Before entering the design build test cycle, there were multiple steps involved to get to our first design order. Here is a breakdown of what our process looked like and what sorts of things went into our first designs: 
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What chassis: Nissle 1917 E.coli
‍Necessary Ingredients: PAPS, 5 enzymes, and Heparosan
‍Heparosan source: Nissle 1917 naturally produces Heparosan
‍PAPS source: Knockout cysH gene in Nissle 1917 genome to increase PAPS presence by removing the PAPS reductase
‍5 enzymes: 5 enzymes will be introduced via plasmids
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Hi if you're a judge please skip this part because it is a whole lot of yap and I know you don’t have much time ;)

‍Escherichia coli (E. coli) can synthesize PAPS; however, its basal levels are typically low because the bacterium also expresses phosphoadenosine phosphosulfate reductase, an enzyme encoded by the cysH gene. This enzyme degrades PAPS by reducing it to adenosine phosphosphate (PAP), thereby limiting the availability of PAPS for sulfation reactions.
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Badri A et.al have demonstrated that deleting the cysH gene significantly increases basal PAPS levels. Building on this finding, we have removed the cysH gene from E. coli Nissle 1917—a probiotic strain known for its safety profile—to enhance intracellular PAPS concentrations. This genetic modification ensures a higher availability of PAPS for the sulfotransferase enzymes involved in heparin biosynthesis.

The main concern for the knockout cassette was ensuring that the cassette would integrate into the correct locus. We also wanted to ensure that the cysH Knock out will have minimal impact on other gene expression so we wanted to try our hardest to avoid unintended effects on adjacent gene expressions.
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Name: BBa_K5254003
Part type: Device
Short description (PCR fragment Chloramphenicol resistance gene flanked with 5’ and 3’ region of cysH gene from Nissle 1917): 
Long description :
Chloramphenicol resistance gene in ID42 parts from 2024 Distribution kit was amplified with primers directing 5’ end and 3’ end of CMR. These primers are also flanked by 40 nt from 5’ (3097442-3097403) and 3’ (3096628-3096667) UTR region of cysH gene (3096668-3097402) in Nissle 1917. PCR product should be CMR flanked with homologous arm to 5’ and 3’ UTR regions of the cysH gene (phosphoadenosien phosphosulfate reductase).

The main problem was expressing these proteins properly in e.coli. Mammalian enzymes can be expressed in a functional manner, but many features of mammalian proteins cannot be reproduced in bacteria. In general, bacterial recombinant proteins often face solubility issues, and on top of that the enzymes we are dealing with are large which can lead to aggregation and loss of function.
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We split the heparosan-heparin pathway into two different plasmids which we called A and B. They were graciously synthesized by Ginkgo Bioworks!

These enzymes are type II transmembrane proteins because they work at the Golgi apparatus in the mammalian cells. However, in E. coli, transmembrane region is not required and even harms the expression of the enzymes. The extracellular domains of these enzymes have been successfully expressed and functional in E. coli ( refer PNAS and Jian Liu paper). Therefore, we designed the plasmid coding of the 6x his and MBP conjugated human NDST2 extracellular domain (from aa 64 to aa 883) and C5 epimerase extracellular domain (from aa 53 to aa617) under the tac promoter/lac operator with LacI integration. We expect MBP conjugation to help the solubilization of the proteins and 6X his for the purification. These enzymes will be induced by IPTG with minimal background expressions. Two Sap I sites are integrated for further procedure such as golden gate methods

This plasmid contains 2-OST, 6-OST1, and 3-OST1. The extracellular domains of these enzymes have been shown to express and solubilize well with the conjugation of MBP( ref PNAS), therefore we exactly follow the design from the previous paper. MBP conjugated Chinese hamster 2-OST extracellular domain (from aa 51 to aa 356) and mouse 6-OST1 extracellular domain (from aa 62 to aa 411) are integrated under the tac promoter/lac operator. 3-OST extracellular domain can be expressed in E. coli without MBP (ref PNAS), then, we also include 6x his tagged mouse 3-OST1 extracellular domain (from aa 48 to aa 311). Two Sap I sites are inserted at the 5’ and 3’ flanking region of enzyme coding regions for further application to combine pGA1 and pGB1 into a single expression vector.

Coding regions for all enzymes are codons optimized for bacterial expression. We designed whole plasmid sequences and Ginkgo Bioworks kindly synthesized the plasmids.

