Team:iTesla-SoundBio/design

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iT-SB 2018 iT-SB 2017

Phase 1
Phase 2
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Introduction

The goal of our project was to recreate the first protein involved in the LAL cascade (Factor C) by using Bacillus subtilis, a gram-positive bacteria, to synthesize the protein of interest. Additionally, we wanted to find an alternative method to detect cleaved factor C. When detecting cleaved Factor C, our goal was to cut down on intermediate steps in the LAL cascade (factor B, clotting enzyme) and skip directly to an engineered reaction that is easily detectable, such as a visual assay. To briefly summarize our project design, it can be explained in two phases.

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

    8. ---------------- 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’s how it could look in terms of design:
    • Since it’s not Factor C we don’t 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
      • Pros: Should work, in theory
      • Cons: Almost certainly difficult/expensive

Option II: We could find another zymogen that can be activated by cleaved Factor C (like Factor B) and attempt to detect that

In an industry lab ran LAL assay the:

  1. The endotoxin binds in the region with the sushi domains
  2. Once binded, it causes the breaking of a specific of serine protease end of the zymogen
  3. The protein end is SPECIFIC for the next factor B

With our modified version:

  1. Zymogen reacts with fluorophore peptide that is similar factor C’s interaction with factor B
    1. In other words, the zymogen “activates” the glowing attribute of modified factor C.

This would require`us to edit our factor C DNA sequence to have a modified end that would cleave/ give a detectable signal when exposed to endotoxin.

Option III: Use western blot technique on a sample with normal factor C and experimental factor C exposed to a sample

    • This should on the basis that Western Blots can be used to note molecular changes in proteins and that cleaved factor C is notable smaller in size meaning that it will appear to be lower in kDa
      • Note that Factor C is 246 kDA and the active form of Factor C is 121.9 kDa
      • Pro: This should reveal how much Factor C was cleaved without need for other proteins and zymogens
      • Pro: It is eliminates the need for horseshoe crab blood
      • Con: while it is a viable option, it isn’t a time effective option- hours compared to just 15 minutes

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

Risks include:

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