Overview
Our team made significant contributions this year by developing a number of tools and resources that aim to give back to the iGEM community and beyond. We optimized several protocols and biological parts to characterize our enzymes and implemented them in a portable blood conversion kit. In addition to this, we created several educational tools for a range of demographics. With these developments, we hope to help advance research, experimentation, and education in synthetic biology.
Assays
In addition to codon optimizing sequences for each enzyme, our wet lab developed detailed protocols for both colorimetric and hemagglutination assays that measure the efficiency of our enzymes. These assays, in conjunction, provide a combination of qualitative and quantitative readouts that measure how efficiently our enzymes cleave the bonds of the A and B antigens and their extended groups. We hope these protocols can be used and modified by other teams that wish to use our enzymes.
Hardware
We designed several tools to contribute to the construction of a portable ABO blood conversion kit. These tools include field-ready blood type-specific lyophilized enzyme vessels that ensure extended enzyme stability, biological plasma and leukocyte reduction membrane filters to separate blood components in the field, and a miniaturized portable electronic lateral flow antigen biosensor to assess the presence of A and B antigens and their extended groups after treatment.
Parts Collection
We developed and documented in the iGEM Registry of Standard Biological Parts a new collection of parts to achieve ABO blood conversion.
Basic Parts Collection
| Name | Part ID | Functional Description |
|---|---|---|
| α-1,3-N-Acetyl galactosaminidase (AmGH36A) | BBa_25A4FF27 | α-1,3-N-Acetyl galactosaminidase cleaves the N-Acetylglucosamine – Galactose bonds found on extended A antigens and base A antigens present on red blood cells. |
| α-1,2-fucosidase (AmGH95B) | BBa_25K5RQXC | α-1,2-fucosidase cleaves the Fucose – Galactose bonds found on H Type 3 antigens present on red blood cells. |
| β-1,3-galactosidase (AmGH35A) | BBa_25GFNYCT | β-1,3-galactosidase cleaves the Galactose – N-Acetylglucosamine bonds found on Gal-A antigens present on red blood cells. |
| β-1,3-N-Acetyl galactosaminidase (AmGH20A) | BBa_25UMC4CD | β-1,3-N-Acetyl galactosaminidase cleaves the N-Acetylglucosamine – Galactose bonds found on ExtB antigens present on red blood cells. |
| α-1,3-galactosidase (AmGH110A) | BBa_25UAXSUG | α-1,3-galactosidase cleaves the Galactose – Galactose bonds found on base B antigens on human red blood cells and α-galactose in pig red blood cells. |
| Aleuria aurantia lectin (AAL) + Bacillus subtilis Nitric Oxide Synthase (bsNOS) | BBa_25JVFV3M | Aleuria aurantia lectin (AAL) binds to human H antigen present on red blood cells. Once bound, Bacillus subtilis Nitric Oxide Synthase (bsNOS) will produce nitric oxide in the presence of arginine and an applied voltage which is used for measuring amount of binding. |
| α-galactosidase | BBa_25GOPTT8 | A cold adapted version of α-galactosidase. Cleaves the Galactose – Galactose bonds found on base B antigens on human red blood cells and α-galactose in pig red blood cells. |
Composite Parts Collection:
| Name | Part ID | Functional Description |
|---|---|---|
| AmGH36A Enzyme | BBa_25GVJ4BC | Insert encoding α-1,3-N-Acetyl galactosaminidase for recombinant expression in E. coli, featuring an mScarlet reporter for visual confirmation and a His-tag for IMAC-based protein purification. |
| AmGH95B Enzyme | BBa_25K8RFNE | Insert encoding α-1,2-fucosidase for recombinant expression in E. coli, with an mScarlet reporter for colorimetric visualization and a His-tag for IMAC protein purification. |
| AmGH35A Enzyme | BBa_25E5Q4LN | Insert encoding β-1,3-galactosidase for recombinant expression in E. coli, incorporating an mScarlet reporter for visual detection and a His-tag for IMAC protein purification. |
| AmGH20A Enzyme | BBa_254NQG3O | Insert encoding β-1,3-N-Acetyl galactosaminidase for recombinant expression in E. coli, with an mScarlet reporter for colorimetric indication and a His-tag for IMAC protein purification. |
| AmGH110A Enzyme | BBa_25HDD6TY | Insert encoding α-1,3-galactosidase for recombinant expression in E. coli, including an mScarlet reporter for visualization and a His-tag for IMAC-based purification. |
| Aleuria aurantia lectin (AAL) + bsNOS | BBa_25Z0V7YA | Insert encoding Aleuria aurantia lectin (AAL) co-expressed with Bacillus subtilis Nitric Oxide Synthase (bsNOS) for quantitative detection, with an mScarlet reporter and a His-tag for IMAC protein purification. |
| Cold Adapted α-galactosidase Enzyme | BBa_25BVHYG0 | Insert encoding α-galactosidase for recombinant expression in E. coli, incorporating an mScarlet reporter for colorimetric visualization and a His-tag for IMAC protein purification. |
Educational Material
We created several valuable educational tools for the purpose of educating the community on the role of blood in general, as well as the importance of blood type compatibility in blood transfusions. By targeting several demographics, our tools were created to educate everyone from elementary students to college students and beyond. We created a children’s book to teach young children about transfusions and the importance of red blood cells in the body, as well as a word search and coloring pages that encourage interaction. For high school students, we designed a presentation and interactive card game activity meant to teach them about blood deserts and how synthetic biology can help address this issue. We created a crochet pattern for a red blood cell and a public perceptions survey to engage with college students and learn about their knowledge/opinions regarding genetically modified blood and medical products. For each event, we gathered feedback from the participants in a comprehensive survey to establish a two way dialogue and measure how effective our teachings were.