Team:Technion-Israel/Results

• Results
• General Overview
• Gels
• Bacillus subtilis Spore
• Mutated ACE2
• Sybodies

General Overview

In this page, detailed information will be provided to guide you through our adventure in the lab. Until this very moment, we have managed to purify and verify the Sybodies as well as created both the Microgel and the hydrogel. Since a relatively large number of experiments have been conducted, it’s reasonable to conclude that our designed “decoy proteins” can certainly be linked with other components as planned and eventually assemble ACT.

Gels

Overview Key Achievements Introduction Aims Results

Various natural and synthetic polymers, such as xyloglucan and dextran, have thermo-responsive properties ^[1]. Hydrogels made of these polymers have been applied in many areas, including drug delivery, tissue engineering since they can perform a quick reversible transformation between liquid and gel under temperature stimulation ^[1]. In search for a polymer that can form an undetectable film on skin surface, Pluronic F-127 was the right candidate, because of its lower critical solution temperature that is around the body temperature (37°C) ^[2].

Furthermore, during the search for carriers of “decoy proteins”, the possibility to use a polymeric material arose, in parallel to the biological one. Nitrilotriacetic acid (NTA) with nickel ions are commonly used in as polymers to purify His-tagged proteins. The NTA has four metal-chelating sites that allow it to hold a stable bonding with metal ions ^[3]. Because of the relatively high affinity and specificity of binding between His-tag and Ni-poly NTA, this polymer was chosen as the polymeric carrier for our designed “decoy proteins” and was named as “Microgel Beads” due to their own physical characteristic and the desired size which is about 6.5μm.

Herein, the Pluronic F-127 based hydrogel was created with a determined gelation time of ~1 minute. Moreover, NTA monomers have been prepared and microscopic images of the Microgel Beads which were composed of these monomers were obtained. Process utilization and the size of the Microgels are limited for now and adjustments must be made to reach the final product.

• Successfully created the target thermo-responsive hydrogel at a concentration of 18% w/v.
• Gelation time of created hydrogel was tested and matched the expectation (~1 min).
• NTA monomers were produced and the amount was relatively large (~11.4gr, 0.23gr was needed per polymerization reaction).
• Microscopic images of the Microgel Beads were obtained.
• The Microgel Beads’ ability to connect to His-tagged proteins was verified using His-tagged-GFP and results were confirmed via microscope and plate reader.

“How are we going to deliver the ‘decoy proteins’?”
This question that has bothered us for a long time since the product was designated to target SARS-CoV-2. With the help of our first public survey, and being inspired by the conventional sunscreens, the proposed product was shaped as a gel-type screen product that can form a thin protective layer which holds the “decoy proteins” with their carriers on the skin. After consulting with Prof. Boaz Mizrahi from the faculty of Biotechnology and Food Engineering in the Technion institute, it was found that a thermo-responsive hydrogel made from Pluronic F-127 (Poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide)) is suitable for the purpose of this project. Thermo-responsive polymers use temperature as their external stimulus to activate solution-gel transition and most of these materials in this family are capable of forming hydrogel at a temperature that is close to body temperature ^[1]. These hydrogels have been used for biomedical purposes, not only because their gelling behavior can be stimulated by temperature, but also due to their ability to swell in situ under physiological conditions ^[4]. These properties have guaranteed that Pluronic F-127 is suitable for the aim of convenient skin administration. This material was from the family of Pluronic copolymers, which also known as PEO-PPO-PEO copolymers ^[1]. Pluronic F-127 was chosen because of its low critical solution temperature (LCST) which is around the body temperature ^[2], and a previous study has also demonstrated that it is suitable for clinical skin administrations ^[5]. Therefore, the chosen concentration of this hydrogel was 18% w/v because according to the results, this concentration would present a product with gelation time of about one minute, which fits the goal of skin applications ^[6]. Moreover, to further verify the created hydrogel’s gelation time for the sake of ensuring that it would be ideal for this project, the variation of temperature at skin surface was taken into consideration ^[6]. Thus, the gelation time was determined at 32℃ and 37℃ in four replicates.

NTA is frequently used as a chelating ligand for metal ions, and Ni-NTA is frequently used to purify His-tagged proteins because this material can bind the histidine residues selectively and effectively via the immobilized metal ions ^[3]. The bonding between poly-histidine residues and metal ions was characterized as metal-ligand coordinative bonds ^[7], which can be generally considered as stable and is also recognized as highly selective ^[8].

In this project, an improved method of preparing the Ni-poly NTA particles was recruited since it enables a higher density of metal ions to be presented on the particles’ surfaces ^[9]. As one of the possible carriers of the “decoy proteins” that targets SARS-CoV-2, Ni-poly NTA matrices were formed and we named them as “Microgel Beads”, due to their spherical characteristic and representing the desired size (Figure 1.1).

First, the NTA monomers, which served as the building blocks of Microgel Beads, were created successfully in a considerable amount. Later, three attempts have been done for fabrication of the Microgel Beads. Although lots of efforts have been invested to optimizing the production of Microgel Beads, the resulting amount of the product was low and after analysis via microscope, it was observed that there was a deviation from the particles’ size (40μm-90μm) and expectation (6.5μm).

Figure 1.1: Illustration of Microgel Beads linked with Sybodies. B- Illustration of Microgel Beads linked with ACE2.

The main focus of this part of the work includes the fabrication of the hydrogel, determination of the hydrogel’s gelation time, creation of Microgel Beads using NTA monomers and Nickel ions, verification of Microgel Beads’ connection with His-tagged proteins and estimation of the beads’ size. In addition, expression and purification of His-tagged mCherry fused to human ACE2 protein using HEK-293F cells were done, since this fused protein is needed in the proof of concept experiment of Microgel Beads to examine whether the connection with beads would interfere the proteins’ functionalities.

1. Hydrogel Fabrication and Gelation Time Examination

   The thermo-responsive hydrogel was created by simply dissolving Pluronic F-127 powder with Double Distilled Water (DDW) (Figure 1.2). Since ACT. is aimed for skin addministration, the chosen concentration of hydrogel was 18% w/v since a previous research has demostrated that at this concentration, it would have a gelation time of approximate one minute at 37℃ ^[6]. The protocol of creating the hydrogel can be found here. Before proceeding to actual gelation time tests, the hydrogel went through a primary transformation test to brifely examine its ability of performing reversible solution-gel transformation. The test was done by transferring a small amount of gel into a glass bottle and placing it in a 37℃ water bath (gel), then putting it on the ice (liquid), as shown in Figure 1.3.

   
   
   

   Figure 1.2: Illustration of Hydrogel preparation

   
   
   
   

   Figure 1.3: A- Solidified Hydrogel after warming at 37℃. B- Liquid Hydrogel after cooling on ice.

