PROS by the Stony Brook University 2022 iGEM Team

Proof of Concept

The overall goal of our project was to express recombinant protein S suitable for administration directly into the human bloodstream. E.coli is one of the organisms of choice for the production of such recombinant proteins. It has been well-established for use as a cell-factory and is, by far, the most popular expression platform. We also aimed to use the Bac-to-Bac baculovirus expression system, which enables the efficient production of recombinant baculovirus for expression testing in insect (SF9) cells. Our lab experiments demonstrated success in designing the recombinant vector and expression of Protein S from E.coli Origami B (DE3) cell strain. Our modeling of constitutive and regulatory gene circuits of E.coli and SF9 provided us an insight into protein and mRNA production kinetics and influenced our wet lab decision-making. Our implementation highlights a potential therapeutic approach in treating protein S deficiency using recombinant human protein S.

Wet Lab

Construction of Recombinant Vector

After a lot of troubleshooting and determining optimal insert and vector concentration ratios for the ligation independent cloning reaction, we successfully cloned Protein S gene into the pET His6 LIC cloning vector (2Bc-t).

Figure 1. PCR amplification of the recombinant vector for insert verification

T7 forward and reverse primers were used to amplify out the MCS, which indicated that our 2 Kb gene of interest was successfully cloned into the vector.

Sequencing Verification of the Recombinant Vector

The following image displays the sequencing result that was performed using the T7 forward primer. The NCBI BLAST tool was used for pairwise alignment of the sequenced DNA vs Protein S insert sequence.

Figure 2. NCBI BLAST pairwise alignment results

The following image displays the sequencing result of the recombinant vector using the T7 reverse primer.

Figure 3. NCBI BLAST pairwise alignment results

Sequencing of our recombinant vector using the T7 forward and reverse primers confirm successful cloning of our insert into the Multiple Cloning Site of the vector using ligation independent cloning.

Expression Test

We induced Origami B cells using 0.5 mM IPTG for expression, and collected pre and post induction samples for conducting a Western blot.

Figure 4. Western Blot result of Origami B expression samples.

As evident from the western blot results, the HRP anti-His 6 antibody successfully detected protein S target band (full length, about 70 kD). The level of expression can be seen increasing with time in sample Ori 3. As expected, the ability of origami B cells to make disulfide bonds and ensure proper folding, greatly enhances protein stability and prevents degradation as opposed to Bl21 strain.

Sample Ori 2 was induced at OD=0.4 and sample Ori 3 was induced at OD=1.1 We collected one pre-induction sample from both Ori 2 and 3 before inducing with 0.5 mM IPTG and 4 post-induction samples at an increment of 1 hour. We expressed sample Ori 2 for about 16 hours and Ori 3 for about 12 hours to collect overnight induction samples.

Well mark-up is as follows:

1. Pre-induction Sample (Ori 2)
2. One hour post-induction (Ori 2)
3. Two hour post-induction (Ori 2)
4. Three hour post-induction (Ori 2)
5. Four hour post-induction (Ori 2)
6. Overnight induction (Ori 2)
7. Empty well (loading dye)
8. Pre-induction Sample (Ori 3)
9. One hour post-induction (Ori 3)
10. Two hour post-induction (Ori 3)
11. Three hour post-induction (Ori 3)
12. Four hour post-induction (Ori 3)
13. Overnight induction (Ori 3)

Based on the experimental results shown above, we were able to effectively express protein S from E.coli Origami B (DE3). Generally, bacterial expression systems are not optimal for secretion of proteins with eukaryotic post-translational modifications and foldings. However, Origami B is an expression strain derived from a LacZY mutant of Bl21 and contains mutations in thioredoxin reductase (trxB) and glutathione reductase (gor) genes. These mutations greatly enhance their ability to make disulfide bonds which prevents protein degradation and increases protein stability. Our parallel induction studies in Bl21 and Origami, demonstrated that Bl21 strains were unable to express protein S because of a failure to introduce the necessary post-translational modifications and protein folding.

After manipulating expression conditions like the concentration of IPTG used for induction and temperature to determine optimal conditions for expressing protein S, we were able to express protein S from Origami B cells.

