Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Emma Marriott, Jessica Lam, Tatum Banayat
Date created: 2013-06-08 11:00:00
Date modified: 2015-05-08 01:14:05
Oxygen-tolerant hydrogenase (with RBS and terminator)
| Types | DnaRegion |
| Roles | engineered_region Composite |
| Sequences | BBa_M36596_sequence (Version 1) |
Description
Hydrogenase is an enzyme that catalyzes the reversible oxidation of H2. This part encodes a hydrogenase actuator taken from the large subunit of the marine bacterium Hydrogenovibrius Marinus, which has been shown to be relatively tolerant to oxygen (Kim et. Al, 2011).This part consists of a strong bicistronic design RBS (found under BioFAB ID: pFAB1655), a codon-optimized HmMBH actuator gene taken from the sequence of the large subunit of hydrogenase found in hydrogenovibrius marinus (original sequence found under F2Z6J6 at uniprot.org), and a high efficiency transcriptional terminator (found under BBa_M36281 in the Registry of Standard Biological Parts).
The mechanism of the part is relatively straightforward: Once induced with the PoPS signal, the actuator will initiate protein translation and express the hydrogenase enzyme. Our actuator was used in conjunction with a rhamnose sensor (e.g. BBa_M36284).
Notes
Considerations of the benefits and convenience of having a bicsistronic design led us to choose this option, in spite of the fact that the bicistronic design is targeted for E. Coli and has not yet been tested in S. Elongatus. Interestingly enough, although the part was originally designed for expression of the hydrogenase protein in cyanobacteria, it has so far only been proved for the expression of hydrogenase protein in E. Coli.In regards to the HmMBH actuator gene, the sequence was optimized to reduce repeats and the number of sites available to restriction enzymes.
Source
Bicistronic RBS:Mutalik VK, Guimaraes JC, Cambray G, Mai QA, Christoffersen MJ, Martin L, Yua A, Lam C, Rodriguez C, Bennett G, et al. Quantitative estimation of activity and quality for collections of functional genetic elements. Nat. Methods. 2013;10:347-353. BioFAB. (http://io.biofab.org:5000/simple/randomized_bds)
Sequence for large subunit Hydrogenase:
Nishihara H, Miyata Y, Miyashita Y, Bernhard M, Pohlmann A, et al. Analysis of the molecular species of hydrogenase in the cells of an obligately chemolithoautotrophic, marine hydrogen-oxidizing bacterium, Hydrogenovibrio marinus. Biosci Biotechnol Biochem. 2001;65:2780???2784. UnitPRO (http://www.uniprot.org/uniprot/F2Z6J6).
High efficiency transcriptional terminator:
Sunil Bodapati, Julia Joung, Daniel Esquivel-Reynoso. Part: BBa_M36281: Hard Information. Registry of Standard Biological Parts. (http://partsregistry.org/cgi/partsdb/part_info.cgi?part_name=BBa%20M36281)
| Sequence Annotation | Location | Component / Role(s) |
| BBa_M36595 Hydrogenase Actuator BBa_M36596 BBa_M36281 | 1,87 94,1926 94,1926 1935,2016 | engineered_region feature/BioBrick feature/dna sequence_feature engineered_region feature/BioBrick engineered_region feature/BioBrick |