   
   

   Since the hydrogel was designated for external skin application and the skin temperature varies according to the sourroundings, two temperatures, 32℃ and 37℃, were chosen to represent the limit of common skin temperature range. This was done in four replicates to increase the measurments’ accuracy. The detailed protocol could be found here. According to the results (Figure 1.4), the gel’s average gelation time at 32℃ was ~63.5 seconds and for 37℃ was ~40.75 seconds. Although there was a deviation between the measured result and expecataion, such a small difference could be caused by manual error since this measurement was not analytical. In conclusion, it is reasonable to say that the 18% w/v hydrogel’s gelation time was about 1 minute in the common range of skin temperature, which makes it suitable for the purpose of ACT.

   
   
   

   Figure 1.4: Gelation time of Hydrogel at 32℃ and 37℃

   
   

   The created hydrogel was planned to be used in the proof of concept experiments of Bacillus subtilis spores and the whole final product. For the verification of spores, the main idea was to figure out whether the spores that displayed the “decoy proteins” on their surfaces would germinate when they were mixed with the hydrogel at different temperatures. Regarding the examination with the Microgel Beads together, the key goal was to examinate whether the hydrogel would interupt Microgel’s ability to connect with His-tagged proteins. Unfortunately, the later part of experiment wasn’t done because of the time and material limitations.

2. Microgel Fabrication

   As mentioned in the introduction, the “Microgel Beads” are polymer of NTA that are linked with Nickel ions. Such a material was chosen because of its ability to perform selective binding with proteins that have His-tags with a high affinity. Detailed procedures of preparing the Microgel Beads could be found here and these steps can be generally summarized into three parts: NTA monomer fabrication, polymerization and nickel attachment. During the time in the lab, two attempts have been made for creating the NTA monomers (Figure 1.5) and three attempts were done for making the final Microgel Beads using these monomers.

   
   
   

   Figure 1.5: A- NTA Product from 1st attempt. B- NTA Product from 2nd attempt.

   
   

   As shown in the photos above, the monomers were created successfully, and a relatively large amount of NTA monomers were obtained (~11.4gr) compared to the amount needed for each round of polymerization (0.23gr). However, the problem was mainly related to the steps after obtaining NTA monomers. In total, the Microgel Beads were prepared three times: the first time with the 1st attempt of monomers while the second and third used monomers from the 2nd attempt. Although the amount of produced Microgel from the first preparation was fairly large (Figure 1.6), the color was close to green and led to the suspicion that the majority of the powders in this tube were nickel salts instead of the desired gel particles. Besides, one of the important steps of creating NTA monomers, which was dripping the mixture of acryloyl chloride and toluene into N,N-bis(carboxymethyl)-L-lysine NaOH solution (Figure 1.7) was done incorrectly since the acryloyl chloride toluene mixture was poured into the beaker instead of adding it one drop at a time. Therefore, the NTA monomers of the 1st attempt (Figure 1.5A) and the first Microgel product were abandoned (Figure 1.6).

   
   
   

   Figure 1.6: : 1st Microgel product

   
   
   
   

   Figure 1.7: : Dripping system used in the NTA monomers’ preparation

   
   

   In the second trial of Microgel Beads creation, the process was improved by changing the power of sonication from 40% to 20%. This change was made because after centrifugation, some of the suspected gel particles were trapped at the interface of water phase and oil phase (Figure 1.8). After discussing with Prof. Boaz Mizrahi, it was found that the sizes of some particles were too small, which meant the sonication power was too strong. Regardless, the amount of resulting product was too little (~0.2mg) (Figure 1.9). To solve the yield problem, in the third attempt, the sonication procedure was replaced by aggressive stirring. Unfortunately, the product yield was even lower.

   
   
   

   Figure 1.8: : Particle Trapped at Interface after Centrifugation

   
   
   
   

   Figure 1.9: : 2nd Microgel product (~0.2mg)

   
   

   Before proceeding to the next step, the created Microgel Beads from the 2nd and 3rd attempt were taken for microscopic analysis to estimate their sizes. Figure 1.10 shows that the Microgel Beads are round, and the one which was circled with white dotted line was a “broken” bead. This “broken” bead led us to believe that the particles were polymers instead of water or air bubbles. To make sure that the particles are three dimensional, another image was taken via Z-Stack (Figure 1.11). Although nice images were obtained using samples from the 2nd Microgel products, no proper image was taken for the product from the 3rd attempt because the sizes of particles varied and some of them were oversized for the microscope. After analyzing all the microscopic photos of particles from 2nd Microgel product, the particles’ sizes were estimated to be in the 40μm-90μm range. According to literature ^[10], the expected size of particles was around 6.5μm. The reason of such a deviation could be caused by the differences in the protocols, since the power of sonication used in the 2nd attempt was 20% instead of 40% that was suggested by Prof. Boaz Mizrahi. Another possible reason is that the sizes of particles increased due to swelling since this polymer can swell in aqueous solutions.

   
   
   

   Figure 1.10: : Microscopic image of Microgel Beads (Product from the 2nd Attempt, circled in white: a “broken” bead)

   
   
   
   

   Figure 1.11: : Height image of Microgel Beads via Z-Stack

   
   

   To conclude this part of the work, the preparation of NTA monomers was successful and the high yield resulted in several trials of polymerization experiments, though extra attention is required at the dripping step. However, our main struggle is improving the yield of final Microgel Beads and restraining their sizes in the desired range.

   Our future plans for the Microgel Beads could be divided into three sections: verification of their connection with His-tagged GFP (detailed in the next section), verification of their connection with fluorescence marked hACE2 and verification of their connection to fluorescence-marked hACE2 after mixing with hydrogel. In addition, only the 2nd Microgel Beads were used in further experiments since their sizes were close to the desired size range.

3. Verification of Microgel Beads via Enhanced Green Fluorescence Protein (eGFP)

   Since Microgel Beads were meant to be the carrier of “decoy proteins” which have His-tags, it is crucial to examine their ability to selectively bind His-tagged proteins. As mentioned in the previous part, three experiments were planned for this verification but only the first one was performed due to time limit, and the results of it would be presented in this section.

   This experiment was conducted according to the protocols and could be concluded as followed: setting GFP blank, incubating the Microgel Beads with GFP solutions, and obtaining results using the plate reader and microscope. As written in the protocol, the Microgel Beads were planned to be tested with GFP solutions at two concentrations (33.33ng/μl, 16.67 ng/μl) since the size of particles were slightly oversized compared to the expectation. Nevertheless, the product was tested with only one concentration (33.33ng/μl) because there was only a tiny amount of Microgel available. The tested samples were taken from the Microgel Beads of 2nd attempt because of their relatively large amount and close to proper sizes.

