Part:BBa_K4427012
pSB1C3-stuffer-all
plasimd containing arof、phea、kdca、adh1
We constructed a large plasmid containing the blue light photosensitive protein EL222 gene, aroF gene, pheA (fbr) gene, KDCA gene and ADH1 gene. The aroF gene can synthesize DS, the pheA (fbr) gene can synthesize CM and PT, and the KDCA and ADH1 genes synthesize KDCA and ADH1 respectively. Upon irradiation with blue light, the LOV structural domain of the EL222 photosensitive protein changes conformation, initiating the synthesis of five catalytic enzymes in four downstream genes expressing DS, CM-PT, KDCA and ADH1. This set of plasmids allows E. coli to synthesise 2-PE using glucose.
We first transformed this plasmid into E.coli (which can be found in Parts) and first confirmed the successful transformation of the plasmid into E.coli by nucleic acid electrolysis plots, (note that thereafter all experimental manipulations were performed under shade) adding the EL222 expression inducer to the E. coli culture medium to get the system started.
DS (3-dcoxy-7-phosphoheplulonate synthas) is an important protease that controls the synthesis of aromatic amino acids by regulating enzyme activity through an allosteric mechanism. The synthesis of PEP (phosphoenolpyruvate) to DAHP (3-deoxy-D-arabino-hept-2-ulosonate 7-phosphate) can be catalysed.
CM-PT (chorismate mutase/prephenate dehydratase) is a key enzyme in the L-Phe anabolic branching pathway, catalyzing the decarboxylation, aromatization and deacylation of prephenic acid (PRE) to produce phenylpyruvate, which then undergoes a pyridoxal phosphate (PLP)-dependent transamination reaction to produce L-Phe.
KdcA (branched-chain alpha-ketoacid decarboxylase) is a ketoacid decarboxylase that converts phenylalanine PPA to PAD, which in turn converts PAD to 2-PE.
Adh1 (alcohol dehydrogenase) converts the above-mentioned PAD catalyzed by KdcA into the final product 2-PE, a geotactic process that uses the genetically catalyzed product PAD of the former enzyme, which is then converted to the final product 2-PE by Adh1.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal suffix found in sequence at 7094
Illegal EcoRI site found at 1
Illegal XbaI site found at 16
Illegal PstI site found at 2400 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1
Illegal SpeI site found at 7095
Illegal PstI site found at 2400
Illegal PstI site found at 7109
Illegal NotI site found at 7
Illegal NotI site found at 7102 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1
Illegal BamHI site found at 1165
Illegal BamHI site found at 7017
Illegal XhoI site found at 1743
Illegal XhoI site found at 7026
Illegal XhoI site found at 8126
Illegal XhoI site found at 9018 - 23INCOMPATIBLE WITH RFC[23]Illegal prefix found in sequence at 1
Illegal suffix found in sequence at 7095
Illegal PstI site found at 2400 - 25INCOMPATIBLE WITH RFC[25]Illegal prefix found in sequence at 1
Illegal XbaI site found at 16
Illegal SpeI site found at 7095
Illegal PstI site found at 2400
Illegal PstI site found at 7109
Illegal NgoMIV site found at 1313
Illegal AgeI site found at 1000 - 1000COMPATIBLE WITH RFC[1000]
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