Engineering

Define the Problem

The engineering design cycle begins with the identification of a prominent problem which our team aims to address. After many days of brainstorming and research, our team discovered a lack of available and inclusive treatments for Exocrine Pancreatic Insufficiency (EPI). EPI is a condition characterized by a deficiency in digestive enzymes including pancreatic lipase, colipase, amylase, and protease. Currently, all prescribed treatments for this condition are sourced from porcine pancreases and thus are associated with various ethical concerns.

Perform Background Research

After identifying our problem, our team aimed to learn more about the condition and current treatments through background research. We learned that EPI is a complication associated with various other conditions, including cystic fibrosis, chronic pancreatitis, type 1 diabetes, and other pancreatic conditions. We also learned more about existing pancreatic enzyme replacement therapies, commonly known as PERTs. We learned that these enzymes are derived from porcine pancreases and are thus not accessible for patients with religious or dietary restrictions. Additionally, we learned that these existing medications are not very effective and are often burdensome on patients, who may have to take up to 30 capsules daily. Our team learned more about our identified problem through online research, as well as discussions with experts in the field (doctors, synthetic biologists, dietitians, etc.).

Identify Solution Criteria

In order to brainstorm and develop a solution, we first needed to identify constraints and specific criteria we wanted our solution to incorporate. Primarily, the key focus of our project was to eliminate the need for animals, such as pigs or cattle, as a source for pancreatic lipase and digestive enzymes. Additionally, we aim for our solution to be more effective than existing treatments, as there is a high financial and pill burden associated with current medications.

Solution Ideation

After identifying what we wanted our ideal solution to look like, we began brainstorming and developing our solution plan. Our team discussed many rough plans and variations for our final design. Our team discussed pros and cons of using constitutive versus inducible promoters/switches, one- versus two-plasmid systems, and different tags and signal peptides for our proteins of interest.

After brainstorming various solution ideas, our team decided on a yogurt formulation containing the B. subtilis probiotic, which would be modified to secrete the desired digestive enzymes. This alleviates the concerns associated with current treatments, as our solution synthetically produces these enzymes, preventing the need for a treatment sourced from animals.

Initial Design

Our novel alternative treatment for EPI planned to utilize the human pancreatic lipase gene (PNLIP) and the procolipase gene (CLPS) to synthetically produce and secrete these enzymes from their chassis, B. subtilis. B. subtilis was chosen as our chassis as it is naturally present in the gut microbiome and is thus non-immunogenic. Additionally, B. subtilis has been found to alleviate pancreatitis-associated complications and has not been found to exacerbate these conditions, unlike other probiotics. We planned to insert the PNLIP and CLPS coding sequences, along with specific promoters and B. subtilis-optimized terminators into our plasmid backbone, pBS1C. As the current treatment is consumed with each meal, we sought our design to be inducible and thus express these enzymes in response to consumption of food. Therefore, our initial circuit design utilized a complex glucose-inducible switch mechanism to induce the expression of PNLIP and CLPS. The glucose-switch mechanism is composed of two key components: a terminator/anti-terminator mechanism and a cre-promoter.

Figure 1. Diagram depicting a brief overview of our circuit design. Without glucose present in the system, PNLIP and CLPS are repressed. When glucose is present in the system, PNLIP and CLPS are expressed.

In the presence of glucose, dephosphorylated GlcT binds to the RNA anti-terminator (RAT) sequence to physically hide the termination sequence, allowing for T7 RNA polymerase mRNA translation. As the promoters preceding the PNLIP and CLPS coding sequences are induced by T7 RNA polymerase, translation of this coding sequence is crucial for expression of lipase and procolipase. Simultaneously, HPrSerP-CcpA complexes form following glucose uptake in B. subtilis (as a result of the carbon catabolite repression pathway). These complexes bind to catabolite responsive elements (or cre sites) and repress the transcription of the lacI gene. This silences LacI production, thus lifting repression on T7 RNA polymerase, PNLIP, and CLPS and enabling the expression of our target genes. In the absence of glucose, GlcT remains phosphorylated; thus, it remains unbound from the RAT sequence, keeps the terminator accessible, and stops T7 RNA polymerase mRNA translation. Additionally, without the formation of HPrSerP-CcpA complexes, lacI is transcribed, producing the lac repressor protein (LacI). LacI blocks transcription of T7 RNA polymerase, PNLIP, and CLPS, effectively shutting down gene expression in low-glucose conditions.