   The microscopic analysis has given us two parts of results, including a rough quantification of connected GFP and some images. Firstly, the green fluorescence intensities of GFP+Microgel samples and pure Microgel negative controls were compared and presented below (Figure 1.12A). To get the value of intensity, an example is shown in Figure 1.12B. Based on the graph (Figure 1.12A), after incubating with GFP, the Microgel’s fluorescence intensity has increased approximately two times, which proves that the beads have connected to the GFP successfully. However, it is obvious that such a measuring method is not analytical and objective enough. Besides, there is also a relatively large amount of GFP trapped inside the polymer particles since there are pores on these spheres’ surfaces. Therefore, we measured the GFP fluorescence by a plate reader. Besides this brief estimation (Figure 1.12), some images were also taken using the microscopy for visual comparison (Figure 1.13). By comparing the images of the Microgel w/o GFP in Figure 1.13, it could be easily told that there is a significant difference between the fluorescence intensities of GFP incubated samples and the control, which meant that the Microgel Beads were capable of binding His-tagged GFP.

   
   
   

   Figure 1.12: A- Comparison of green fluorescence intensity of Microgel Beads incubated with and without GFP via optical microscopy. B- Example of determining the green fluorescence intensity via microscopy, the particle’s fluorescence intensity was represented by the peak value of the graph.

   
   
   
   

   Figure 1.13: A, B- Microscopic images of Microgel Beads (negative control) taken at 490nm light and were standarized to an intensity of 5*10⁴. C, D- Microscopic images of Microgel Beads+GFP (490nm light, standarized to an intensity of 5*10⁴.

   
   

   The plate reader was used to verify the Microgel Beads’ ability to bind His-tagged proteins in a more analytical way. Since directly measuring the fluorescence of a dense sample of Microgel+GFP might result in low accuracy, we chose to use an indirect method. This indirect measurement was aimed to use the difference in fluorescence intensities of pure GFP solution and supernatant of Microgel+GFP which contains unbound GFP to represent the amount of connected GFP. According to the results (Figure 1.14), the average value of green fluorescence in the supernatant (Post-treatment) was almost half of the intensity of the original GFP solution (Pre-treatment), which could led to the conclusion that the Microgel Beads created in the lab were able to bind the His-tagged proteins as expected.

   
   
   

   Figure 1.14: Comparison of green fluorescence intensities between GFP solution (Pre-treatment) and supernatant (Post-treatment) that contains unbound GFP (460nm excitation and 510nm emission)

   
   

   After proving the Microgel’s ability to connect with His-tagged proteins, our future plan regarding this part involves combining the created Microgel Beads with fluorescent marked human ACE2 protein to figure out whether the connected proteins’ functionalities would be altered. Then, the Microgel would be connected with the designed “decoy proteins” to construct the final product.

4. Side Task: hACE2-mCherry-tdPP7-His Protein Expression in HEK-293F Cells

   Production of fluorescent marked human ACE2 protein were necessary since this material couldn’t be obtained commercially and was needed for one of the proof of concept experiments of the Microgel Beads. The used plasmid that encodes human ACE2 protein with fluorescent marker and His-tag hACE2-mCherry-tdPP7-His (Target, Figure 1.15A) and a second plasmid, hACE2-tdPP7-His (Control, Figure 1.15B), was used as a control for the experiment. This Control was used because this plasmid has been used for ACE2 protein expression in HEK-293F cells and the proteins were also purified successfully in the lab. And the Target is what we actually wanted to obtain. Both plasmids were designed and cloned by Dr. Sarah Goldberg, a Research Assistant from Prof. Roee Amit’s lab in the Faculty of Biotechnology and Food Engineering in the Technion.

   
   
   

   Figure 1.15: A- Gene map of hACE2-mCherry-tdPP7-His (Target). B- Gene map of hACE2-tdPP7-His (Control).

   
   

   The chosen cells for transfection were the HEK Freestyle 293F cells, and the protocol for cell culture could be found here. The whole process includes cell culture, transfection and protein purification. This was done with the help of Dr. Sarah Goldberg and Or Willinger from Prof. Roee Amit’s lab. To check whether the cells were transfected properly, samples were taken for plate reader and Fluorescence-activated cell sorting (FACS) at three time points: one, two and five days after transfection. Results were presented as followed (Figure 1.16). As shown in Figure 1.16A, the mCherry fluorescence intensity has increased significantly at Day 2, which implied that the transfection process has succeeded. However, since the FACS results could not provide useful information regarding amount of fluorescent protein that were secreted into the medium, it is necessary to include plate reader analysis of the medium. This step is important because eventually, the proteins would be purified from the medium. According to the result in Figure 1.16B, the fluorescence intensity of transfected cells’ supernatant, which represents the cell medium, has also increased dramatically at Day 2. Therefore, by combing the results from FACS and plate reader, it could be concluded that the cells were transfected successfully and have started to express the plasmid and explode the proteins to medium two days post-transfection.

   
   
   

   Figure 1.16: Figure 1.16: A- FACS Results at 1, 2, and 5 Days Post Cell Transfection (Percentage of Live & Single Cells was based on total cell count of 10⁴ while percentage of mCherry Expressing was based on the number of cells that were alive and single.). B- Plate Reader Results at 1, 2, and 5 Days Post Cell Transfection (560nm excitation and 612nm emission; Blank: FreeStyle Medium; Target: hACE2-mCherry-tdPP7-His.).

   
   

   Five days post-transfection, the proteins were purified from the transfected cells’ growth medium for both the Target (hACE-mCherry-tdPP7-His) and Control (hACE2-tdPP7-His). To increase purification yield, this process was split into two parts using different methods: His-affinity agarose (Figure 1.17) and MagneHis Protein Purification System (Figure 1.18). Firstly, samples were taken for concentration measurement (Table 1.1 and Table 1.2) using Nanodrop spectrophotometer in order to select the elution solution that has the highest protein concentration. According to the Nanodrop’s results, the His-affinity agarose’s purification efficiency was higher than its counterpart and in general, the products’ protein concentrations were low. To ensure that the purified proteins of the Target set were with fluorescence as desired, samples were taken for plate reader and the elution buffer was used as control. As shown in the figure (Figure 1.19), compared to the control which was the elution buffer, the elution samples from His-affinity agarose purification method had a significant fluorescence, which indicates the presence of desired proteins. By combining the above-mentioned results, from products purified by His-affinity agarose, the third elution product (E3) of Target and second elution product (E2) of Control were chosen to be loaded in the SDS-page with all the products from MagneHis Protein Purification System as well. After analyzing the images of SDS-page gel (Figure 1.20, Figure 1.21), negative result was found for the elution product of MagneHis Protein Purification System while for its counterpart, bands showed up for both Control and Target, and the sizes were close to expectation.

   To sum up this part, both proteins for the Target and Control were successfully purified from the growth medium of transfected cells although the concentrations were low.

