking Gel).


    Observation and Inferences:

    The protein is getting eluted in Flow-Through and not binding to the column.


    Fig: SDS image of BC2 after Ni-NTA purification


    Probable reasons for failure:
    • The 6X His-Tag is incorporated in the 3D structure of the protein which does not expose it resulting in a lower affinity of our protein to the Ni-NTA column.
    • The Ni-NTA resin is not of good quality.

    Troubleshoot:
    • In order to confirm if the resin was the problem, we used a Qiagen Ni NTA resin provided by a Ph.D. guide who frequently uses it to purify his protein of interest. This still provided the same results which imply that the resin was not the problem.
    • We also tried incubating our crude lysate with the Ni-NTA resin on a rotor for an hour to facilitate a greater extent of binding of our protein to the beads. But the protein was becoming eluted inflow through.

    Conclusion:

    As we did not see any positive results by changing the resin, we confirmed that the 6X His-Tag present in our protein of interest was incorporated within its 3D structure which prevented its proper binding to the Ni-NTA Column. Upon consulting our Ph.D. advisors, we decided to perform Urea Dialysis wherein we would first denature our protein with 8M urea. This would allow the 6X His Tag to be exposed such that our protein of interest could bind effectively to the column.


    Urea Dialysis:
    • The pellet of secondary inoculum was resuspended in an extraction buffer containing 8M Urea, followed by purification using Ni NTA resin and dialysis.
    • In dialysis, the chemically denatured protein is refolded to sufficiently decrease the denaturant concentration and allow protein refolding. We did stepwise dialysis, the denatured protein solution was first brought to equilibrium with a high denaturant concentration (buffer containing 6M Urea), then, the concentration was decreased and brought to equilibrium at a medium concentration (buffer containing 4M Urea) and, then, further decreased and brought to equilibrium at a low concentration (buffer containing 2M Urea). At last protein, the solution was brought to equilibrium with a buffer containing 0M urea.

    Results:

    During the last phase of Dialysis, we observed suspended precipitates in the dialysis bag which had formed due to aggregation of our protein. The solution was centrifuged and the supernatant was separated into a separate tube. The nanodrop result for the supernatant exhibited a very low concentration of 30 ug/mL (total volume = 4 ml).


    Improvement I:
    • One of the probable reasons for aggregation could have been due to the long exposure of our protein to urea. It is known that at medium denaturant concentrations the proteins often take the inactive form during the refolding process due to the reformation of aggregates and the presence of other misfolded species.
    • Thus, we switched to a faster and more efficient way of removing urea from our protein solution. The method used was Buffer Exchange where the protein solution with urea was concentrated and diluted with the buffer (without Urea) over and over thus decreasing the concentration of urea. Urea gets diluted so much that it wouldn’t interfere with the downstream activity of the enzyme.
    • Even after Buffer Exchange, as we concentrated our Enzyme solution, we observed white precipitate suspended in the solution. The solution was immediately centrifuged and the supernatant was separated. Nanodrop of the supernatant still showcased very little concentration which implied that most of the proteins were lost as aggregation.

    IMPROVEMENT II:
    • We conducted an extensive literature study to find new methods for purifying proteins like ours. It was at this stage that we came across a suggestion in Research Gate provided by Reinhold Horlacher (currently working at Trenzyme GmbH, Germany) who had also encountered a similar problem.
    • We tried a new method of ethanol precipitation as suggested by Reinhold: 9 volumes of ice-cold ethanol (100%) are added to one volume of buffer containing the protein in 8M urea. The sample is incubated for 2h at -20°C and then spun down to pellet down the precipitate/protein. The pellet is washed with 90% ice-cold ethanol after which it is resuspended in 1X PBS, 0.1%SDS.
    • While concentrating our protein, we did see aggregation when concentrated below 2 mL. Thus, we changed the composition of the buffer by adding stabilizers such as 10% glycerol and increasing the NaCl concentration to 200 mM [ 1X PBS, 0.1%SDS, 200mM NaCl, and 10% glycerol] (Glycerol acts as a protein stabilizer by enhancing the hydrophobic interactions as a consequence of an increase in the solvent ordering around the proteins)
    • The nanodrop of the new protein showed a concentration of 3.2 mg/ml of protein which was appreciably good.

