Difference between revisions of "Part:BBa M36284"

(New page: 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-cistr...)
 
 
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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.
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Our device is an actuator that produces ADH1B following an RNA polymerase per second (PoPS) signal from a sensor. This actuator begins with a high-expressing bi-cistronic ribosome binding sequence (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 human ADH1B (BBa_M36280), which we have codon optimized for high expression in E. Coli. 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 (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.

Latest revision as of 03:40, 11 December 2011

Our device is an actuator that produces ADH1B following an RNA polymerase per second (PoPS) signal from a sensor. This actuator begins with a high-expressing bi-cistronic ribosome binding sequence (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 human ADH1B (BBa_M36280), which we have codon optimized for high expression in E. Coli. 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 (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.