   
   
   

   Figure 1.17: Protein Purification via His-affinity Agarose

   
   
   
   

   Figure 1.18: Protein Purification via MagneHis Protein Purification System

   
   

   Table 1.1: Concentrations of Purified Proteins Measured by Nanodrop spectrophotometer (His-affinity Agarose, calibrated by elution buffer, the values of samples that were used for further processes are underlined)

   Name	Eluted Product (E1) Concentration (mg/ml)	Eluted Product (E2) Concentration (mg/ml)	Eluted Product (E3) Concentration (mg/ml)	Eluted Product (E4) Concentration (mg/ml)	Eluted Product (E5) Concentration (mg/ml)
   Target	0.259	1.026	1.161	1.110	1.069
   Control	0.615	1.379	1.339	1.218	1.117
   BSA 1:10	1.406

   Table 1.2: Concentrations of Purified Proteins Measured by Nanodrop spectrophotometer (MagneHis Protein Purification System, calibrated by elution buffer)

   Name	Concentration of Eluted Product (mg/ml)
   Target	0.117
   Control	0.076
   BSA 1:100	0.118

   
   
   

   Figure 1.19: Plate reader results of samples post-purification

   
   
   
   

   Figure 1.20: SDS-Page result of purified proteins from His-affinity agarose (Target: hACE2-mCherry-tdPP7-His; Control: hACE2-tdPP7-His; Protein Standard: BSA 1:10; Protein Ladder: PM2600 ExcelBand)

   
   
   
   

   Figure 1.21: SDS-Page result of purified proteins from MagneHis Protein Purification System (Target: hACE2-mCherry-tdPP7-His; Control: hACE2-tdPP7-His; Protein Standard: BSA 1:100; Protein Ladder: PM2600 ExcelBand)

   
   

   Due to the low concentrations of the purified proteins, a concentration step was needed. Prior to the actual concentration, a dialysis (Figure 1.22) was required since the buffers used in the purification contained imidazole which would lead to unavailable His-tags. As a result of the SDS-page analysis, only the third elution product from the His-affinity agarose purification was used. The protein concentrations were again measured via Nanodrop (Table 1.3). Post-concentration samples’ concentrations were re-measured after the concentration process (Table 1.4). Unfortunately, although the concentrations of Control and the BSA were lifted successfully, the concentration of Target has failed.

   
   
   

   Figure 1.22: Dialysis of Purified Proteins

   
   

   Table 1.3: Concentrations of Purified Proteins Measured by Nanodrop (Post-dialysis, calibrated by dialysis buffer)

   Name	Protein Concentration (mg/ml)
   Target	0.179
   Target’ *	0.015
   Control	0.246
   BSA 1:100	0.005

   (* The sample of Target was split into two since the volume of sample increased significantly after the dialysis. These two sets of samples were combined again during the concentration.)

   Table 1.4: Concentrations of Purified Proteins Measured by Nanodrop (Post-concentration via Amicon, calibrated by dialysis buffer)

   Name	Protein Concentration (mg/ml)
   Target	0.108
   Control	1.117
   BSA 1:100	0.013

   
   

   As a summary to all the work in this part, two plasmids were transfected into HEK-293 cells and the proteins were successfully purified. However, since the proteins’ concentrations were low and the concentration using Amicon did not work well, the products could not be used in the proof of concept experiments for Microgel Beads. One of the possible solutions for obtaining the Target proteins in a higher concentration might be growing more transfected cells to increase the initial protein concentration prior to the purification.

Bacillus subtilis Spore

Overview Key Achievements Introduction Aims Results

Due to accumulated knowledge of many studies over the years, and the fact that its genome can be easily manipulated, B. subtilis bacteria has become one of the most widely used strains in the world of synthetic biology. Moreover, unlike its common counterpart, E. coli, B. subtilis is considered "Generally Recognized as Safe" (GRAS) and is used even in the food industry.

In the search for a carrier that will display the “decoy proteins” on its surface, one of the most attractive options was to display them on a membrane of living B. subtilis bacteria. In that way, the living bacteria would become the front-line producers of those “decoy proteins” and one of the dominant components in the final product against the viruses. While searching for methods to express heterologous proteins on the membranal surface of a vegetative B. subtilis, we came across a special technology that involves the use of B. subtilis spores. B. subtilis spore surface display (BSSD) technology uses the fusion of spore coat protein (or Cot) with heterologous proteins for displaying them on the spore’s coat with high activity and stability.

Here, we present a method for the expression of our heterologous “decoy proteins” on the surface of the B. subtilis PY79 spores using BSSD technology. By fusing the spores coat protein (CotC or CotG) to “decoy protein” we will be able to decrease the binding of the viruses to the human cells, thus reduce the chance of infection. During our lab work, we were able to induce sporulation on the B. subtilis PY79 strain, as well as design, construct and integrate pBS1C _SYB15 and pBS1C _SYB68 plasmids to the B. subtilis genome. The integration of pBS1C _SYB68 into the B. subtilis PY79's genome was successfully verified by Colony-PCR. Germination of the B. subtilis PY79 spores in the hydrogel was tested as well and it was found that our hydrogel is a less convenient environment for B. subtilis PY79 spores’ germination compared to water.

• Sporulation of B. subtilis PY79
• pBS1C _SYB15 and pBS1C _SYB68 plasmids construction
• pBS1C _SYB15 and pBS1C _SYB68 plasmids integration into the amyE locus of the B. subtilis.
• Incorporating of B. subtilis in thermo-responsive hydrogel

Bacillus subtilis, a gram-positive bacteria that can be found in different environments, has been the model bacteria for many iGEM groups and researchers in the field of synthetic biology over the years. The development of the synthetic biological toolbox in recent years has made the B. subtilis one of the most productive hosts for enzymes and biochemical products alongside Escherichia coli (E. coli), which is another common host in the world of synthetic biology ^[10]. The industrial need for the ongoing production of highly utilized recombinant proteins has increased over the years and made B. subtilis one of the most widely used bacteria thanks to extensive knowledge and ease of genetic manipulation.

Under significant extreme conditions, such as nutrients depletion in the environment, many vegetative gram-positive bacteria, including B. subtilis, can perform sporulation as described in Fig. 2.1 In this form, the bacteria can survive for a long time under extreme conditions. During this process, an asymmetrical division of genetic materials takes place from a mother cell into smaller daughter cells called pre-spores. At the end of this process, the pre-spore cells become endospores and are released into the surrounding medium. The pre-spore organized into three main layers: an amorphous undercoat, a lamellar layer, and a striated electron-dense outer coat. In parallel with the later formation, a proteinaceous layer forms around the surface of the outer spore coat ^[11].

