Parts

Our project, "Yeast Medics," is built upon a sophisticated synthetic biology system designed to dynamically sense and treat diabetic wounds. The core of this system is the engineered Yarrowia lipolytica Po1h, which we have programmed with multiple genetic circuits. The key parts we used and constructed can be categorized as follows:

1. Chassis and Selection Markers

We utilized the Yarrowia lipolytica Po1h as our chassis. To enable plasmid selection and maintenance, we employed parts like BBa_2584OLTJ (Ura3 gene) for uracil autonomous synthesis and BBa_25PAFKYX (KanR) for kanamycin resistance in E. coli cloning steps.

2. Expression Cassettes

The therapeutic functions are driven by specialized expression cassettes. These include constitutive, inducible, and protein-fusion parts. These parts could control the expression of therapeutic proteins under specific conditions.

2.1 Constitutive promoter

BBa_25Q513SS (hp4d promoter) drives the continuous expression of the antimicrobial peptide Pexiganan (BBa_25ZL42F5) in the PPB medium.

2.2 Inducible promoter

Glucose inducible BBa_25DWY2L9 (PGK promoter) controls the expression of the anti-inflammatory cytokine IL-4 (BBa_25328220) in response to high glucose levels in the wound.

Heat inducible: The promoter from BBa_25WAG6JK (HSP90) controls the expression of the pro-angiogenic factor VEGF (BBa_25F8XQQG) upon local heating via infrared light.

2.3 Protein tag:

BBa_25EYN5ZA (6xHis-tag) was fused to proteins like IL-4 and VEGF to facilitate detection and purification.

3. Surface Display System

To anchor Pexiganan to the yeast cell wall, we employed a GPI-anchoring system. This involves a fusion protein containing the BBa_25PO9JTJ (signal peptide) for secretion and the BBa_25FXECR4 (GPI anchor domain) for covalent attachment to the cell wall.

3.1 Homologous Arms & Assembly:

Parts like BBa_254YC05O and BBa_25Z4VEH6 (both of them are zeta sequences but in different length) were used for genomic integration of expression cassettes into the Y. lipolytica chromosome, ensuring stable inheritance.

3.2 Terminators:

Essential parts like BBa_250XAGTD ensure proper termination of transcription for each gene.

4. Plasmids

4.1 Plasmid backbone:

Our expression system uses the integrated plasmid pINA1317 (BBa_258PCOD1)

4.2 Promoter Test Plasmids:

For the glucose-inducible promoter PGK and high temperature-inducible promoter HSP90 screened by us, we inserted eGFP into pINA1317 and directly replaced the promoters with PGK (BBa_257SU7R8) and HSP90 (BBa_252Z87Z2). After inducing eGFP expression, the green fluorescence intensity was observed under fluorescence microscope.

4.3 Expression Bioactivity Test Plasmids:

For the three target genes, Pexiganan used a surface display plasmid (BBa_25VB2NC8) modified with pINA1317, and IL-4 and hVEGF were labeled with 6xHis tags at the C end and inserted into pINA1317 together (BBa_25W0LDYC, BBa_25ZQ6DXN).

In summary, our collection of BioBrick parts enables the construction of a "living therapeutic factory" that can sense wound signals and execute a programmed sequence of anti-microbial, anti-inflammatory, and pro-regenerative actions, all encapsulated within a smart hydrogel delivery system, L-HBC.

Parts Summary Table