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GOAL: Create one construct containing the 4 enzymes necessary for carbon fixation
Pyruvate Synthase (PFOR)
Pyruvate Carboxylase (PYC)
Oxaloacetate Acetyl Hydrolase (OAH)
Acetate-CoA Ligase (ACS)
The POAP Cycle
The POAP Cycle
A synthetic carbon fixation pathway designed to convert carbon dioxide molecules into a non-toxic byproduct using engineered enzymatic steps
Sequencing Data
Sequencing Data
Plasmid map of the four-enzyme POAP construct, showing the arrangement of PYC, OAH, PFOR, and ACS enzymes, assembled via Gibson Assembly into a single vector for in vivo expression.
Assembly Method
Used Gibson Assembly to join three DNA fragments:
PYC-OAH construct
PFOR-ACS insert
Vector backbone
Enzyme Order:
Enzymes were arranged as PYC → OAH → PFOR → ACS.
OAH was placed upstream to enhance its expression, based on prior research showing it needs constant replenishment in vitro.
Challenges
Challenges
Challenges in implementing the four-enzyme construct include distinguishing construct-derived acetate consumption from background media levels, managing cellular metabolic burden, and ensuring balanced enzyme expression, all critical for validating POAP cycle function in vivo.
Made by Hayley Sperinde
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