Fig. 2.1: Illustration of the sporulation process

The outer coat layer of the spore is a source of great interest as a tool for research and application in many fields such as biotechnology, synthetic biology, and molecular biology. The reason for this is the ability to use several dozen proteins that make up the outer coat of the spore (Cot proteins) as an anchor to other heterologous proteins and display them on the outer surface of the spore ^[12]. For example, Negri et al. were able to express fragments of Clostridium difficile (C. diff) flagellar cap protein (FliD protein) on the surface of B. subtilis spores using many Cot proteins, such as CotC and CotG, to produce a FliD-based vaccine against C. diff ^[13]. Another example came from the 2016 iGEM group from Freiburg, which worked on the introduction of functional glutathione S-transferase (GST) and an epitope-specific nanobody for targeted drug delivery ^[14]. The technology used by most researchers is called "Bacillus subtilis spore surface display" (BSSD) which involves a fusion of the target protein to one of the Cot proteins, transforming the relevant plasmid into the B. subtilis and encouraging sporulation. Verma et al. ^[15] showed a functional expression of one of our chosen “decoy proteins” (Angiotensin-converting enzyme 2 (ACE2)) in Lactobacillus paracasei. Based on the extensive work that is mentioned above, we decided to combine the ideas to create a B. subtilis spore that expresses the “decoy proteins”, which can bind to the virus's spikes, as described in Fig. 2.2 These spores will be an integral component in our final hydrogel-based product, meant to decrease virus' infectivity.

Figure 2.2: Illustration of the binding between the virus’s spike protein with “decoy protein” that will be expressed on the spore's surface of B. subtilis PY79.

• Design the final plasmid that includes the gene encoding the coat protein fused to the gene encoding one of the "decoy proteins".
• Transformation and integration of the fusion protein gene into the B. subtilis PY79 genome.
• Induction of sporulation on the B. subtilis PY79.
• Testing for the expression of the protein complex.
• Germination test in the hydrogel environment.
• Test the ability of the Cot-decoy protein complex to bind to the virus's spikes.

Plasmid Construction

Initially, six different sequences were designed to be incorporated into the genome of B. subtilis as describes in Fig. 2.3 Inspired by the articles mentioned above10 ^{[11] [12] [13] [14] [15]}, a 33 bases linker was attached between the Cot protein genes and the gene of interest. The decision to use the pBS1C_LacZ as a backbone was based on a study of Radeck et al. in which this vector was specified as capable of undergoing integration into the Amy-E locus (encoding for α-amylase) of B. subtilis.

Fig. 2.3: Designed plasmids for integration into the B. subtilis PY79's genome: pBS1C_CotC_Syb15, pBS1C_CotC_Syb68, pBS1C_CotC_ACE2, pBS1C_CotG_Syb15, pBS1C_CotG_Syb68, pBS1C_CotG_ACE2.

The first cloning step involved linearization of the vector pBS1C_LacZ, removal of the LacZ-encoded sequence, and insertion of corresponding edges to the G-block sequences encoding the “decoy proteins” via PCR reaction and Gibson Assembly (Fig. 2.4)

Fig 2.4: First cloning steps – Revers PCR and Gibson Assembly.

After each plasmid transformation into E.coli Top10, 2 different tests were performed to verify the insertion of the sequences. The first one was a colony-PCR test, in which two different primers were used- one that overlaps with the backbone (pBS1C) and the other that overlaps with the sequence encoding for each one of the decoy proteins (ACE2 / Sybody15/ Sybody68) separately. Promising clones were then sent for another verification by Sanger sequencing.

After a few attempts, we were able to get both pBS1C_Sybody15 and pBS1C_Sybody68 as seen in Fig. 2.5 & 2.6 with a 100% match to the planned sequences. Regarding the ACE2 sequence, several attempts have been made with changed parameters to insert it into the pBS1C backbone using the Gibson assembly method, but no positive result has been obtained. In this case, a second option was planned, which included the use of restriction enzymes, but so far, we have not been able to perform it, due to lack of time. At this stage of the experiment, we decided to proceed only with the pBS1C_Sybody15 and pBS1C_Sybody68 plasmids.

Fig. 2.5: 1K NEB Ladder. Black arrows represent bands in the desired sizes. Expected results: 2’.1-2’.8, pBS1C_Syb15 ~ 600 bp. Negative control ~ no bands.

Fig. 2.6: 1K NEB Ladder. Black arrows represent bands in the desired sizes. Expected results: 3.1-3.4, 3’.5-3’.8: pBS1C_Syb68 ~ 600 bp. Negative control ~ no bands.

The pBS1C backbone we worked with also contained the mRFP encoding sequence that we removed in a subsequent step, in which we used pre-designed specific restriction sites to insert the sequence encoding the Cot proteins as seen in Fig. 2.7.

Fig. 2.7: Cot protein insertion and mRFP removal.

Unfortunately, we were unable to insert the designed sequence encoding the coat protein even after several restriction attempts under different conditions. That could be due to low insert/backbone ratio, low inserts concentration (Cot proteins), as a result of relatively low recovery from low-melting agarose gel. Due to a lack of time, we were unable to test all parameters.

Integration test

Using an integrative vector in bacillus strains is common and intended to prevent instability of recombinant plasmids as a result of structural rearrangement ^[16]. One of the methods to make a vector integrative is to use a gene encoding to α-amylase (amyE locus). In this method, the desired gene can be integrated within amyE locus by utilizing the natural mechanism of homologous recombination, by flanking the gene of interest on both sides in two halves of the amyE gene designated amyE-front and amyE-back ^[17] as seen in Fig. 2.8.

Fig. 2.8: Integration to AmyE locus

Here, we used the described method to integrate pBS1C_Syb68/15 plasmid into the genome of B. subtilis. A colony-PCR test was performed after plasmid transformation and integration. The results in Fig. 2.9 show the success of pBS1C_Syb68 integration in several colonies.

Fig. 2.9: Log Ladder. Black arrows represent bands in the desired sizes. Expected results: 1-17: PY79_Syb68 - ~600 bp.

Genomic sequencing of the bacillus, which was pre-designed for the final plasmid (pBS1C_CotC/G_ACE2/Sb#15/Sb#68), is another option for testing the integration for further verification.

Sporulation

Sporulation is a process that gram-positive species can undergo, including B. subtilis bacteria, when they are facing harsh environmental conditions, such as the depletion of nutrient sources. The coat of B. subtilis spores composed of more than 20 polypeptides, which confers resistance to lysozyme and too harsh surrounding environments. During sporulation, the bacteria form a rigid shell that maintains the ability to survive under environmental stress ^[18][19].

Encouraging sporulation is an integral part of our idea. Since the "decoy proteins" can only be expressed when the bacteria undergo sporulation as described in Fig. 2.10, an adjustment of the working conditions is required to make sure that the majority of the population will be in the form of spores, rather than vegetative bacteria. Therefore, as a preliminary step, a protocol corresponding to the B. subtilis 168 WT was tested, and adjustments were made to adapt it to our chosen strain - PY79.

