Coding

Part:BBa_K2300002

Designed by: Ari Edmonds   Group: iGEM17_Macquarie_Australia   (2017-10-08)


HydG (2017)

Sequence and Features

Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1327
  • 1000
    COMPATIBLE WITH RFC[1000]

Overview

The original HydG (BBa_K1998010) (http://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).

All genes within this plasmid are sequences obtained from Chlamydomonas reinhardtii and codon optimised to be expressed in Escherichia coli.

Biology & Literature

In all [FeFe] hydrogenases, the H-cluster includes a di-iron subcluster that contains azadithiolate, three CO, and two CN− ligands. During the assembly of the H cluster, the HydG radical SAM enzyme lyses the substrate tyrosine to yield CO- and CN- ligands. These diatomic products serve as a precursor for eventual H-cluster assembly (Dinis et al., 2015).

HydG may also facilitate the transfer of the intact (CO)x(CN)y synthon to its cognate acceptor for the assembly of the H-cluster (Driesener et al., 2013).

Part Verification

The entire Hydrogen Gas Producing Gene Cluster (BBa_K2300001) was sequenced and confirmed once it had been ligated together. This included the HydG part.

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.

HydrogenProduction

Fig 1. 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.

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

Dinis, P., Suess, D.L., Fox, S.J., Harmer, J.E., Driesener, R.C., De La Paz, L., Swartz, J.R., Essex, J.W., Britt, R.D. and Roach, P.L. 2015. X-ray crystallographic and EPR spectroscopic analysis of HydG, a maturase in [FeFe]-hydrogenase H-cluster assembly. Proceedings of the National Academy of Sciences, 112, 1362-1367.


Driesener, R.C., Duffus, B.R., Shepard, E.M., Bruzas, I.R., Duschene, K.S., Coleman, N.J.R., Marrison, A.P., Salvadori, E., Kay, C.W., Peters, J.W. and Broderick, J.B., 2013. Biochemical and kinetic characterization of radical S-adenosyl-L-methionine enzyme HydG. Biochemistry, 52, 8696-8707.


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


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