Part:BBa_M36284

Our proposed gene sequence is an actuator that produces ADH1B following an RNA polymerase per second (PoPS) signal from a sensor. This sensor will be followed by a high-expressing bi-cistronic ribosome binding sequence provided by BIOFAB (BBa_M36282). Specifically, our group chose to use BD2 because it offers consistently high to medium expression of the gene of interest. After BD2, we have inserted the gene sequence of ADH1B (BBa_M36280), which we constructed using the protein sequence from protein database UniProt that was cross referenced with NCBI. Since we are inserting a eukaryotic gene into a prokaryote, we optimized the DNA sequence for E. Coli using a codon optimizing chart, selecting for highly transcribed genes as our objective is to produce large amounts of the enzyme. Our ADH1B gene was followed by a histidine tag consisting of 6 histidines that can be used to purify the enzyme with nickel affinity chromatography. Then we put a stop codon TAG that terminates translation. Lastly, we placed a transcription terminator, part apFAB391 provided by Biofab (BBa_M36281), that terminates transcription 99% of the time because our objective is to transcribe ADH1B so we would want to prevent transcribing other proteins on the same plasmid to conserve energy.