Fig. 2.10: Illustration of B. subtilis spore surface display of ACE2 using CotC/CotG protein.

The B. subtilis PY79 strain is characterized by a knockout of two genes, which are responsible for the production of proteases – proteins that impair other protein bindings to the outer spore coat. These bacteria were diluted and incubated in a Difco Sporulation Medium (DSM) that stimulates sporulation ^[20]. After several adjustments that included increasing the number of washing steps, we were able to reach ~90% spores out of the general population Fig. 2.11.

Fig. 2.11: B. subtilis PY79 spores under a microscope. Spores are shown as white spheres.

Integration of the Bacillus in the hydrogel

Although spores can survive for a long time in their dormant state, once the conditions in the environment become more favorable for the growth of the vegetative bacteria, the spores will germinate and become living and functioning bacteria ^[21]. In that scenario, the decoy proteins expressed on the surface of the spore will no longer display and the effectiveness of the product will be significantly reduced.

Many components and conditions trigger germination. For example, the presence of nutrients in the living environment such as L-alanine, L-valine, and L-asparagine, high pressure of several thousand atmospheres, etc.

In this part of the project, the stability of the spores, and the germination rate were examined given the aqueous environment and hydrogel one. The effect of skin temperature (~37°C) and refrigerator storage temperature (4°C) on the stability of the spores were also examined. As a control, samples incubated in water were examined, since high water content is a significant factor in the swelling of the spore and the completion of the germination process.

The first test performed is based on the turbidity in O.D_600nm. Spores exhibit light refraction that can be detected using a spectrophotometer set to O.D_600nm ^[22]. During germination, the spore changes its physical shape resulting in lower O.D values. As can be seen in Fig. 2.12, even after 1-hour incubation no significant decrease in the spores’ amount was observed in different environments and temperatures, which emphasizes the stability of the spores in our product.

Fig. 2.12: The change in turbidity (O.D600 values) relative to initial measurement over time

To further examine the germination process, a pyridine-2,6-dicarboxylic acid (DPA) release test was performed using O.D420nm turbidity test. The core region of the spore is rich in that spore-specific molecule (DPA) and poor in water ^[22]. During germination, the same molecules are released into the environment and can serve as a tool for testing the germination rate. In Fig. 2.13, you can see the difference in concentration between the aqueous environment and the hydrogel environment after about an hour. While the DPA concentration reached ~ 250 g/mL in the aqueous environment, its equivalent in the gel showed concentrations that were ~10 times smaller. Therefore, it can be concluded that the hydrogel environment does not constitute a good basis for the germination process and that the integration of the spores in the gel raises the possibility of obtaining a stable product.

Fig. 2.13: DPA concentration in both medium (water and hydrogel) at different temperatures

The structure of endospores makes it difficult for chemicals to penetrate, making them difficult to stain. Some dyes penetrate the spore using steam. Malachite green is one of the dyes that stains endospores in green, as can be seen under the microscope ^[23]. The greenish color does not come out of the stained spores, even after rinsing with water. In contrast, it is easy to remove the green Malachite dye in vegetative cells. For vegetative bacteria, Mercurochrome is frequently used, which stains the cells in a red-purple color ^[24]. By using those dyes, we were able to tangibly see all the results described under the microscope. Fig. 2.14 shows the significant differences in the concentration of vegetative bacteria in water and gel after one hour, when more vegetative bacteria were observed in water. These results are consistent with the results of the DPA test described above.

Fig. 2.14: Spores and vegetative cell assessment in water and hydrogel. (A) Spores in water, time zero; (B) Spores in water after 1-hour incubation; (C) Spores in Hydrogel, time zero; (D) Spores in Hydrogel after 1-hour incubation. Spores were stained with Malachite green (greenish spheres) and vegetative B. subtilis PY79 cells with Mercurochrome (red-purple rod shapes).

Future plans

During the construction of the plasmids, we encountered difficulties in reaching the final constructs. The first hurdle was the construction of the pBS1C_ACE2 plasmid. After several unsuccessful attempts using Gibson assembly, it was decided to proceed only with the pBS1C_Syb15/68, as they managed to pass the initial stage of cloning. To ligate the ACE2 sequence, a contingency work plan was planned in advance for further work. This plan involves the use of restriction enzymes instead of Gibson assembly to attach the ACE2 to the pBS1C backbone.

The final plasmid assembly including fusion of the decoy proteins with the sequence encoding the coat proteins was designed using restriction enzymes.

The expression of the proteins on the spores was designed to be tested by marking the c-Myc with a fluorescent anti-Myc followed by FACS measurement.

In the final stages, the affinity of the “decoy-proteins” for the spike proteins was designed to be tested using affinity tests such as Bio-layer interferometry (BLI) real-time test or Surface Plasmon Resonance (SPR) ^[25].

Mutated ACE2

Overview Key Achievements Introduction Aims Results

Angiotensin-converting enzyme 2 (ACE2) is an enzyme found in cell membranes of cells in the lungs, arteries, heart, kidneys, and intestines. its role is to catalyze the hydrolysis of angiotensin II into angiotensin (1-7) and therefore lower blood pressure. In addition to its natural role in the body, ACE2 is a cellular receptor for SARS-CoV-2 (and other coronaviruses). The infection by SARS-CoV-2 happens through the connection between the ACE2 extracellular peptidase domain (PD) and the SARS-CoV-2 spike proteins’ receptor-binding domain (RBD) ^[26].

The goal of this sub-project is to produce one of the “decoy proteins”, an improved ACE2. The protein that we aim to produce will play a role in the product as "the bait". The main purpose of producing an improved ACE2 is to enable an interaction with higher affinity between the protein and the virus's spike proteins compared to the native one. This will be achieved by changing different amino acids (AA) that are related to the spike - ACE2 binding to achieve a higher affinity connection. In this way, we can increase the chances of capturing the virus and prevent infection.

Here, we managed to produce linear fragments out of p416-FEC-nCore1 plasmid. Those fragments contain 20-40 overlap base-pairs that enable us to use them in "Any Gene Any Plasmid" (AGAP) cloning method ^{[27] [28]}. In addition, transformation was conducted with the linear fragments and a designed gBlock that contains a mutated ACE2 sequence into W303 yeasts strain. We observed growth of yeast on the reaction plate, but due to the contamination that was found on the plate, the experiment was terminated. Unfortunately, due to the lockdown, we did not achieve our goals, but in the future, we aim to proceed by repeating the procedures and take special caution regarding a sterile work environment.

• Producing "R-16 cut" fragment by PCR reaction.
• Producing F-9 fragment with additional His-tag by PCR reaction.
• Transformation of 3 linear fragments to W303 yeasts strain.
• Adding His-tag to p416-FEC-nCore1 plasmid.
• Transformation of p4160-FEC-mCore1 with His tag plasmid to top10 E. coli.

