USAFA - iGEM 2024

Overview

The USAFA iGEM team contributions can be split into three categories for use by future iGEM teams (Figure 1).

_{Figure 1: USAFA Contributions to other iGEM teams}

1: The Contribution of A New Part and Data on an Existing Part

In 2022, the USAFA iGEM team used silicatein genes from the marine sponge Latrunculia operinae and the freshwater sponge Ephydatia fluvialis with the goal of conferring radiation protection to bacterial cells performing urease-based calcium carbonate biocementation. Upon further collaboration with the University of Virginia, we found that a truncated and codon-optimized silicatein gene from Suberites domuncula could catalyze biocemenation by itself.

For 2024, we focused on using the new basic Part:BBa_K5199003 (silicatein alpha) and combined it with the Ice Nucleating Protein (INP) for external expression. The resulting composite part is Part: BBa_K5199004. We also contributed to the iGEM registry by adding data and insight into >E. coli viability for Part BBa K1890001. The previous team was not able to get E. coli INP SIL to grow and produce viable colonies, but we were able to get E. coli INP SIl to grow with our updated part sequence and by waiting to add the ortho silicate until after log phase growth. (Link to BBa_K5199003) (Link to BBa_K5199004) (Link to BBa_K1890001)

To make this part, a pET-28a (+) TF-silicatein-α plasmid gifted from Bryan Berger's lab was cut with BamHI and HindIII to remove the TF part. INP (Ice Nucleation Protein) was cloned out of pET-28a INP OspA (USAFA 2023 iGEM Team) with INP specific primers. BamHI and HindIII sites were added to INP via PCR.T4 ligase was used to ligate the INP fragment into the BamHI and HindIII sites of the pET-28a-silicatein-α plasmid. The final plasmid was transformed into E. coli BL21 DE3 cells. Protein expression was induced with a T7 RNA polymerase and isopropyl ß-D-1-thiogalactopyranoside (IPTG) induction system. A key advantage of expressing silicatein-α externally in E. coli was its facilitation of silica dioxide synthesis, which helps reduce ammonia byproducts, making the biocementation process more biosafe.

Our 2024 research aimed to optimize biocementation using the composite of silicatein-α and INP genes in E. coli. Preliminary results indicated that while the INP-silicatein-α bricks were less structurally sound compared to S.pasteurii bricks, the biocementation process showed promise for dust mitigation and plant growth applications. Overall, our work contributed to a biosafe biocementation process through the composite expression of INP and silicatein-α in E. coli.

2: Brick Molds- Enhancing Biocementation Efficiency

Since 2022, the USAFA iGEM team has been refining our brick molds for biocementation. Initially, it took up to 30 minutes to pipette bacteria and cement solutions into the molds (Figure 2). This year, we developed a new 3D-printed mold design that significantly reduced pipetting time to about one minute, thanks to its taller design, which adds a liquid reservoir to allow bacterial cultures and solutions to slowly drip through the sand (Figure 2). This improvement has enhanced the efficiency of our E. coli-based biocementation, allowing us to compare its strength to traditional methods using S. pasteurii. We are sharing these optimized molds to help other teams achieve similar efficiencies. Please reach out if you would like the 3D mold design code.

_{Figure 2: Comparison of old (right) and new (left) brick mold designs}

3: Systems Engineering- Optimizing Team Diversity and Efficiency

Systems Engineering involves designing, integrating, and managing complex systems to ensure that all components work together efficiently to achieve their goals. The USAFA iGEM Team applied systems engineering principles to form a highly effective group to successfully prepare and compete at the iGEM Jamboree.

Efficiency in the Lab: The iGEM team was divided into three specialized groups: the Biomineralization/Ammonium team, the Dust Mitigation/Plants team, and the Biocement Brick team. Each group focused on different applications of the genetically engineered E. coli. Their primary goal was to develop cementation capabilities while minimizing harmful byproducts like carbon dioxide and ammonium. The focused structure allowed each group to conduct laboratory work efficiently, contributing to the overall goal of biosilicification.

Efficiency with Deliverables: Each iGEM deliverable was assigned a team lead, along with supporting members. By assigning specific tasks to designated leads, we ensured that all requirements were met well before their deadlines, streamlining the workflow and maximizing productivity.

Communication: To maintain team cohesion and ensure progress, we held bi-weekly meetings for three months. These meetings allowed the team to address challenges, review progress, and maintain a high level of communication among all 21 members. This consistent interaction helped align our efforts, contributing to our success in reducing the environmental impact of biocementation.

Group Retreat: We concluded the project with an on-campus all day iGEM retreat, during which we finalized deliverables, reviewed lab work, and updated our project wiki. The retreat also featured a team hike, which emphasized the environmental impact of soil erosion and reinforced the importance of our biosafe silicatein-α application for soil stabilization.

As a contribution to other iGEM teams, we have provided a checklist with steps that enable other organizations to incorporate systmes engineering into their teams to build efficient, cohesive groups that achieve concrete objectives.

Click here for the Systems Engineering Checklist