  4. Chitinase Assay:


    DNS Assay Method

    1. Experiment I:

      Design:

      Initially the experiments were designed by using crude enzymes due to complications in protein purification.


      Build:

      DNS assay was conducted with crude enzyme where 2.5 mL of Colloidal Chitin was mixed with 0.5 ml of the enzyme. Additionally, 2.5 mL of 1X PBS was also added to maintain pH at 7.3. 500uL was taken in intervals of 1,2,4,6 hour etc. The controls for the experiment were:

      i)Enzyme Control: instead of the enzyme, an equal amount of buffer was added to the reaction mixture.
      ii)Substrate control: instead of the substrate, an equal amount of buffer was added to the reaction mixture.
      iii) Denatured Enzyme Control: The enzyme was heat-denatured at 100℃ before incubating with the substrate.


      Test:

      Although the test sample showed an increase in OD540 during each interval, the substrate control, as well as denatured enzyme, also showed a random increase in OD540 after each interval. Hence the results of the test could not be validated further.


      Improvement:

      Since the data from the previous experiment was not conclusive. Our Ph.D. mentor suggested verifying the enzyme has some activity. This was done by incubating the substrate and enzyme at different ratios. It was assumed that with a higher enzyme ratio, the enzyme activity should be higher.


    2. Experiment II:

      Design:

      Since the absolute value of protein concentration could not be determined, we wanted to know if we could indirectly measure the activity of the crude enzyme. In order to verify the crude enzyme does show some activity, we designed yet another experiment with the guidance of a Ph.D. student.


      Build:

      Keeping the substrate concentration constant, the volume of the enzyme was changed in the following manner: 1:1, 1:3, 1:5. Appropriately the respective controls were also set up with enzyme lysate being replaced with lysate of Bl21(DE3) with an empty pET28a vector. The control, as well as the test samples, were incubated at the following temperatures: 30℃,40℃,50℃,60℃. If there was a trend that showed an increasing OD540 value as the enzyme: substrate ratio increases then the enzyme does have some activity.


      Test:

      Both the control as well as test samples showed an increasing trend with an increasing ratio. The control samples showed almost equal or even higher values of OD540 than the test samples.


      Improvement:

      We now wanted to know if the crude enzyme itself was contributing significantly to the OD540 values. For this, we designed yet another experiment.


    3. Experiment III:

      Design:

      Since the OD540 values were very less in the previous experiment, we wanted to know if the crude enzyme itself was contributing significantly to the OD540 data without the reaction even happening.


      Build: .

      We carried out an experiment where we selectively add only enzyme solution instead of the substrate to prevent the reaction. The OD540 was measured for test samples along with appropriate controls. The assay was carried out at 50℃ and only two ratios were used; 1:3 and 1:5. The additional volume was made up with the 1X PBS buffer.


      Test:

      The new data (without substrate) was comparable to the previous data (with substrate). This proved that any quantitative analysis done with crude enzyme was unreliable and hence it was not possible to measure the activity of the enzyme.


      Improvement:

      It was understood that we couldn’t move further with the assays by using crude enzymes. Thus, the team tried various methods for purifying the protein of interest (BC2). It was only after a thorough literature review that we were finally able to troubleshoot and purify BC2.

      After purifying our protein. We had a thorough discussion with Dr. Binod Parmeshwaran, NIIST, Thiruvananthapuram who had experience in performing chitinase assays. He suggested preparing fresh colloidal chitin using a modified protocol provided by him.


    4. Experiment IV:

      Design:

      Since we were not able to purify the wild-type chitinase in time, we decided to follow the DNS assay as mentioned in the paper from which the sequence had been borrowed.


      Build:

      According to the protocol, 1mL of enzyme solution (5ug of protein) was added to 1 mL of 1%(w/v) Colloidal Chitin. Appropriate blanks were set up and the experiment was performed in triplicates. The solutions were incubated for 10 mins at 40℃ after which 3 mL of DNS was added and heated to 100 ℃ in a boiling water bath. After heating, they were centrifuged at 7000 RPM for 10 minutes. 200 μL from each solution was loaded into a 96 well plate and their absorbance at 540 nm was measured using a 96 well plate reader.


      Test:

      The OD540 values were within the range of 0.0- 0.003 which were very small. This happened because the control itself had a high absorbance value at the specified OD.