In our project, we tried to find a variant of human ACE2 protein that has higher affinity to the spike protein compared to the W/T. In order to achieve this goal, we created a library of 93,311 options of mutant ACE2 proteins based on four different studies ^{[29] [30] [31] [32]}. Efforts have been invested to identify amino acids (AA) in the ACE2 sequence that are related to its binding to the SARS-CoV-2 S protein.

16 AA were identified under that criteria:
N330, K353, G354, D355, F356, R357, A396, A25, T27, K31, Y41, Q42, L79, M82, Y83, P84.

Figure 3.1: The identified AA (red) one ACE2.

To create the library, we replaced those AA with other AA that maintain their bio-chemical characteristics (Aliphatic, Aromatic, Acidic, Basic, Hydroxylic, Sulfur containing, Amidic). Those changes gave us total of 97,202 variants.

As part of the efforts to find the most suitable variant, we planned to narrow down the size of the library using the ROSETTA bioinformatics modeling software ^[33]. Due to the lack of time, we started the lab work with 3 initial variants, in parallel to the modeling work.

The three variants we worked with are:

1. X1 isoform, which is the most common ACE2 isoform in human lungs ^[14], as our positive control.
2. Variant V1 as our library’s representative, which is taken from literature ^[13]. This variant was shown to have high affinity to the spike protein.

   	Mutations deriving from the X1 isoform
   V1	H47A, T105Q, Q338P, A399L

3. As the negative control, an ACE2 variant which was shown to be non-binding to the spike protein, according to literature.

   	Mutations deriving from the X1 isoform
   V-neg	Q89N, L92E, T105S

After creating the final variant library, we aim to screen the library by a protein interactions model and then transform the sequences into yeast by heat shock. Finally, we plan to analyze the resulting proteins and optimize our design to the best. Results are obtained via SDS-page and FACS analysis.

• Purifying ACE2 protein with Nickel column.
• Purifying YFP protein with Nickel column.
• Analyzing the ACE2 size and perform affinity test for the mutant ACE2 protein.

Our lab work started with cloning a plasmid backbone that will contain the ACE2 variants. We extracted an existing plasmid (p416-FEC-nCore1, ~6000 bp) out of Saccharomyces cerevisiae W303 strain that we received from Prof. Roee Amit's lab. This backbone contains a yeCitrine fluorescent protein gene that will be replaced with the ACE2 variants later.

To allow extraction of the ACE2 protein from yeast, a His-tag sequence was added by performing a PCR reaction. A restriction map was created to verify the plasmid sequence, after several failures in PCR reactions.

Figure 3.2: 1 Kb Ladder. Black arrows represent bands in the desire size. PCR expected results - ~6 Kb for reaction and no bands on the control. 1 Cut restriction expected results - 6 Kb. 2 Cut restriction expected results - 5 Kb + 1 Kb. Control for restriction – Circular plasmid.

Once we verified the restriction map of the plasmid, we realized the issue might be the primers. Therefore, we used a different polymerase that is designed to work on difficult sequences - PrimeSTAR GXL polymerase (GXL).

Using the GXL polymerase we prepared a circular p4160-FEC-mCore1 plasmid with His-tag via PCR reaction, PNK and ligation. The circular plasmid was transformed to E. coli top10 to produce a yeCitrine protein fused to a His-tag.

Figure 3.3: Transformation of the circular plasmid to E. coli top10 as shuttle vector.

We attempted to verify our cloning using Colony PCR. Unfortunately, the results were not as expected – 700 bp instead of 3400 bp. Thus, we decided to send the colonies for sequencing. The sequencing showed that during ligation of the blunt end PCR product, a few base-pairs were dropped from the plasmid which lead to the unexpected size. We realized that a different strategy was required.

However, due to the lack of time, we decided to put this part of the project on hold and continue to other parts.

Alongside the yeCitrine purification, we started working on "Any Gene Any Plasmid" (AGAP) cloning method. This method enables us to produce the desired plasmid by transforming linear fragments that contain 20-40 bp overlaps into yeast, which will be closed into a circular plasmid by in vivo homologous recombination ^[11][12].

Figure 3.4: Illustration of AGAP homologous recombination method.

We produced the linear fragments for the AGAP method by performing several reactions. The 1st fragment "R-16 cut" was amplified from p416-FEC-nCore1 plasmid using PCR reaction followed by restriction (yeCitrine protein was cut out). The 2nd fragment "F-9" was also amplified from p416-FEC-nCore1 plasmid. And the 3rd fragment was ordered as gBlock.

Results of the PCR reactions are exhibited in pictures below.

Figure 3.5: Log Ladder. Black arrows represent bands in the desired sizes. Expected results: F9 – 4300 bp; R16 – 1600 bp; Negative Controls – no bands.

Figure 3.6: Log Ladder. Black arrows represent bands in the desired sizes. Expected results: X1, V1, Vneg G-Blocks – 2600 bp; Negative Controls – no bands.

The AGAP method requires a large amount of product (~1 µg) from each fragment. Therefore, many PCR reactions were needed to allow the R-16 cut fragments to reach this amount.

After getting sufficient amounts, we transformed the fragments into W303 type YA619 yeast strain. While yeast grew on the plate, mold grew on it as well, meaning we could not use the yeast colonies. At this point, we could not repeat the experiment because of the lockdown in our country.

Our future plans are to perform another transformation of the fragments into yeast, which will express the desired variant. Then, we aim to extract the proteins and purify them with Nickel column. Afterwards, we will verify the size of the protein extracted with SDS-page analysis and measure the affinity to the spike protein by Fluorescence-activated cell sorting (FACS) using a fluorescently labeled spike protein and an antibody targeting the human ACE2.

Sybodies

Overview Key Achievements Introduction Aims Results

Nanobodies are the variable domain of camelid heavy chain antibodies ^[34]. Despite their small size, nanobodies exhibit full capacity to bind proteins selectively and were chosen due to their good properties and ability to simultaneously associate with the RBD ^[35].

In this part, the results that we got through creating the Sybodies are presented. The Sybodies are destined to be attached to the Microgel Beads as binders for the SARS-CoV-2’s spike proteins. The creation of the Sybodies was divided into three parts: cloning the desired plasmid containing one of the Sybody’s sequences, over express it in designated over expression bacteria, and extraction of the proteins from the cells. During the time we had in the lab, we successfully created over-expression plasmid containing the Sybodies’ sequence with additional His-tag. The sequence of the ligated plasmid was verified via colony PCR and Sequencing. Additionally, the first proof of concept for Sybody’s expression and extraction has achieved via SDS-PAGE and Western Blot.

• Cloning a ligated plasmid into TOP10 E.coli and verifying its sequence.
• Expression and Extraction of the Sybodies as proteins from KRX E.coli cells.

