Team:Technion-Israel/Engineering
Research begins with an idea; the design cycle then begins.
In the diagram, the “learning” step and the “improvement” step are emphasized since we believe they are what sets apart a good engineering process from a great engineering process. In research as in life, we fall so we can learn to pick ourselves up. With each failure, we learned from our mistakes and with each success we thought about ways to improve our results for the next time.
B. subtilis Spore Surface Display
Research
We first conducted a literature review searching for suitable "decoy proteins" against SARS-CoV-2. This review led us to the cell entry mechanism of SARS-CoV-2 (the hACE2 receptor), and to information about the Sybodies (Sb#15 and Sb#68) [1] [2] [3]. For surface display, B. subtilis spore display (BSSD) was found to be the most suitable technique for displaying functional heterologous proteins anchored to bacteria [4][5] .
Design
6 different plasmids were designed to be integrated to B. subtilis (B. subtilis) PY79. Featuring CotC or CotG as anchor proteins, linker and one of the decoy proteins: ACE2, Sb#15 and Sb#68 [2] [4] [5].
Engineering the vector
The cloning was planned as follows:
- The “decoy proteins” will be cloned to a linearized pBS1C_lacZ vector using Gibson assembly and transformed to E. coli TOP10 cc.
- Sequences which encode the Anchor (Cot) proteins will be inserted to the vector using restriction enzymes.
- The final vector will be inserted to B. subtilis PY79’s genome
All steps are verified by colony PCR and sequencing with optimized conditions for each plasmid
Decoy proteins
Test
The cloning of Sb#15/68 and the pBS1C_LacZ vector was verified using colony PCR. However, colony PCR did not yield positive results for the ACE2 insertion.
Re-test
We planned another verification test using restriction enzymes in order to examine the cloning by detecting the unique restriction pattern of the plasmid (restriction map). However, the pattern was not detected.
Re-plan
Due to the limitation of parameter-changing in the Gibson assembly method, the Colony PCR’s conditions were the ones to be adapted: temperatures, duration time and number of cycles were changed several times. Yet, the cloning of ACE2 and pBS1C_LacZ was not verified.
Re-test
Transformation of pBS1C_syb15/68 and pBS1C_ACE2 was checked by Sanger sequencing. The results confirmed that the vector was transformed into the bacteria but without the ACE2 fragment. However, positive cloning of the Sybodies and the vector was verified.
Research
Another cloning attempt using restriction enzymes was planned.
Re-Plan
Since the combination of the Sybodies has showed substantial inhibition of SARS-CoV-2’s interaction with ACE2 [1], we decided to continue with pBS1C_syb15 and pBS1C_syb68 to the second cloning step - adding the anchor proteins to the final plasmid.
Insertion into B. subtilis genome
Plan
Genomic insertion of pBS1C_syb15 and pBS1C_syb68 into B. subtiis using corresponding insertion protocols and colony PCR.
Test
After insertion, colony PCR was conducted to verify the insertion of pBS1C_syb68 to B. subtilis’s genome. The insertion of pBS1C_syb15 was not verified.
Research
Other verification methods, such as amylase test and colony PCR are being investigated at the moment for future research and development.
Anchor protein cloning
Plan
Insertion of the anchor proteins, CotC and CotG into the pBS1C_ACE2/syb15/syb68 vector using restriction enzymes. The anchor proteins and vectors will be digested, separated using electrophoresis, and ligated.
Test
No anchor proteins were detected in electrophoresis gel.
Re-Plan
A larger amount of anchor proteins was digested.
Re-Test
Anchor proteins were detected in electrophoresis gel.
Verification
Colony PCR results did not confirm the ligation.
Re-Plan
Ligation temperature and duration were adjusted. Since the Cot protein sequences are short (~250-700bp), larger amounts of DNA fragments were ligated.
Verification
Verification attempt using restriction enzymes was done. Results of restriction map were inconclusive.
Re-Test
Suspected colonies from restriction-map attempt were sent for Sanger sequencing. But the sequencing results implied that the anchor proteins and vector were not cloned.
Re-Plan
Different approaches, such as using Gibson assembly, have been investigated.
Sporulation
Research
Sporulation was conducted for B. subtilis PY79’s bacteria, With the goal of testing the germination ratio of B. subtilis PY79 in the chosen hydrogel of the final product.
Plan
Three germination tests were performed:
- Analyzing germination protocol
- Assessment of Dipicolinic acid (DPA)
- Spore Staining
Test
Germination and DPA tests verified the stability of spores in the hydrogel compared to water. However, staining test indicated that there was a large amount of living bacteria presented in the gel at time zero.
