Team:Technion-Israel/Design

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ACT. Overviews

ACT. is a hydrogel-based skin-screen containing proteins that serve as “decoy proteins”, aimed to block infection of host cells by the SARS-CoV-2 virus. For better understanding and visualisation, the product can be described as a three-part system: a fishing rod, a hook, and a bait (while the fish is the virus).

As the bait that entices the SARS-CoV-2, we use two “decoy proteins”: mutated ACE2 and Sybodies. As the hooks that carry the baits, we recruit two protein carriers: Bacillus subtilis spores or Microgel Beads containing Ni+ molecules. To make the product spreadable on our skin, in other words, as the fishing rod that holds them all together, we use a thermo-responsive hydrogel.


Fig. 1: Product design
The “decoy proteins” (Sybodies in purple, ACE2 in green) will be bound to one of the carriers (Microgel Beads in blue, Bacillus subtilis spore in yellow), those complexes will be integrated to the hydrogel base to create ACT.


The Baits

Mutated ACE2

Angiotensin-converting enzyme 2 (ACE2) is an enzyme that is attached to the cell membranes of cells in the lungs, arteries, heart, kidneys, and intestines. ACE2, additionally to its natural role in the body, is a cellular receptor for SARS-CoV-2 (and other coronaviruses). This is done through the connection between the ACE2’s extracellular peptidase domain (PD) and the spike protein’s receptor-binding domain (RBD)[1].

In our product, we tried to find a mutation of human ACE2 protein that has a stronger affinity, compared to the human WT, to the spike proteins. We planned to implement an intelligent design method: creating a library of the possible mutations, screening the library by protein interaction models, cloning the resulting sequences into yeast, analyzing the resulting proteins and then optimizing our design until the best result is achieved.


Fig. 2: Library Reduction and Verification Processes


The initial library contained 97,202 variants and was based on four different studies[2][3][4][5], all tried to identify amino acids (AA) composing the ACE2 sequence that are related in some way to the bonding with Spike protein (S). 16 AA were found to fit this criterion. We changed each of these AA to a different AA but with the constraint of not changing the AA sidechain group (Aliphatic, Aromatic, Acidic, Basic, Hydroxylic, Sulfur containing, Amidic). Thus, we got all the possible mutation combinations.

We reduced the library size to 2162 variants, using the Rosetta bioinformatics software (as explained and detailed at the Modeling page). If the number of variants were to be lowered even more, the next step would be reducing the new library by cloning and purifying the proteins by using the following four steps:

  1. Fusing a 6xHistidine tail (His-tagged) to a high yield protein plasmid applicable for yeast (416-FEC-mCore1).
  2. Cloning this plasmid (containing different variants) to a Saccharomyces cerevisiae W303 yeast strain by using a method known as “Any Gene Any Plasmid" (AGAP). This new method is reported to be faster and more efficient than simple cloning. It uses homologous recombination in vivo and includes transformation of linear fragments with homologous sites to each other[6][7].
  3. Purifying the protein variants from the yeast.
  4. Scanning their affinity to the virus’s spike protein by Fluorescence-activated cell sorting (FACS) using florescence-tagged spike protein and an antibody targeting human ACE2.

Fig. 3: AGAP – The engineering of the p416-FEC-mCore1-Histag-ACE2
[1] The p416-FEC-mCore1 was amplified to create two linear fragments. [2a] Those fragments, alongside with a G-block, were transformed into yeast. [2b] Inside the yeast, the plasmid was closed by in vivo recombination.


We intended to initially test the protein purification method by using the same system with yeCitrine (Yellow Fluorescent Protein) instead of the variants derived from our mutation library.

Sybodies

Sybodies, which stand for synthetic nanobodies, are synthetic camelid-like single-domain antibodies with specific and high affinity to only one cognate target. Normally, they are easy to produce, stable, have soluble behavior in aqueous solutions and can refold reversibly without additional improvements. All those properties are ideal for a practical binder, especially for prokaryotic expression[8]. In this part of our project, we produced Sybodies from E. coli cells, in order to incorporate them in the Hydrogel.

The sequences of the Sybodies used in our project were taken from the study “Sybodies targeting the SARS-CoV-2 receptor-binding domain”[9] , that describes the creation of three Sybody libraries targeting the receptor-binding domain (RBD) of SARS-CoV-2. We chose two sequences based on their high association constant (Ka) and low dissociation constant (Kd).