Most antibodies are composed of two heavy chains and two light chains while both chains contribute to the two identical antigen-binding sites. In addition to these conventional antibodies, camelids and sharks also produce antibodies composed only the heavy chains ^[18]. These peculiar heavy chain antibodies contain only a single variable domain and two constant domains. The single variable domain is called nanobody or single domain antibody ^[36]. Although their sizes were small, nanobodies are capable to bind proteins selectively and with high affinity ^[37] with full capacity. The differences between the three types of antibodies can be seen in the figure below.

Figure 4.1: Antibodies, Nanobodies and Sybodies

Antibodies are widely used for the detection and manipulation of proteins in diverse research fields. However, the number of validated antibodies is limited and there is an unmet need for binding proteins tailored to fulfill desired technical and biological requirements ^[38]. Creation of synthetic nanobody can solve this problem. Sybodies, which stand for synthetic nanobodies, are easy to produce, stable, soluble in aqueous solutions and can perform reversible refolding without the need for additional improvements. All those properties are ideal for a practical binder, especially for bacterial expression ^[19]. Various researches that used synthetic nanobody for detection can be found ^{[12] [40] [41]} as well as a variety of protocols for creating them ^{[22] [42] [43] [44]}. One significant example of synthetic nanobody’s novel application is the cancer treatments. Chimeric antigen receptor-modified T cells (CAR T-cells) are highly researched and used in various clinical trials ^{[45] [46] [47] [48]}.

Sybodies were chosen as “decoy proteins” for the SARS-CoV-2 due to the advantages listed above. During the search on Sybodies, we came across the work of Dr. Justin D. Walter et al, in which 3 Sybody libraries targeting the receptor-binding domain (RBD) of SARS-CoV-2 were created and tested. The sequences for Sybody number 15 (Syb15) and Sybody number 68 (Syb68) were chosen for our project. Those sequences are two of the top six Sybodies’ sequences with the best properties tested in this article, such as kinetic parameters for interactions between Sybodies and spike proteins of SARS-CoV-2. Moreover, those two specific Sybodies were chosen because of their ability to simultaneously associate with the RBD ^[25]. Using both proteins together in our product can, potentially, increase the overall avidity to the spike proteins and by that increase the efficiency of the product. We have consulted with Dr. Walter regarding this choice and he supported it.

In our project, the Sybodies are used to target the RBD of the SARS-CoV-2 spike proteins. Those Sybodies contain additional His tag that allows to bind them to the Microgel Beads and will be suspended in the Hydrogel later. Together, all three parts complete the ACT. product.

One of the advantages of Sybodies is that they can be produced easily in bacteria once their sequence is determined. In this project, the production of the Sybodies is composed of few steps. First, the Sybodies’ sequences and the pET9D backbone were restricted and ligated together. The ligated plasmid was transformed into TOP10 E.coli for sequence verification and amplification. Then, the amplified plasmid was transformed into KRX competent cells to overexpress the proteins. The cell content was extracted and the proteins themselves were purified. After that, a verification for the extracted proteins’ size and structure was done. The final step planned for this part was verification of the Sybodies’ functionality. This experiment was not preformed due to a lack of time.

The main goal of this part of the project is to produce functional Sybody proteins, that will be connected to Microgel Beads and serve as binders for the SARS-CoV-2 spike proteins. The production of the Sybodies was divided into two stages (cloning and expression), after which there would be another step of functionality test for the proteins. All the process is described in the schema below, in Figure 4.2.

Figure 4.2: Sybodies creation process

Cloning a ligated plasmid:

The first step in the process of creating the desired proteins is to create a whole plasmid containing both the plasmid backbone and one of the Sybodies’ sequences.

This step began with amplifying the pET-9D backbone and inserting into it a Sybody sequence with additional His-tag. The insertion was done by restriction and ligation. This ligated plasmid was transformed into TOP10 E.coli in order to amplify it and its sequence was verified by colony PCR and Sanger sequencing. In Figure 4.3 & 4.4, the gels of the colony PCR are shown, for both Sybodies. In the end of this step we had two verified plasmids containing each Sybody’s sequence.

Figure 4.3: Colony PCR gel for Sybody 15 (2-Log ladder. Black arrows represent bands in the desired sizes. Expected results: 1-19: pET9d_syb15 – 555bp).

Figure 4.4: Colony PCR gel for Sybody 68 (2-Log ladder. Black arrows represent bands in the desired sizes. Expected results: 1-18: pET9d_syb168 – 773bp)

Expressing the Sybodies in KRX cells and purification:

The second step in the process is creating the Sybodies as proteins.

This part began with overexpression of the ligated plasmid in KRX competent cells, to produce a large amount of Sybody proteins. The transformation into KRX competent cells was done by heat shock. Then, the bacteria were grown until 0.8-1.0 OD was reached. Afterward, the overexpression was induced by adding the inducer rhamnose into the cells’ growth medium. Extraction of the cell content was done by Homogenizer and the purification of the Sybody proteins was done using a Nickel column. In this stage, purification Nickel-bound beads from two different companies were used (Cube Biotech and Promega) to compare their efficiency and as duplicates. Eventually, to verify the proteins’ presence in the purified solution, SDS-PAGE and Western blot were performed. Pictures of the gels from both the SDS-PAGE and Western blot are shown below, in Figure 4.5 & 4.8.

Figure 4.5: SDS-PAGE for the extract of KRX cells producing Sybody 68 (Excel band PX2600 marker. Black arrows represent bands in the desired sizes. Expected results: Sybody 68 – 14.26 kDa). E- Elution, F- Flow, W- Wash

Figure 4.6: SDS-PAGE for the extract of KRX cells producing Sybody 15 (Excel band PM2600 marker. Black arrows represent bands in the desired sizes. Expected results: Sybody15 – 13.12 kDa)

Figure 4.7: SDS-PAGE for the extract of control KRX cells (Excel band PM2600 marker).

Figure 4.8: Western Blot with anti his-tag antibody (PageRuler ladder. Black arrows represent bands in the desired sizes. Expected results: Sybody 15 – 13.12 kDa; Sybody 68 – 14.26 kDa).

Future plans:

The last step in this part of the project is to prove the Sybodies’ functionality: their ability to capture the SARS-CoV-2 virus. This will be done by inducing and measuring a reaction between the Sybodies and the spike proteins, as shown in Figure 4.11. This was planned to be achieved with SARS-CoV-2 Spike Protein-Coated 96 wells Plates via ELISA procedure. In this procedure the produced proteins are reacted with the SARS-CoV-2 spike protein bound to the commercial plate. The amount of protein reacted with the plate measured by absorbance of light. Unfortunately, because of the COVID19 situation and the lockdowns in Israel, we did not manage to do this test.

After completing the Sybodies’ testing, verification of the interaction between the Sybodies and the SARS-CoV-2’s spike protein on the surface of the Microgel Beads will be done by the Microgel group.

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