Team:iTesla-SoundBio/Design

InterLab Human Practices Collaborations

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

In order to produce the factor C protein, we need the DNA sequence that was also codon optimized for Bacillus subtilis. Since we had a limitation to the length of our geneblock, we had to split it into two fragments (Fragment I and Fragment II), and we split it so that both can be digested by BglII restriction enzyme. To start the cloning process, we begin with only Fragment I, then move on to Fragment II after ligating FI into pSB1C3.

1) Acquire the amino acid sequence from factor C and translate it into codons. Codon optimize it for Bacillus subtilis.
a)After acquiring the full sequence for the factor C gene, split into two fragments to meet IDT’s limits on sequence length. Fragments were created with BglII cut sites that allow them to be joined together via restriction digest and ligation.
2) Digest Fragment I and psb1C3 with EcoRI and PstI
a) This step creates the necessary sticky ends for us to insert Fragment I into the backbone.
3) Ligate Fragment I and psb1C3
a)This would put the first half of the factor C gene into Fragment I, leaving only Fragment II for insertion.
4) Digest the Fragment I and psb1C3 construct with BglII and PstI
a)As with step 2, this step creates the necessary sticky ends in the backbone for us to insert Fragment II.
5) Digest Fragment II with BglII and PstI
a)This creates the necessary sticky ends in Fragment II for ligation into the Fragment I + pSB1C3 backbone.
6) Ligate Fragment II into the Fragment I and psb1C3 construct
a)This results in the entire factor C gene being inserted into pSB1C3.
7)Transform into E. coli
a)This allows replication of the plasmid for later extraction.

---------------- 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
a)This step gives us usable quantities of the factor C + pSB1C3 plasmid.
9)Digest with BamHI and SacI to isolate factor C, then gel extraction and purification of the factor C band
a)In removing and isolating the factor C sequence from pSB1C3, we prepare to insert it into a Bacillus integration vector.
10)Digest pAX01 with BamHI and SacII
a)This creates sticky ends on the pAX01 integration vector, preparing it for the insertion of factor C.
11)Ligate pAX01 and factor C
a)This creates a plasmid that should integrate the factor C sequence into the Bacillus genome.
12)Transform into B. subtilis 168
a)This puts the factor C gene into an organism that can properly express it, allowing us to work with the factor C protein itself.
13)His-tagged protein purification of factor C
a)This facilitates the usage of factor C in future experiments.

Verification: Western blot with Bacillus subtilis that has the pax01 factor C insert. One will have Bacillus that has been exposed to xylose (inducing the promoter in pAX01) and the other will not be induced.

Note: This flowchart allows us to plan future days based on the success/failure of prior experiments and clearly illustrates what we need to do and when to do it at any given point.

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:

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.

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.
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