The first step in the process of creating functional Sybodies was to insert their sequence, with an additional fused His-tag, into an over-expression plasmid. The selected plasmid for this purpose was pET-9D, due to its T7 promotor, a commonly used promoter for over-expression of proteins[10].


Fig. 4: The final design of the plasmid pET-9d-Syb
The Sybodies are in purple, the His-tag is marked in gray and restriction sites that are used for the cloning (with BamHI and NcoI enzymes) are marked with black lines. Plasmid A contains Sybody #15 while plasmid B contains Sybody #68.


The second step was transformation and over-expression of the Sybodies with KRX competent cells. Single Step (KRX) Competent Cells are E. coli K12 derivative cells designed for improved protein expression. Following a protein purification step, the Sybodies’ presence was verified by SDS-PAGE and Western blot methods. Those methods allow to verify that the proteins were created in the right size and structure as Sybodies are expected to be.

The final step in this process is to verify the viability of the created Sybodies. The proteins’ ability to bind the SARS-CoV-2 Spike proteins was tested with SARS-CoV-2 Spike protein-Coated commercial 96 wells Plates with ELISE procedure.

The Hooks

Bacillus subtilis spore

In this part of the project, we aimed to successfully display the mutated ACE2 or Sybodies 15 & 68 (“decoy proteins”) on the surface of Bacillus subtilis PY79 spores. Spore surface display offers a rather new and useful way to display stable functional proteins for long periods of time without the use of chemicals[11]. While sporulating, the bacteria expresses spore coat protein (Cot) C/G. We use this to our advantage by fusing these Cot proteins, with a linker spacer, to one of the “decoy proteins”. These proteins are then displayed on the surface of the Bacillus subtilis spores and are available to interact with the virus's spike proteins.

Initially, the design for Bacillus subtilis spore surface display vector was planned based on four main studies containing overviews on the subject and lists of vectors with characteristics that match the purpose of the displayed proteins and their expected sizes[12][13][14][15]. For site-specific genomic integration into the Bacillus, a backbone plasmid was used as shuttle vector between E. coli and Bacillus subtilis[16]. Additionally, for validation, c-Myc tag was added downstream to the gene of “decoy protein”.


Fig. 5: The final design of the plasmids Pbs1c-Cot-Decoy
The Cot proteins (in yellow), the linker area (in green) and the decoy sequence (in red). Plasmid A contains Sybody #15 as the “decoy protein” while plasmid B contains Sybody #68 as the “decoy protein”, and plasmid C contains the human ACE2 sequence after codon optimization for Bacillus subtilis, as the “decoy protein”.


The cloning was done using E. coli TOP10 as the engineering chassis. After removing some fragments, several cell lines were created. Each cell line contained a different “anchor protein” (either Cot C or G) linked to a different “decoy protein” (either ACE2, Sb#15, or Sb#68). Our screening processes throughout the steps were based on antibiotic resistance and florescence (mRFP expression).

We then transformed the final plasmids to competent Bacillus subtilis PY79 cells and induced sporulation. To test the expression of the fused proteins on the spores’ surfaces we planned to use a colocalization assay based on fluorescent antibodies that target the different tags that were fused to the “decoy proteins” and to the spike protein (c-Myc tag and His tag respectively).

To ensure an affixed expression of spore display in the final product, we planned to validate the spores’ stability in the hydrogel environment. Therefore, we conducted several experiments to convey the applicability of using spores in the hydrogel in term of germination kinetics

Three germination tests were performed in water and in hydrogel:

  1. Analysis of germination in B. subtilis spores using optical readout– Spores exhibit light refraction that can be detected using a spectrophotometer set to 600 O.D. During germination, the spore loses this attribute resulting in decreasing of O.D. values.
  2. Dipicolinic acid (DPA) Measurement– DPA is used during sporulation to lower the water content of the cell and to preserve DNA. The spore excretes DPA into the media during germination, therefore, an increase in DPA concentration corelates to higher germination rates[17].
  3. Spore Staining The results indicated that B. subtilis spores are stable and have a lower germination rate in the hydrogel, as compared to water. This corresponds to a steady and mild decrease in O.D values and very slow increase of DPA concentration.
    However, the staining test indicated that there was a large amount of living bacteria present in the gel at the initial time point. Thus, the spore purification protocol needs to be modified.

Since the proteins are only active on sporulated bacteria, we wanted to find a way to make germination as least likely to happen as possible (especially in our gel). Therefore, in the future, we aim to use a non-germinating strain created and used by iGEM Munich 2012 team and iGEM Freiburg 2016 team[18][19].