We chose to add His-6 and HA tags, respectively, to the PNLIP and CLPS coding sequences with for protein purification purposes. Additionally, as these proteins are not naturally secreted by our selected chassis, we also flanked our coding sequences with signal peptide coding sequences (described in detail later in this page) so they are flagged for secretion from the cell. However, during the time of design, we were unsure as to which conformation or order of the signal peptide, tag, and coding sequence is most optimal for our intended design. Thus, we synthesized two variations of each coding sequence with its signal peptides in order to determine which was more optimal in our intended design. These two variations are: signal peptide – tag – coding sequence versus signal peptide – coding sequence – tag.

Given the various components of our design and the high variability we sought in our constructs to permit ease of testing and determination of more optimal part conformations, we decided to develop two “storage plasmids” using Gibson Assembly, the more effective method for combining various inserts into one plasmid backbone. We used pBS1C for our plasmid backbone and intended to digest certain parts and re-ligate to form constructs for testing. Our storage plasmids, distinguished by their names “A” and “B” are as described briefly below:

Gibson Plasmids

Gibson A Plasmid	Gibson B Plasmid
1A: signal peptide - His6 - PNLIP 2A: signal peptide - HA - CLPS 3A: T7RNAP 4A: glucose-metabolite binding site	1B: signal peptide - PNLIP - His6 2B: signal peptide - CLPS - HA 3B: RNA anti-terminator (RAT) 4B: glucose-metabolite binding site

Figure 2. Diagrams depicting each of the components described above for the Gibson A plasmid and the Gibson B plasmid.

Part 1: Lipase (BBa_K5178012)

Note: As indicated above, we synthesized two versions of this part (Part 1A and Part 1B). Part 1A contained a sequence with the His6 Tag after the signal peptide and before the lipase coding sequence, whereas Part 1B contained a sequence with the His6 tag after the lipase coding sequence. These two iterations allowed us to determine which conformation would permit more efficient secretion and protein folding of the lipase enzyme.

• PT7lac Promoter: The PT7lac promoter is a chimeric part proven to function in B. subtilis and was engineered as a part of an inducible promoter system involving the lac repression protein and T7 RNA polymerase (T7RNAP)[1]. In the presence of LacI, transcription is repressed at PT7lac, minimizing leakiness in the expression of the gene of interest. When no LacI is present, the polymerase binds to this promoter region and actively initiates transcription[2].
• RiboJ: Insulation with RiboJ increases gene expression at RNA and protein levels[3]. Two parts comprise RiboJ: the sTRSV ribozyme and a 23-nt hairpin. The self-cleaving mechanism of sTRSV causes the remaining sequence to form a terminal hairpin[3,4]. RiboJ shields downstream transcription from upstream factors4, providing stability and increased accuracy to gene transcription. B. subtilis is often considered to be leaky in inducible promoter systems and controlling gene transcription is not always accurate; RiboJ is one part that serves to minimize this leakiness[1].
• MF001 RBS: This ribosome binding site is commonly-used in and optimized for B. subtilis. It has precedence in the literature from which the circuit and construct was inspired[1].
• Human pancreatic lipase gene (PNLIP): As this is the main enzyme lacking in Exocrine Pancreatic Insufficiency (EPI) patients[5,6], the main goal of our project was to engineer bacteria to secrete this enzyme as an alternative enzyme replacement therapy[7]. Thus, this coding sequence, codon-optimized for B. subtilis, permits expression of the protein.
• Lipase signal peptide: As lipase is not naturally secreted from B. subtilis, the protein must be tagged with a signal peptide to be secreted from the cell. The signal peptide allows for the protein to be flagged for secretion. The sequence for the signal peptide was determined through reverse translation from the amino acid sequence of a signal peptide with similar function identified in literature[8,9].
• His6 Tag: The His6 tag allows for the protein to be purified using immobilized metal affinity chromatography (IMAC) due to its high affinity for immobilized metals which provides a simpler purification compared to other methods[10]. The His6 tag also allows us to use anti-His6 antibodies to test for presence rather than anti-lipase antibodies which allows us more financial flexibility.
• B0015 Terminator: This terminator is commonly used in and optimized for B. subtilis. It has precedence in the literature from which the circuit and construct was inspired[1].