Unfortunately, our SF9 experiments were difficult to finish on our limited timeline. However, we were able to computationally model the amount of possible protein production using the baculovirus system, and were able to simulate conditions to enable scaling up production to an industrial scale. The results of our modeling suggests that experiments with SF9 could be continued and would significantly aid in expressing homologous protein S to treat patients with either a genetic or acquired protein S deficiency.

Model Results

Our project aims at expressing recombinant protein S in SF9 and E. coli cells. We aimed to model constitutive and regulatory genetic circuits for both these cell lines by taking into consideration the kinetics of gene expression, interactions between the Polyhedrin promoter binding protein and the Polyhedrin promoter, and the inducible lac operon regulating the T7 promoter. For modeling our gene circuits for E. coli, we derived a system of ODEs for comparing protein production in constitutive vs regulatory circuits. We conducted a literature search to find the rate constants for our ODEs and modeled the circuits in MATLAB. The Baculovirus Expression System for producing recombinant proteins in SF9 cells is widely used and has multiple benefits over traditional prokaryotic systems for producing human proteins. We derived a system of ODEs to model SF9 constitutive gene circuits and the regulatory circuit which models the interactions of the PPBP and the polyhedrin promoter. This interaction and its effect on protein production through baculovirus expression systems is not well studied. We hoped to provide some insight into SF9 gene expression kinetics and analyze protein S production in eukaryotic vs prokaryotic expression systems. More details on our mathematical modeling can be found on the Model page.

Implementation

We envision that our project, PROS, will be a long-term treatment therapy for patients with protein S deficiency, with the potential to act as a short-term therapeutic for patients suffering from related disorders including COVID-19 and traumatic brain injury (TBI). To achieve this, we aimed to produce recombinant human protein S with all of its post-translational modifications and excluding introns, using genetically engineered E.coli and SF9 insect cells. This protein will be identical to human protein S, and can be directly administered to patients intravenously. Our Proposed Implementation page provides details on the prescription plan and method of delivery of PROS therapy.

Diagnostics

Protein S deficiency can be either congenital or acquired, and its diagnosis has several components including patient history, identification of symptoms and clinical testing of the levels and activity of protein S. Current diagnostics techniques include ELISA, Latex particle-based Agglutination assays and functional assays like APTT and PT-based functional protein S assays. In a partnership with iGEM Ashei-Ghana, we designed a biosensor to directly enable detection and quantification of protein S in blood samples. The design involves engineering aptazymes that can specifically target and bind to protein S in blood samples. More details about the design of the model, specified by Ashei-Ghana, can be found on our Diagnostics page. We also worked to help streamline the diagnostic procedure for protein S deficiency. This involved establishing a comprehensive and cost-effective algorithm to help in protein S assessment. This algorithm cuts the cost of testing by more than 40%, and can be distributed to healthcare providers to implement.

Conclusion

Overall, the goal we set for our project has been achieved. We were able to express recombinant protein S. There is definitely scope for improvement such as including a quantitative analysis of the production of our protein, optimization of the protein structure and function, testing of our injectable design, and scaling up production to an industrial level. We also would like to further explore using the Bac to Bac expression system, which shows great promise.

However, despite these drawbacks, by using an innovative and thorough approach of dynamic modeling, in coordination with an extremely comprehensive entrepreneurship analysis, and taking into account the opinions and ideas of communities who suffer from protein S deficiency, we were able to demonstrate the feasibility of industrialization of our project, and the potential widespread implications. We were successful in creating a platform that can be further built upon to manufacture and deliver our PROS injectable as a therapeutic solution for protein S deficiency and related disorders.

Overall, producing natural proteins for human use and finding alternative treatments to current therapies are only some of the possible applications of synthetic biology. We hope our project can serve as an example of the benefits of synbio and help further the imaginations of synthetic biologists, and the implementation of the field for widespread global benefit. The end of our competition cycle marks the beginning of more widespread efforts. So far, we have demonstrated that our therapy is feasible and achievable, even as a small team of 11 undergraduate students.