Overview

1. Background

2. Design Iterations

3. Constructing Parts

Background

In order to decide what enhanced properties we wanted our probiotic to have, we conducted additional background research on the molecular basis of Maple Syrup Urine Disease. We learned that mutations in the BCKDH enzyme complex, which is responsible for breaking down branched chain keto-acids (BCKAs), causes the buildup of BCAAs and BCKAs to toxic levels in MSUD patients.

We decided to have our probiotic bacteria overexpress BCKDH to break down excess BCAAs before they are absorbed into the bloodstream. We were initially drawn to E. coli Nissle, a well studied probiotic strain of E. coli commonly used in synthetic biology and by iGEM teams, as a chassis. However, we learned that the human BCKDH enzyme is a very large complex consisting of multiple copies of each of 4 subunits encoded by different genes. E. coli lacks this enzyme complex or similar machinery to break down BCAAs, so we would need to import all these sequences into E. coli, which would be rather difficult and potentially unstable. As a result, we instead turned to looking at existing bacteria that already possess the BCKDH complex, and we found B. subtilis.

B. subtilis is a gram-positive bacterium commonly found in soil (1). B. subtilis is also a native inhabitant of the GI tract and a commonly used probiotic with proven health benefits (2). One unique feature of B. subtilis is that it is able to form spores and become metabolically dormant to survive in adverse environments (source??), and many probiotics consist of B. subtilis in spore form. This allows it to survive the transit through the acidic environment of the stomach, and then germinate in the nutrient-rich small intestine.

The bkd operon in B. subtilis encodes the BCKDH enzyme complex. Combined with a transaminase and various other downstream enzymes, B. subtilis is able to fully metabolize BCAAs down to the basic substrates of central metabolism, such as acetyl CoA and succinyl CoA. As B. subtilis already has the bkd operon within its genome, we can simply use homologous recombination to replace the operon’s promoter with a constitutive one in order to upregulate it. We also identified various other genes in B. subtilis responsible for importing and exporting BCAAs in order to enhance B. subtilis’s ability to uptake and retain BCAA’s.

Below is a simplified model of the relevant genetic circuitry within B. subtilis. Our genes of interest are

1. bcaP: a BCAA symporter
2. ilvE and ilvK which encode BCAT: BCAA transaminase, which catalyzes the first step in the breakdown of BCAAs
3. bkd operon: an operon that encodes branched chain keto-acid dehydrogenase (BCKDH), the enzyme complex that catalyzes the second step in the breakdown of BCAAs
4. azlC and azlD: BCAA exporters in the azlBCDEF operon

Design Iterations

Our project had three main iterations:

1. Basic Proof of Concept

In this first iteration, we wanted to prove that overexpressing these critical genes could lead to a measurable increase in BCAA uptake, so we designed parts for constitutive expression of these genes.

Design
The four main components of this system are

1. Constitutive expression of the bcaP importer to import more BCAAs into the cell
2. Constitutive expression of ilvE/ilvK transaminase to break down BCAAs into alpha keto-acids
3. Constitutive expression of the bkd operon to break down alpha keto-acids
4. Knockout of azlCD exporters to prevent BCAAs from leaving the cell

Testing
To transform B. subtilis with our DNA, we planned to have our parts integrate directly into the genome through homologous recombination. To confirm that we were able to execute this, we also designed parts that expressed mApple, a red fluorescent protein, and KanR, a protein for kanamycin resistance. These same parts were also used in E. coli to check that our promoters were working as expected.

In total, our cloning and testing steps included the following experiments:

• Gibson cloning and traditional cloning to construct plasmid backbones
• Golden Gate to construct plasmids from our parts
• Colony PCR of both plasmid and genomic DNA to quickly check our Golden Gate products, as well as to check integration of our DNA into the B. subtilis chromosome
• Restriction digests to check our Golden Gate products prior to sequencing
• Transformation of E. coli to replicate our plasmid DNA
• Transformation into B. subtilis

Following successful transformation, we then assayed the ability of our B. subtilis to break down BCAAs, as well as B. subtilis’s natural rate of BCAAs.

We performed the following experiments to assay B. subtilis:

• Test homologous recombination into B. subtilis with kanamycin selection marker
• BCAA concentration assay to determine the sensitive range of our assay and the best starting concentration of BCAAs with which to perform subsequent assays
• BCAA time point assay to compare the rate of BCAA breakdown between wild-type and probiotic B. subtilis

2. Recombinase switch for constitutive expression

This next design iteration keeps the basis of overexpressing genes but adds in additional regulatory controls. As we are engineering B. subtilis to overexpress many large protein complexes, it is critical to maintain fitness of the organism while it is being manufactured or traveling within the GI tract. After much discussion with our mentors, we decided to induce the expression of a recombinase to switch on constitutive expression of these proteins at the right time.

What is a recombinase?
There are two types of recombinases: tyrosine recombinases (ex. Cre) and serine recombinases (ex. PhiC31). Recombinases recognize certain sequences in the DNA (ex. loxP and attP/B), and act on the DNA in between them. Generally, this takes the form of either an excision or a reversion of the intervening sequence, depending on the directions in which the flanking sites are facing. Excision is useful for permanently turning on or off a gene, while an inversion can function as a switch that can be adjusted depending on the condition of the cell.

Design
We placed the Cre recombinase under the inducible control of a pTet/tetR/ATC system in order to turn on the constitutive expression of bcaP, ilvE/ilvK, and the bkd operon in the presence of doxycycline.

Cre recognizes specific loxP sites in DNA, in our case the lox71 and lox66 sites. There are two ways in which Cre can function, depending on how these sites are oriented:

1. If the loxP sites face the same direction, the region of DNA between the sites is excised as a circular piece of DNA.
2. If the loxP sites face opposite directions, the region of DNA in between the sites is inverted.

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