Microgel Beads

The Microgel is commonly used for separation and purification of proteins fused to a His-tag. But in this project, it was recruited as a possible carrier of our “decoy proteins” against SARS-CoV-2.

Preparation of the Microgel can be summarized and depicted in three steps[3]:

  1. Fabrication of nitrilotriacetic acid (NTA) monomers
  2. Polymerization
  3. Nickel attachment.

As for the proof of concept of the Microgel Beads, several aspects were taken into consideration: size, functionality, and safety.

Size: We planned to use a Scanning Electron Microscope (SEM) and an optical microscope for the sake of getting images of the Microgel Beads to verify their features, including the sizes of particles and pores.

Functionality: This part of work was furthered divided into two parts: beads only and mixture of beads and hydrogel. The plan was designed this way since it is necessary to assess the Microgel Beads’ ability to connect His-tagged proteins with and without the presence of hydrogel. In this way, the connecting capability could be ensured, and the interference of the hydrogel to this property could be checked. The above-mentioned experiments would first be done with His-tagged green fluorescent protein (GFP) before examination with His-tagged mCherry-hACE2 protein.

Safety: The concerns regarding safety came from three aspects: skin penetration, nickel release and possible skin damage from Microgel Beads. First of all, since the aim of our project was to create a product which can form a thin layer on skin surfaces, it is crucial to validate the particle sizes of the product and do skin penetration tests. Previous research has already demonstrated that particles larger than 1 micron would barely penetrate the skin[20]. To determine the product’s actual skin penetration effect, we planned to apply porcine ear skin test method[21]. Secondly, due to the fact that there were nickel ions bound on Microgel Beads, it is also important to evaluate the percentage and rate of released nickels because free nickel ion is known to be a common allergen. To assess the release of nickel ions from the product, we planned to use an artificial sweat assay[22]. Last but not least, although the actual Microgel Beads were made of NTA polymer, there is no actual evidence to guarantee the safety of this material in skin applications[23]. Though we have consulted with several experts and received positive answers regarding this usage, we still planned to test it via Human Repeat Insult Patch Testing (HRIPT) in the future, in accordance with the FDA guidelines[21][24].

The above-described processes could be summarized as followed:

  1. Size: SEM & Optical Microscope;
  2. Functionality:
    • GFP connection for Microgel Beads & Microgel Beads + Hydrogel;
    • mCherry-hACE2 connection for Microgel Beads & Microgel Beads + Hydrogel;
  3. Safety: Porcine Ear skin test (skin penetration), Artificial sweat test (nickel release), HRIPT (NTA skin irritation);

The Fishing Rod

Thermo-Responsive Hydrogel

The thermo-responsive hydrogel is a polymer that can reversibly transform between liquid and solid (gel). When refrigerated (4oC) the hydrogel is liquid and once applied to the skin (37oC) it solidifies. This polymer was chosen for our project because the final product is expected to be applied on the skin and form a thin film. Previous research has already demonstrated that this hydrogel is suitable for clinical skin applications[25].

To prepare the hydrogel in a manner that would fit the project’s requirements, Pluronic F-127 was selected due to its low critical solution temperature which is around the body temperature[22][23]. In addition, considering the fact that the final product is aimed for skin administration, its gelation time would be critical for achieving this goal. In order to make the hydrogel fulfill this requirement, we went through the article and decided the most suitable concentration of Pluronic acid based on the results of that study.


Fig. 6: Illustration of Pluronic F-127 hydrogel creation.