Part 2: Procolipase (BBa_K5178006)

Note: Similar to Part 1, we synthesized two versions of this part (Part 2A and Part 2B) as well. Part 2A contained a sequence with the HA Tag after the signal peptide and before the procolipase coding sequence, whereas Part 2B contained a sequence with the HA Tag after the procolipase coding sequence. These two iterations allowed us to determine which conformation would permit more efficient secretion and protein folding of the procolipase enzyme, which then becomes colipase.

• PT7lac Promoter: The PT7lac promoter is a chimeric part proven to function in B. subtilis and engineered as a part of an inducible promoter system involving the lac repression protein and T7RNA polymerase[1]. In the presence of LacI, transcription is repressed at PT7lac, minimizing leakiness in the expression of the gene of interest. When no LacI is present, the polymerase binds to this promoter region and actively initiates transcription[2].
• RiboJ: Insulation with RiboJ increases gene expression at RNA and protein levels[3]. Two parts comprise RiboJ: the sTRSV ribozyme and a 23-nt hairpin. The self-cleaving mechanism of the ribozyme causes the remaining sequence to form a terminal hairpin[3,4]. RiboJ shields downstream transcription from upstream factors[4], providing stability and increased accuracy to gene transcription. B. subtilis is often considered to be leaky in inducible promoter systems and controlling gene transcription is not always accurate; RiboJ is one part that serves to minimize this leakiness[1].
• MF001 RBS: This ribosome binding site is commonly-used in and optimized for B. subtilis. It has precedence in the literature from which the circuit and construct was inspired[1].
• Pancreatic procolipase (CLPS): In the presence of bile salts (which are prevalent in the digestive system), lipase requires a cofactor (colipase) to bind to and catabolize lipid macromolecule structures[11]. Colipase is initially secreted as procolipase, which is then cleaved and transformed into colipase[12]. Thus, this coding sequence is crucial for procolipase to be expressed.
• Procolipase signal peptide: As procolipase is not naturally secreted from B. subtilis, the protein must be tagged with a signal peptide to be secreted from the cell. The signal peptide allows for the protein to be flagged for secretion. The sequence for the signal peptide was determined by reverse translation of the nucleotide sequence of a signal peptide with similar function identified in literature[8,9].
• HA Tag: The HA tag permits efficient and simple affinity purification of the tagged protein. Additionally, the HA tag allows for the protein’s presence to be detected using anti-HA antibodies[13], which are a much lower financial burden than anti-procolipase antibodies.
• B0015 Terminator: This terminator is commonly used in and optimized for B. subtilis. It has precedence in the literature from which the circuit and construct was inspired[1].

Composite Part 3: T7RNAP and Terminator/Anti-terminator Sequence for Glucose-Mediated Switch Mechanism

Part 3A (BBa_K5178017

Note: Part 3a has two components, Part 3A.1 and Part 3A.2. Part 3A.2 will be used in the final construct, whereas Part 3A.1 was synthesized purely for testing purposes.

• Part 3A.1 (BBa_K5178014)

  • P43 Promoter: The P43 promoter is a strong, constitutive active promoter. It has been optimized for B. subtilis[14].
  • Lac Operator: The lac operator is the sequence at which LacI binds to prevent transcription in the lac operon mechanism, first described in E. coli[15]. It has been demonstrated to function in B. subtilis in literature[16].
  • MF001 RBS: This ribosome binding site is commonly used in and optimized for B. subtilis. It was referenced commonly in the literature from which the circuit and construct was inspired[1].

