Difference between revisions of "Part:BBa K5165000"

Latest revision as of 09:56, 2 October 2024

Polyhydroxybutyrate (PHB) depolymerase

PHB depolymerase is an esterase from the Talaromyces funiculosus species involved in the hydrolysis of polyhydroxybutyrate, a microbial polyester that can be produced from renewable resources.

We verified the expression of this protein by linking GFP to this new part via a GS linker (part ). Cells were transformed into BL21 cells and selected for using antibiotic (amplicillin) selection plates. To verify that our new part, PHAZ_TALFU – which is a PHB depolymerase that catalyzes the hydrolysis of PHB into monomers of BHB, and thus crucial for our project's main objective, we linked PHAZ_TALFU with GFP (part BBa_K2459011) using a GS linker sequence. E.coli BL21 cells were successfully transformed with our expression plasmid (pET 32a+) containing the parts PHAZ-TALFU + GFP. Different concentrations of IPTG were used as an inducer molecule and GFP fluorescence was compared to untransformed wild type BL21 e. coli cells. Fluorescence was measured 16 hours after induction.

The purpose of this experiment was to verify PHAZ_TALFU expression via GFP signal as a positive control using a simple system. Fluorescence was measured using FLUOstar Omega microplate reader (BMG Labtech, Ortenberg, Germany).

Later this part was added to a new composite part (BBa_K5165002), with the objective to produce BHB in the extra cellular environment of cultured e. coli cells. The results below show that this new part successfully depolymerised excreted PHB and BHB was detected in the extra cellular environment via colorimetric assay.

Polyhydroxybutyrate (PHB) can be depolymerized to form the high-value product beta-hydroxybutyrate (BHB). This construct accomplishes BHB formation through two steps in E. coli: 1) PHB synthesis 2) PHB depolymerization The construct is designed so that the host cell prioritizes the production of the enzymes involved in PHB synthesis. Quorum sensing mechanisms, regulated by the lux system, are responsible for transitioning the cell into stage two for PHB depolymerization. Such a two-part system allows for optimized production in each step separately, which is essential for BHB synthesis as the initiation of step two depends on the products of step one.

The first part is regulated by the repressible promoter pTet. PHB synthesis is driven by three enzymes that are characterized in the part BBa_K934001: phaC, phaA, and phaB. The phaCAB enzymes, accompanied by the strong RBS BBa_B0034, constitute the main component of part one. BBa_K2260002 codes for a protein tagging the PHB for secretion, and BBa_K4998027 codes for LuxI, an Acyl-Homoserine Lactone (AHL) synthase enzyme. LuxR, working in tandem with AHL to regulate quorum sensing, is constitutively expressed in the construct. As time progresses, the concentration of both phaCAB enzymes and AHL-luxR complexes increase.The AHL-luxR complexes in turn induce the downstream lux promoter pLux, initiating the start of stage two.

In stage two, the pLux promoter regulates the synthesis of the PHB depolymerase, encoded in the part BBa_K5165000, and the TetR repressor. The TetR repressor inhibits the upstream pTet promoter, ceasing the production of stage one proteins, focusing the cellular processes on depolymerizing the PHB molecules produced from stage one.

The results indicate a high level of BHB in the extracellular fluid of cells containing Plasmid 1 (self-sustaining system) indicating endogenously produced AHL was upregulating the expression of PHAZ_TALFU in comparison to trace amounts found the extracellular space of cells transformed with plasmid 2. Using the standard curve of BHB concentration (y = 0.4651x - 00221) we determined the average concentration of BHB in the extracellular space of cells transformed with plasmid 1 to be 1.347 umol/mL compared to just 0.138 umol/mL in the extracellular space of cells transformed with plasmid 2 (which needed to be induced for expression).

The concentration of BHB was determined using a standard curve for BHB as seen below.(y = 0.4651x - 00221)

The image below qualitative data from β-Hydroxybutyrate colorimetric assay. Tube 1 contains kit reagents + a known quantity of standard solution (2 umol/mL). Tubes 2, 3, & 4 contains kit reagents + supernatant of cell environment for BL21 cells transformed with plasmid 1, plasmid 2, and wild type (untransformed cells) respectively.

Sequence and Features