      Improvement:

      Since the OD540 values were very less, we decided to increase the time of incubation from 10 mins to 1 hour. While repeating the experiment with increased time, we observed that the OD540 value was lower than the control value implying that the previous values might have been an instrumental error. This could have happened due to the following reasons:

      i) Our protein might have degraded
      ii) The DNS reagent might be contaminated
      iii) The other components of the reaction mixture itself have a higher OD540 reading that would mask the lower reading of the enzyme-substrate reaction.


    5. Experiment V:

      Design:

      The protein sample was subjected to Circular Dichroism (CD) which showed us that the protein still had its secondary structures intact. Hence we tried to resolve the issue with DNS by preparing a fresh DNS solution.


      Build:

      A background noise experiment was carried out with new as well as old DNS samples. This would measure how each component of the reaction mixture responded to the OD540 values.


      Test:

      It was observed that all the samples with new DNS showed a much lower background value than with the old DNS reagent. This would mean that we could get better OD540 readings with the actual assay. The buffer and water did not show any significant OD540 values. This implied that the buffer and water are not contributing significantly to the OD540 values during actual experimentation.


    6. Experiment VI:

      Design:

      In order to confirm that the OD increase was indeed due to enzyme activity and not a random event, we decided to design yet another experiment.


      Build:

      The DNS assay was performed with different amounts of protein (5 µg, 100 µg, and 200 µg). The expected results were that as the amount of protein increases the same would be reflected in their OD540 values.


      Test:

      It was observed that all three samples had a lower value than the control and hence the values were negative.


      Improvement:

      Since we were loading only 200 µL out of 4 mL, we thought that the N-Acetylglucosamine (NAG) released would be diluted a lot such that it would not be detected properly by the instrument. Hence we decided to lower the total reaction volume from 4 mL to 400 µL while maintaining the ratios in which each component of the reaction mixture was added.


    7. Experiment VII:

      Design:

      As the detectable product - NAG, was being diluted a lot, the final reaction volume was reduced from 4 mL to 400 µL while maintaining the ratios in which each component of the reaction mixture was added.


      Build:

      200 µL of the enzyme was added to 200 µL of the substrate and incubated for 10 mins at 40℃. After incubation, the amount of NAG released was quantified by the DNS method (200 µL of DNS was added instead of 3 mL).


      Test:

      This time we did observe a higher OD change from the control than ever before. We went ahead with calculating the activity for our enzyme using the OD540 values. We found out that the scale of the standard graph was not appropriate to correlate smaller quantities of NAG. Hence we decided to make another standard NAG curve within the range of 50-300 µg/mL of NAG. After plotting the new standard curve we were able to effectively calculate the enzymatic activity of our protein.


  1. Abhishek Singh Rathore, Rinkoo D. Gupta, "Chitinases from Bacteria to Human: Properties, Applications, and Future Perspectives", Enzyme Research, vol. 2015, Article ID 791907, 8 pages, 2015. https://doi.org/10.1155/2015/791907
  2. Madan K, Madan M, Sharma S, Paliwal S. Chitinases: Therapeutic Scaffolds for Allergy and Inflammation. Recent Pat Inflamm Allergy Drug Discov. 2020;14(1):46-57. doi: 10.2174/1872213X14666200114184054. PMID: 31934842; PMCID: PMC7509760.
  3. Laniado-Laborín R, Cabrales-Vargas MN. Amphotericin B: side effects and toxicity. Rev Iberoam Micol. 2009 Dec 31;26(4):223-7. doi: 10.1016/j.riam.2009.06.003. PMID: 19836985.
  4. Brian Park, PharmD. "Olorofim Receives Breakthrough Therapy Designation For Invasive Fungal Infections".MPR,2019, https://www.empr.com/home/news/drugs-in-the-pipeline/olorofim-receives-breakthrough-therapy-designation-for-invasive-fungal-infections/.
  5. Wiederhold NP. Review of the Novel Investigational Antifungal Olorofim. J Fungi (Basel). 2020;6(3):122. Published 2020 Jul 30. doi:10.3390/jof6030122
  6. "Measure Microbial Growth Using The „OD600“". Bmglabtech.Com, 2021, https://www.bmglabtech.com/measure-microbial-growth-using-the-od600/.