Difference between revisions of "Part:BBa K2300002"
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<html><center><img src="https://static.igem.org/mediawiki/parts/f/f2/Show_gel_for_all_genes.png" alt="HydrogenProduction" height="45%"width="70%"></center></html> | <html><center><img src="https://static.igem.org/mediawiki/parts/f/f2/Show_gel_for_all_genes.png" alt="HydrogenProduction" height="45%"width="70%"></center></html> | ||
| − | <b>Fig 1.</b> Agarose gel (1%) electrophoresis of single (EcoRI) and double (Eco-RI with PstI) digests of parts. | + | <center><b>Fig 1.</b> Agarose gel (1%) electrophoresis of single (EcoRI) and double (Eco-RI with PstI) digests of parts. |
Left: Lane 1 contains a 1kb ladder. Lanes 2 and 3 show single (~10,700bp) and double (~8700bp with ~2000bp) digests respectively of the composite Hydrogen Gas Producing Gene Cluster plasmid (HGPGC). Lanes 4 and 5 show single (~7400bp) and double (faint ~5400bp with ~2000bp) digests of hydEFG. Lanes 6 and 7 show single (~5400bp) and double digests (~3400bp with ~2000bp) of fer/hyd1. | Left: Lane 1 contains a 1kb ladder. Lanes 2 and 3 show single (~10,700bp) and double (~8700bp with ~2000bp) digests respectively of the composite Hydrogen Gas Producing Gene Cluster plasmid (HGPGC). Lanes 4 and 5 show single (~7400bp) and double (faint ~5400bp with ~2000bp) digests of hydEFG. Lanes 6 and 7 show single (~5400bp) and double digests (~3400bp with ~2000bp) of fer/hyd1. | ||
| − | Right: Lane 1 contains a 1kb ladder. Lanes 2 and 3 show double digests (~1900bp with ~2000bp) and single digest (~3900bp) of hydG. | + | Right: Lane 1 contains a 1kb ladder. Lanes 2 and 3 show double digests (~1900bp with ~2000bp) and single digest (~3900bp) of hydG.</center> |
===Protein information=== | ===Protein information=== | ||
Revision as of 02:25, 1 November 2017
HydG (2017)
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1327
- 1000COMPATIBLE WITH RFC[1000]
Overview
The original HydG (BBa_K1998010) (https://parts.igem.org/Part:BBa_K1998010) was not submitted in 2016 as the DNA sequence had a mutation present. HydG was re-synthesised by Macquarie iGEM 2017. HydG is a radical S-adenosyl methionine (SAM) enzyme which functions as part of the [FeFe] hydrogenase maturation enzyme complex. All organisms with [FeFe] hydrogenase contain homologues of this gene (Mulder et al., 2010).
Biology & Literature
Part Verification
To confirm the efficacy of the ribosome binding sites in our parts we used the Salis Lab Ribosome Binding Site calculator from Penn State University. The results from this were that our ribosome binding site had a translation initiation rate of 1324.3.
Left: Lane 1 contains a 1kb ladder. Lanes 2 and 3 show single (~10,700bp) and double (~8700bp with ~2000bp) digests respectively of the composite Hydrogen Gas Producing Gene Cluster plasmid (HGPGC). Lanes 4 and 5 show single (~7400bp) and double (faint ~5400bp with ~2000bp) digests of hydEFG. Lanes 6 and 7 show single (~5400bp) and double digests (~3400bp with ~2000bp) of fer/hyd1.
Right: Lane 1 contains a 1kb ladder. Lanes 2 and 3 show double digests (~1900bp with ~2000bp) and single digest (~3900bp) of hydG.Protein information
HydG
Mass: 63.74 kDa
Sequence:
MSVPLQCNAGRLLAGQRPCGVRARLNRRVCVPVTAHGKASATREYAGDFLPGTTISHAWSVERETHHRYRNPAEWINEAA
IHKALETSKADAQDAGRVREILAKAKEKAFVTEHAPVNAESKSEFVQGLTLEECATLINVDSNNVELMNEIFDTALAIKE
RIYGNRVVLFAPLYIANHCMNTCTYCAFRSANKGMERSILTDDDLREEVAALQRQGHRRILALTGEHPKYTFDNFLHAVN
VIASVKTEPEGSIRRINVEIPPLSVSDMRRLKNTDSVGTFVLFQETYHRDTFKVMHPSGPKSDFDFRVLTQDRAMRAGLD
DVGIGALFGLYDYRYEVCAMLMHSEHLEREYNAGPHTISVPRMRPADGSELSIAPPYPVNDADFMKLVAVLRIAVPYTGM
ILSTRESPEMRSALLKCGMSQMSAGSRTDVGAYHKDHTLSTEANLSKLAGQFTLQDERPTNEIVKWLMEEGYVPSWCTAC
YRQGRTGEDFMNICKAGDIHDFCHPNSLLTLQEYLMDYADPDLRKKGEQVIAREMGPDASEPLSAQSRKRLERKMKQVLEGEHDVYL
References
Mulder, D.W., Shepard, E.M., Meuser, J.E., Joshi, N., King, P.W., Posewitz, M.C., Broderick, J.B. and Peters, J.W., 2011. Insights into [FeFe]-hydrogenase structure, mechanism, and maturation. Structure, 19(8), pp.1038-1052.
Posewitz, M.C., King, P.W., Smolinski, S.L., Zhang, L., Seibert, M. and Ghirardi, M.L., 2004. Discovery of two novel radical S-adenosylmethionine proteins required for the assembly of an active [Fe] hydrogenase. Journal of Biological Chemistry, 279(24), pp.25711-25720.
Vancouver