• Part 3A.2 (BBa_K5178015)

  • T7RNAP gene: The T7 RNA polymerase gene (“T7RNAP gene”) is derived from bacteriophage T7[17]. T7RNAP has a high and specific processivity, with maximal expression of the transcribed gene(s) of interest[1,17]. Additionally, T7RNAP is highly specific toward the T7 promoter[17], preventing unwanted transcription of other genes in our device with non-T7 promoters. It has been shown that T7RNAP binds strongly to PT7lac and is effectively repressed by LacI[1], reinforcing our inducing mechanism.
  • B0015 Terminator: This terminator is commonly used in and optimized for B. subtilis. It has precedence in the literature from which the circuit and construct was inspired[1].

Part 3B (BBa_K5178000)

Note: will replace Part 3A.1 to create a composite part (BBa_K5178016)

• Lac Operator: The lac operator is the sequence at which LacI binds to prevent transcription in the lac operon mechanism, first described in E. coli[15]. It has been proven to function in B. subtilis. in literature[16].
• PptsG Promoter: The PptsG promoter is a native promoter of B. subtilis that is utilized before the ptsG gene within the phosphotransferase system. This is a strong promoter that contains a RAT sequence and a terminator sequence, both of which are used in a mechanism that responds to extracellular glucose (as explained below)[18].
• RAT Terminator/Anti-Terminator: The RAT sequence and the terminator sequence both follow the PptsG promoter. The RAT sequence forms a mRNA anti-terminator structural hairpin that exposes the terminator sequence so that the following PptsG promoter and the respective coding sequence are inhibited. When there is extracellular glucose present, the phosphotransferase system is activated, and a downstream molecule (phosphorylated GlcT) becomes dephosphorylated. The dephosphorylated GlcT will bind to the RAT sequence, causing a conformational change so that the terminator is inaccessible, and the promoter sequence is available. This will lead to the transcription of the subsequent coding sequence[19].
• MF001 RBS: This ribosome binding site is commonly used in and optimized for B. subtilis. It was referenced commonly in the literature from which the circuit and construct was inspired[1].

Composite Part 4: Cre-site Mechanism for Glucose-Mediated Switch Mechanism

Note: Two versions of this part were synthesized to determine which was more efficient. Part 4A sourced the sequence of the amyE cre site from a database, and Part 4B sourced the sequence of the amyE cre site from literature. GFP, flanked by restriction enzymes, comprised Part 4c; this part was used only to test the relative fluorescent expression of Parts 4A and 4B, and it did not become part of our final construct.

• 4A: BBa_K5178001
• 4B: BBa_K5178002

• P43 Promoter: The P43 promoter is a strong, constitutive active promoter. It has been optimized for B. subtilis[14].
• amyE cre site: Utilization of the carbon catabolite repression (CCR) pathway in B. subtilis allows for a glucose-inducible circuit[20]. Through the uptake of glucose, the protein HPr becomes phosphorylated and binds to Carbon Control Protein A (CcpA)[21]. The HPrSerP-CcpA complex then binds to catabolite responsive elements (cre sites) of DNA sequences[22]. Cre sites can be located upstream or downstream relative to the promoter, and they activate or inhibit gene transcription, respectively[21]. The amyE cre site was selected from over 50 known cre sites from the B. subtilis genome for its high affinity and close distance of +4 bp to the TSS of the gene of interest[21]. The amyE cre site is bound and transcription is strongly repressed when glucose is present.
• MF001 RBS: This ribosome binding site is commonly used in and optimized for B. subtilis. It was referenced commonly in the literature from which the circuit and construct was inspired[1].
• LacI gene: lacI is a well-characterized gene first described in E. coli that encodes for the lactose repressor protein (LacI). LacI is a DNA-binding transcription factor that strongly represses transcription[23]. LacI was expressed successfully in B. subtilis as a part of the inducible promoter system[1] and serves to repress T7RNAP expression in our device system by binding to our incorporated lac operator sequence.
• B0015 Terminator: This terminator is commonly used in and optimized for B. subtilis. It has precedence in the literature from which the circuit and construct was inspired[1].
• GFP: One of the most common fluorescent markers used in analyzing expression strength, GFP helps us to assess the two cre site sequences and determine which is more effective. The sequence[24] was codon-optimized for B. subtilis.