References
  1. Perlman S, Netland J. Coronaviruses post-SARS: Update on replication and pathogenesis. Nat Rev Microbiol. 2009;7(6):439-450. doi:10.1038/nrmicro2147
  2. Procko E. The sequence of human ACE2 is suboptimal for binding the S spike protein of SARS coronavirus 2. bioRxiv Prepr Serv Biol. 2020;2:21-24. doi:10.1101/2020.03.16.994236
  3. Devaux CA, Rolain JM, Raoult D. ACE2 receptor polymorphism: Susceptibility to SARS-CoV-2, hypertension, multi-organ failure, and COVID-19 disease outcome. J Microbiol Immunol Infect. 2020;53(3):425-435. doi:10.1016/j.jmii.2020.04.015
  4. Inducible K, Natesh R, Schwager SLU, Sturrock ED. Crystal structure of the human angiotensin-converting enzyme – lisinopril complex. Nature. 2003;421(1995):551-554.
  5. Wan Y, Shang J, Graham R, Baric RS, Li F. Receptor Recognition by the Novel Coronavirus from Wuhan: an Analysis Based on Decade-Long Structural Studies of SARS Coronavirus. J Virol. 2020;94(7):1-9. doi:10.1128/jvi.00127-20
  6. Joska TM, Mashruwala A, Boyd JM, Belden WJ. A universal cloning method based on yeast homologous recombination that is simple, efficient, and versatile. J Microbiol Methods. 2014;100(1):46-51. doi:10.1016/j.mimet.2013.11.013
  7. izasa E, Nagano Y. Highly efficient yeast-based in vivo DNA cloning of multiple DNA fragments and the simultaneous construction of yeast/Escherichia coli shuttle vectors. Biotechniques. 2006;40(1):79-83. doi:10.2144/000112041
  8. Muyldermans S. Nanobodies: Natural Single-Domain Antibodies. Annu Rev Biochem. 2013;82(1):775-797. doi:10.1146/annurev-biochem-063011-092449
  9. Walter JD, Hutter CAJ, Zimmermann I, et al. Sybodies targeting the SARS-CoV-2 receptor-binding domain. bioRxiv. 2020. doi:10.1101/2020.04.16.045419
  10. Tabor S, Richardson CC. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Biochemistry. 1985;82(February):1074-1078.
  11. https://www.pnas.org/content/pnas/82/4/1074.full.pdf
  12. Guoyan Z, Yingfeng A, Zabed HM, et al. Bacillus subtilis Spore Surface Display Technology: A Review of Its Development and Applications. J Microbiol Biotechnol. 2019;29(2):179-190. doi:10.4014/jmb.1807.06066
  13. Iwanicki A, Piątek I, Stasiłojć M, et al. A system of vectors for Bacillus subtilis spore surface display. Microb Cell Fact. 2014;13(1):30. doi:10.1186/1475-2859-13-30
  14. Lin P, Yuan H, Du J, Liu K, Liu H, Wang T. Progress in research and application development of surface display technology using Bacillus subtilis spores. Appl Microbiol Biotechnol. 2020;104(6):2319-2331. doi:10.1007/s00253-020-10348-x
  15. Radeck J, Kraft K, Bartels J, et al. The Bacillus BioBrick Box: generation and evaluation of essential genetic building blocks for standardized work with Bacillus subtilis. J Biol Eng. 2013;7(1):29. doi:10.1186/1754-1611-7-29
  16. 2012 M. How do Sporulation & Germination Work? https://2012.igem.org/Team:LMU-Munich/Germination_Stop/Knockout. Freiburg 2016. Knock-Out. https://2016.igem.org/Team:Freiburg/Knockouts.
  17. Brock Biology of microorganisms 12th edn
  18. iGEM team Munich 2012, How do sporulation & Germination Work, 2012
  19. iGEM team Freiburg 2016, Safety: Knock-Out, 2016
  20. Lademann J, Schaefer H, Otberg N, Teichmann A, Blume-Peytavi U, Sterry W. Penetration von Mikropartikeln in die menschliche Haut. Der Hautarzt. 2004;55(12):1117-1119. doi:10.1007/s00105-004-0841-1
  21. Jacobi U, Kaiser M, Toll R, et al. Porcine ear skin: an in vitro model for human skin. Ski Res Technol. 2007;13(1):19-24. doi:10.1111/j.1600-0846.2006.00179.x
  22. Midander K, Pan J, Wallinder IO, Heim K, Leygraf C. Nickel release from nickel particles in artificial sweat. Contact Dermatitis. 2007;56(6):325-330. doi:10.1111/j.1600-0536.2007.01115.x
  23. SCCS. Scientific Committees opinion on Trisodium nitrilotriacetate (NTA). 2010
  24. Nash JF, Kern P, Integrated Safety Testing and Assessment of Topical Drug Products: FDA public workshop. 2018; Washington, DC.
  25. Lufton M, Bustan O, Eylon B, et al. Living Bacteria in Thermoresponsive Gel for Treating Fungal Infections. Adv Funct Mater. 2018;28(40):1801581. doi:10.1002/adfm.201801581




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Department of Biotechnology & Food Engineering
Technion – Israel Institute of Technology
Haifa 32000, Israel

    • igem2020.technion@gmail.com