Re-Plan
Sporulation protocol requires modification in order to achieve a product consisting of at least 90% spores. In the future, we hope to use the non-germinating B. subtilis mutant strain that was synthesized by the 2012 Munich iGEM group [6] to avoid possible germinations.
Sybodies – planning, cloning, and purifying
As mentioned in the Sybodies Result page and the Design page, Sybodies were chosen as decoy proteins against SARS-CoV-2 due to their advantages as binders. As far as we know, we are the first and only iGEM team to use these synthetic nanobodies in the iGEM competition.
We came across the Sybodies while searching for a way to destroy the virus once it attaches to the gel in our product. To learn more about the ACE2-Spike protein binding mechanism, we consulted with Assistant Prof. Yotam Bar-On from the Technion and Dr. Ron Diskin from the Weizmann institute. Both experts suggested us to look for novel antibodies and Dr. Diskin advised us to expand our research to the “bioRxiv” preprints.
We were able to find two possible proteins for our project: Sybodies [3] and Human mAb 47D11 [7]. Both options are antibodies while the first is a nanobody (more information in the Sybodies Result page) and the latter is a human antibody. The advantage of the Human mAb 47D11 antibody is that it would not compete with the binding of the spike protein to the ACE2 receptor expressed at the human cell surface [7], so it can be used together with our ACE2 baits. The disadvantage is the lack of information about it. Nanobodies, on the other hand, are highly researched and are reported to be functionally expressed in bacteria [8] [9] [10]. Because of this, we chose to continue with the Sybodies.
The most relevant work we encountered is the preprint prepared by Walter et al [3]. After careful consideration and consultation with the writers, we chose two Sybody sequences. Detailed information can be found in the Sybodies Result page. At this point, we started to design our genetic constructs and entered the lab with a well written plan and complete protocols. The general plan is described in Fig.1.
After performing restriction and ligation cloning on the Sybody sequences and the PET-9d backbone, bacterial growth was observed in the control plates. The immediate culprits that came to mind were the restriction enzymes. We repeated the cloning process with enzymes from another laboratory and different suppliers, but it did not solve the problem. The next possible issue that came to mind was a possible issue with the plasmid. To check it, we ran a restriction verification test of the plasmid and saw that it exhibited the expected patterns on the gel. We decided to continue with our colonies to colony PCR, to see whether the Sybody sequences have been ligated into the plasmid backbone successfully despite the growth in the control plates. As shown in the Sybodies Result page, the ligation of the sequences was indeed successful. Our conclusion was that the backbone had a high tendency to perform self-ligation even though different restriction enzymes were used. In addition to colony PCR, the ligated plasmid sequences were also confirmed by Sanger sequencing. The next step in the Sybody creation was transformation into KRX E. coli, a strain of E. coli suitable for over-expression of proteins [11]. After protein extraction and purification, the Sybodies’ presence and size were examined by running an SDS-PAGE and a Western-Blot, both tests resulted in positive verifications. The complete results can be found in the Sybodies Result page and both of Sybody 15 and Sybody 68 were registered as new Parts in the iGEM part registry.
- Brielle ES, Schneidman-Duhovny D, Linial M. The SARS-CoV-2 Exerts a Distinctive Strategy for Interacting with the ACE2 Human Receptor. Viruses. 2020;12(5):497. doi:10.3390/v12050497
- 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
- Zimmermann I, Egloff P, Hutter CAJ, et al. Generation of synthetic nanobodies against delicate proteins. Nat Protoc. 2020;15(5):1707-1741. doi:10.1038/s41596-020-0304-x
- 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
- 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
- iGEM team Munich 2012, How do sporulation & Germination Work, 2012
- Wang C, Li W, Drabek D, et al. A human monoclonal antibody blocking SARS-CoV-2 infection. Nat Commun. 2020;11(1):2251. doi:10.1038/s41467-020-16256-y
- De Genst E, Silence K, Decanniere K, et al. Molecular basis for the preferential cleft recognition by dromedary heavy-chain antibodies. Proc Natl Acad Sci. 2006;103(12):4586-4591. doi:10.1073/pnas.0505379103
- Holliger P, Hudson PJ. Engineered antibody fragments and the rise of single domains. Nat Biotechnol. 2005;23(9):1126-1136. doi:10.1038/nbt1142
- Muyldermans S. Nanobodies: Natural Single-Domain Antibodies. Annu Rev Biochem. 2013;82(1):775-797. doi:10.1146/annurev-biochem-063011-092449
- Promega Corporation: Hartnett J, Gracyalny J, Slater MR. The Single Step (KRX) Competent Cells: Efficient Cloning and High Protein Yields. Promega Notes. 2006;94(August):27-30
