Difference between revisions of "Team:iTesla-SoundBio/Design"

Phase 1

Overview: Produce and purify Factor C from Bacillus subtilis

1. Acquire the amino acid sequence from factor C and translate it into codons. Codon optimizes it for Bacillus subtilis
• After acquiring the full sequence for the Factor C gene, split into two fragments to meet IDT’s limits on sequence length. The end of the first fragment and the end of the second fragment had a shared cut site of the BglII restriction enzyme.
2. Digest Fragment I and psb1C3 with EcoRI and PstI
3. Ligate Fragment I and psb1C3
4. Digest the Fragment I and psb1C3 construct with BglII and PstI
5. Digest Fragment II with BglII and PstI
6. Ligate Fragment II into the Fragment I and psb1C3 construct
7. Transform into E. coli

---------------- At this point we have a plasmid with the entire factor C gene in E. coli-----------------

8. Miniprep E. coli for the Factor C gene
9. Digest with BamHI and SacI to isolate factor C
• Gel extraction and purification of the factor C band  
10. Digest pAX01 with BamHI and SacII
11. Ligate pAX01 and Factor C
12. Transform into B. subtilis 168
13. His-tagged Protein Purification of factor C

Verification: western blot with Bacillus subtilis that has the pax0I Factor C insert. One will have Bacillus that has been induced by xylose and the other will not be induced.

Phase 2

Objective: Find an alternative procedure using the Factor C we produced to replace the LAL assay that is as effective, cheaper, and does not need horseshoe crab blood compared to industry options for endotoxin tests.

Option I: We could synthesize all the relevant proteins for the natural coagulation cascade

• Here is how it could look in terms of design:
• Since it is not Factor C we do not have to transfer it to Bacillus subtilis
• 1) Get the DNA sequence and add a His-tag for Factor B,
• 2) DIgest with enzymes so that we can ligate it into psb1C3
• 3) Transform into E. coli
• 4) His-tagged Protein Purification of factor C
• 5) repeat with clotting enzyme, and substrate
• 6) Once all the other compounds have been isolated, we can mix them with factor C
• 7) Advantages and Disadvantages:
1. Advantage: As all components of the LAL coagulation cascade are present in this solution (the only major difference being that they were produced synthetically), it could work if none of those components have difficult-to-replicate post-translational modifications.


2. Disadvantage: Given that we were unable to replicate even just factor C this iGEM season, trying to replicate the entire cascade would be extraordinarily challenging.
3. Disadvantage: While recombinant factor C is reasonably well-documented, recombinant factor B and the proclotting enzyme are not. This could lead to significant challenges (for instance, if either required post-translational modifications) which we may not be equipped to handle. For this reason, Option I is not particularly viable.

Option II: Identification of another zymogen that can be activated by cleaved factor C (like Factor B) and attempt to detect that.

• Industrial Lab-Run LAL Assay:
• 1) Endotoxins bind to the “sushi domains” (otherwise known as short consensus repeats, or SCRs; sushi domains are protein domains comprising one three-stranded 𝛽 sheet and two other, separate 𝛽 sheets) in factor C.
• 2) Once bound, they cleave factor C into an H chain, A chain, and B chain, converting it into its active form.
• 3) The active form of factor C activates Factor B. Factor B activates a proclotting enzyme, which in turn cleaves coagulogen to create a gel.

• Proposed Modified Assay:
• 1) Factor C reacts with a fluorophore peptide in a way similar to its interaction with factor B
• a) In other words, instead of activating factor B, factor C would activate a protein that emits a detectable signal.
• b)This is a technique already in use: the recombinant factor C (rFC) assay, sold by numerous pharmaceutical companies, does exactly this.

Option III: Use a Western blot (an application of gel electrophoresis that separates proteins by size) to compare uncleaved factor C with sample-exposed experimental factor C

• 1) As the H, A, and B chains of cleaved factor C are much smaller than uncleaved factor C (uncleaved factor C is 123 KDa, while the H, A, and B chains of activated factor C are 80, 7.9, and 34 KDa respectively), a Western blot should reveal whether or not cleavage of factor C took place in a given sample.
• 2) Advantages and Disadvantages:
• a) Advantage: As the only protein necessary for this procedure is factor C, it eliminates the need for extraneous protein synthesis/acquisition.
• b) Advantage: The efficacy of this technique has been verified by researchers at Japan’s Kyushu University.
• c) Disadvantage: Time-inefficient: Western blotting a sample might take hours, while the commercial LAL assay requires only fifteen minutes.




Option IV: We did consider using DFP because an article stated that activated factor had a site that was sensitive to it. However, after asking Dr. Thomas Novitsky, we realized DFP is extremely toxic and dangerous to handle which we did not want to risk.

Risks Include:

• 1) Intense miosis, ciliary spasm, headache
• 2) It hydrolyzes so rapidly that contact with eye droppers during application can inactivate the drug substance