New Parts
WT E.coli Nissle 1917 naturally produces 3′-phosphoadenosine-5′-phosphosulfate (PAPS) and Heparosan. Taking advantage of this, we introduced 4 sulfotransferases (NDST II, 3-OST, 6-OST, 2-OS) and an epimerase (C5 epimerase) to complete the synthesis pathway of heparin in E. coli. To prevent the natural degradation process of PAPS within the bacteria, we designed and tested a knock-out cassette to facilitate a targeted gene disruption in the Nissle genome. Our homologous recombination replaced cysH, the gene that codes for the enzyme that degrades PAPS, with a selectable antibiotic resistance gene CmR.
We’ve amassed a part collection containing the genes to complete the heparin synthesis pathway. To learn more about our part collection, navigate to this page which contains the complete descriptions for these sequences. This outstanding part collection can provide the iGEM community members with a versatile knock-out cassette as well as the complete enzyme pathway of heparin synthesis!
- Nissle CysH Knockout Strain → BBa_K5254018
- Plasmid A(NDST II, C5 Epi) → BBa_K5254012
- Plasmid B(2-OST, 6-OST, 3-OST) → BBa_K5254013
Wetlab
Innovative SDS page gel stained with Alcian Blue
Since our final goal is to have our product be applied in multiple settings to address the massive stress challenges, we finished a few rounds of engineering cycles and amassed valuable experience in terms of cyanobacteria culturing in different conditions. Based on that, we built an online available tool for people to easily estimate relative OD of their green algae or cyanobacteria using just a photo, which can be beneficial for other iGEM competitors to evaluate the growth of their cyanobacteria. Read more about it on our Model page and access the source code on Github.In order to confirm the presence of bacterial Ecorin Heparin, we designed an innovative testing strategy involving an SDS page gel with Alcian Blue staining
[2]. Since heparin is an acidic carbohydrate, we utilized Alcian blue dye (which naturally stains very acidic carbohydrates) to detect its presence in the gel. This dye is usually used to detect acidic proteoglycans such as glycosaminoglycans
[1], and has never been used before to detect heparin in the iGEM competition. Read more about this on our Wetlab page!
Universal sulfotransferase Kit(functional assays for enzymes and CysH KO)__
In order to test our designs, we developed a functional assay using a universal sulfotransferase kit Another issue we uniquely resolved is the expensive prices of the
- Universal sulfotransferase kit measures the amount of PAP signal
- Showing sulfotransferase works in a functional assay
- Adding low, medium, high amounts of PAPS into our chassis(purified enzymes) so our signal should be a low medium high read out.
Human Practice & Education
For Human Practices, we organized an event at a locally hosted summer program to expose and introduce young students to the concept of enzymatic reactions. To do this, we wrote and ran a protocol for the interaction between the bromelain enzyme in pineapples and the proteins in milk. Through an interactive lecture-esque style session, we educated them about our heparin project as well as the basics of molecular enzyme interactions. We received positive responses from the students, and it was a successful, community-building event!
Future of Ecorin
Our Contribution/Approach
We are the first iGEM team to genetically engineer Escherichia coli Nissle 1917 to produce heparin. Over the course of the summer, Boston-Boslab has made the following strides:
- Introduced critical enzymes for heparin biosynthesis:
Sulfotransferases: NDST II, 3-OST, 6-OST, and 2-OST.
C5 epimerase, which converts glucuronic acid to iduronic acid in heparin.
- Successfully knocked out the cysH gene to prevent PAPS degradation, which increases the sulfation donor in vivo.
- Created the first-ever all-in-one heparin-producing bacterial strain, which we’ve named Ecorin.
- Successfully knocked out the cysH gene to prevent PAPS degradation, which increases the sulfation donor in vivo.
- Created the first-ever all-in-one heparin-producing bacterial strain, which we’ve named Ecorin.
What makes our process unique/novel?
- All-in-one system: Our Ecorin strain condenses every essential aspect of heparin biosynthesis into a single bacterial cell. This allows continuous heparin production through a simple cell culture process. Unlike other methods relying heavily on in vitro stages, our approach minimizes complexity and maximizes efficiency by operating entirely in vivo.
- Simplification of steps: Traditional biosynthetic heparin production involves complex, multi-step processes that require in vitro enzyme purification and synthetic intermediates. By recreating the mammalian heparin biosynthesis pathway in a bacterial host, we streamline the entire production.
- Bacterial advantages: Although producing heparin in mammalian cells might seem more straightforward because of their natural role, our Ecorin strain provides a safer, more reliable, and more scalable option. This raises fascinating questions about bacterial evolution and the potential for other organisms to perform similar tasks.
How we can inspire other iGEM teams?
We hope to set a foundation for future iGEM teams to build upon our work in several ways:
- Genome recombination with Lambda Red: Using the Lambda Red system in E. coli Nissle 1917 has enabled us to perform efficient homologous recombination for genome editing. This non-pathogenic strain offers a safer genetic engineering tool, opening the door for future applications in synthetic biology.
- Versatile hybrid systems: Our approach combines genetic modification of E. coli with plasmid transformation, making it a versatile platform for various biomanufacturing challenges.
Future Vision: Where do we go from here?
- Purification: Our current focus is on sulfation, but we recognize that downstream purification is crucial. We’re in the process of developing a purification protocol to isolate the final heparin product. This phase remains hypothetical as we continue advancing toward this goal. Our main goal for the 2024 iGEM competition was to prove that sulfation could be accomplished in bacteria.
- Scaling up: Beyond proof-of-concept, we aim to scale up production and explore partnerships to manufacture heparin at a commercial level.
- Entrepreneurship: We envision the possibility of marketing our Ecorin system or its heparin production pipeline, potentially opening doors for partnerships and entrepreneurship.