This approach will provide Nissle with high PAPS content and NDST2 expression simultaneously. NDST2 expression will not be regulated by the lac operon; instead, NDST2 is integrated just downstream of the 5' UTR of the CysH CDS. Therefore, its expression levels should be similar to that of PAPS reductase (CysH), which may be more relevant to PAPS generation. Appropriate expression levels of NDST2 relative to PAPS may be suitable for successful N-sulfation. The successful knock-in of NDST2 could also demonstrate the feasibility of knocking in the entire pathway. Modifying the bacterial genome by inserting genes could surpass the plasmid approach, as genome integration does not carry risks such as plasmid loss. Additionally, we may maintain exogenous gene expression under physiological regulation.

The concerns with the knock-in cassette were similar to those of the knockout cassette design: ensuring that the NDST2 gene was being integrated into the correct position while minimizing its impact on neighboring gene expressions. However, we were also aware that NDST2 is a relatively large protein, and thus we anticipated more challenges with this design compared to our knockout construct.

Keeping these concerns in mind, we used PCR methods to subclone the NDST2 and KanR genes from the template plasmid. The template plasmid contains the full-length cDNA for NDST2, so we needed to truncate the NDST2 CDS. This limited the flexibility in designing the forward primer, as we required a specific region of NDST2 without deleting the cysH UTR regions.

NDST2 extracellular domain and Kanamycin resistance gene en bloc in MGC Mouse Ndst2 cDNA was amplified with primers directing 5’ end and 3’ end of NDST2 extracellular domain and Kanamycin resistance gene. These primers are also flanked by 40 nt from 5’ (3097442-3097403) and 3’ (3096628-3096667) UTR region of cysH gene (3096668-3097402) in Nissle 1917. PCR product should be NDST2+KanR flanked with homologous arm to 5’ and 3’ UTR regions of the cysH gene (phosphoadenosine phosphosulfate . Successful recombination will achieve cysH gene knockout and NDST2 integration. This cassette will replace exactly cysH gene locus, therefore NDST2 expression will be regulated by an intrinsic promoter.

Warning: We never actually received this order due to complications.

This part is composed of a maltose-binding protein (MBP) sequence and the sequence of the NDST2 protein. A lac operator was used to control the expression of the part, such that a lac repressor can be integrated if gene expression regulation is necessary. The MBP has been widely used to enhance the solubility of the recombinant proteins, improving protein folding as well as preventing aggregation. This design was obtained from Choe’s 2017 paper, “Prokaryotic soluble expression and purification of bioactive human fibroblast growth factor 21 using maltose-binding protein”, in which human fibroblast growth factor 21 (hFGF21) was attached to several different tags, including MBP, and was expressed in E. coli. The MBP-tagged hFGF21 far outperformed the other tags in terms of solubilizing the protein. Each enzyme has a His tag attached. The terminator of the design is removed due to the complexity limitations of the IDT system, but it’s still in use with the Amp Golden Gate. Additionally, due to the Dou 2015 paper, NDST II was clarified to be truncated, codon-optimized, and also cut without transmembrane regions.

1. TIME IS MONEY. Time moves by quickly, and it’s over before you even realize it. This means that BUDGETING your time is crucial.

2. Don’t assume that DNA orders will arrive quickly, especially larger ones. We learned that there are often many issues with ordering, including complexity and other factors beyond your control.

3. Order as soon as you're ready! I can’t emphasize this enough. No matter how smart you are or think you are, your design will never be perfect. It’s better to place an order when you have a solid design and work out the details later through experimentation. Delaying orders and striving for perfection is a WASTE OF TIME.

4. Design is like an engine starter; it gets the wheels moving, builds momentum, and sets the stage for either success or failure.

Once all ingredients are acquired. The two systems— plasmids and Knockout Cassette— added together give multiple options to approach heparin synthesis: in-vivo, in-vitro, or ex-vivo.

Our team attempted to try all three and thus during the summer we carried out all three projects in parallel with each other. We speculated that the most successful method would be the ex-vivo strategy. To test for final heparin product we carried out two types of gel staining: alcian blue stain and toluidine blue stain.

Where will we go from here?

‍Our designs and this project focuses more on the actual biosynthesis of heparin in E.coli. In this year's IGEM competition, we focused more on proving sulfation is possible and that heparin production in e.coli is in fact possible. Our project focused on the biomanufacturing process and identifying and testing that we have created heparin products. The next step will be to purify our product and test its quality and compare against existing heparin like enoxaparin etc.

Despite not purifying heparin products yet, we believe our designs strongly contribute to the movement of sustainability in the heparin field. We believe that while medicine and technology continue to grow and evolve in unbelievable speeds, this field has remained surprisingly stagnant relying on methods that are surprisingly outdated and relatively inefficient for such a crucial medication.