Team:UChicago/Parts
Overall Design Goals
Full FASyn Operon (7520 base pairs)
Composite part consisted of Fab individual enzymes (D, H, Z, I, F), TesA enzyme, and acpP enzyme. This composite part is also divided into 6 individual fragments, where each enzyme spans across overlapping fragments. 1 Gibson assembly is constructed by assembling all individual enzymes after they have been flag tagged; another Gibson assembly is constructed by assembling all individual fragments.
Individual genes in FASyn Operon
FabD - ACP transacylase with 2 substrates (ping pong). 3083 base pairs proceeded by ribosome binding site.
FabH - ketoacyl- ACP synthase with 2 substrates (ping pong). 3158 base pairs proceeded by ribosome binding site.
FabZ - dehydratase with 1 substrate. 2671 base pairs proceeded by ribosome binding site.
FabI - enoyl- ACP reductase with 2 substrates (sequential displacement). 2981 base pairs proceeded by ribosome binding site.
FabF - ketoacyl- ACP synthase with 2 substrates (ping pong). 3461 base pairs proceeded by ribosome binding site.
TesA - thioesterase with 1 substrate. 2831 base pairs proceeded by ribosome binding site.
acpP - Acyl carrier protein. 2447 base pairs proceeded by ribosome binding site.
Alk Operon (10,556 base pairs)
Composite part consisting of V. Fischeri LUX operon Genes ABDCEG and Chlorella variabilis NC64A Fatty Acid Photodecarboxylase gene. Gibson Assembly is used to combine lux operon fragment, NC64A fragment and iGEM backbone.
Lux A - Encodes for the alpha subunit of the heterodimer Luciferase
Lux B - Encodes for the beta subunit of the heterodimer Luciferase
Lux D - Diverts fatty-acyl groups from Fatty Acid Biosynthesis to be used for luminescence
Lux C - Uses ATP to activate diverted fatty-acyl group in an active complex
Lux E - Reduces activated fatty-acyl complex into an Aldehyde using NADPH
Lux G - Reduces flavin mononucleotide into reduced flavin mononucleotide using NADH
Luciferase catalyzes the oxidation of the reduced flavin mononucleotide and aldehyde via oxygen consumption to produce blue light.
NC64A - Activated by blue light, the fatty acid photodecarboxylase converts long chain fatty acids into long chain alkanes.
Exporter Operon (2817 bp)
The Exporter plasmid contains the gene which encodes for the passive importer channel protein AlkL. This channel can import long chain alkanes. Other features of the plasmid include the SigA promoter and chloramphenicol resistance.
AlkL - AlkL encodes for a transmembrane beta-barrel protein which has an alkane-philic domain in its center. It naturally functions as a passive importer channel.