Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Sunil Bodapati, Julia Joung
Date created: 2011-11-29 12:00:00
Date modified: 2015-05-08 01:14:04
Human Alcohol Dehydrogenase 1B actuator
| Types | DnaRegion |
| Roles | engineered_region Device |
| Sequences | BBa_M36284_sequence (Version 1) |
Description
Our proposed gene sequence is an actuator that produces ADH1B following an RNA polymerase per second (PoPS) signal from a sensor. The sensor will be followed by a bi-cistronic ribosome binding sequence provided by BIOFAB (BD2). This type of ribosome binding sequence contains a ribosome binding site followed by the DNA sequence TAATG, which terminates translation of the first part with stop codon TAA. A second ribosome can bind in the sequence and start translation of the gene with start codon ATG. The bi-cistronic ribosome binding sequence prevents the protein???s ribosome binding site from forming a hairpin loop with another part of the sequence, which would reduce the amount of translation. 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, 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 provided by Sharp et al. (1988), 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, 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.Notes
We maximized expression by using a highly expressing bicistronic RBS. We needed a way to purify the enzyme post production. The histidine tag allows us to perform nickel affinity chromatography to isolate purified protein.Source
Bicistonic RBS - BioFab: apFAB563ADH1B - partsregistry: BBa_M36666
Histidine Tag - 6 histidines
Stop Codon - generic TAG
Terminator - BIOFAB: apFAB391
| Access | Instance | Definition |
| public public public | BBa_M36282 BBa_M36280 BBa_M36281 | BBa_M36282 BBa_M36280 BBa_M36281 |
| Sequence Annotation | Location | Component / Role(s) |
| BBa_M36282 BBa_M36280 BBa_M36281 | 1,88 97,1239 1248,1329 | BBa_M36282 BBa_M36280 BBa_M36281 |