Difference between revisions of "Part:BBa K2300001"
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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. | ||
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Discuss Clark electrode and Octopus here. | Discuss Clark electrode and Octopus here. |
Revision as of 08:47, 30 October 2017
Hydrogen Gas Producing Gene Cluster
The final composite part to create hydrogen
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1792
Illegal NheI site found at 5180
Illegal NheI site found at 5390
Illegal NheI site found at 5834
Illegal NotI site found at 3584 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 4232
Illegal NgoMIV site found at 8188
Illegal AgeI site found at 5147
Illegal AgeI site found at 6790 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 6015
Illegal BsaI.rc site found at 3635
Illegal BsaI.rc site found at 3747
Biology & Literature
The Macquarie Australia iGEM team have successfully transformed E. coli (DH5α) with a hydrogenase gene cluster capable of converting glucose into hydrogen gas. This was achieved with our main bio-brick submission, the Hydrogen Gas Producing Gene Cluster.
This gene cluster translates to a complex consisting of the [FeFe] hydrogenase enzyme (Hyd1) (Mulder et al., 2011), Ferredoxin, Ferredoxin-NADP+-Reductase (FNR) and the maturation enzymes (HydEF and HydG). All gene codes were sourced from the eukaryote Chlamydomonas reinhardtii. In the original organism these enzymes represent the final step in the photosynthetic pathway utilised for energy transduction from sunlight. By transforming E. coli with this Hydrogen Gas Producing Gene Cluster, these enzymes work cohesively to convert glucose into hydrogen gas whilst avoiding the detrimental emissions formed during current hydrogen gas production processes. We hope that bacteria transformed with this bio-brick will become a viable source of hydrogen and contribute to the growing number of zero emission alternative renewable fuels providing electricity to address the global energy crisis.
Part Verification
The entire Hydrogen Gas Producing Gene Cluster was sequenced and confirmed once it had been ligated together.
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.
Discuss Clark electrode and Octopus here.
Protein information
Ferredoxin
Mass: 13.0 kDa
Sequence:
MAMRSTFAARVGAKPAVRGARPASRMSCMAYKVTLKTPSGDKTIECPADTYILDAAEEAGLDLPYSCRAGACSSCAGKVAAGTVDQSDQSFLDDAQMGNGFV
LTCVAYPTSDCTIQTHQEEALY
Ferredoxin NADP+ Reductase (FNR)
Mass: 38.27 kDa
Sequence:
MQTVRAPAASGVATRVAGRRMCRPVAATKASTAVTTDMSKRTVPTKLEEGEMPLNTYSNKAPFKAKVRSVEKITGPKATGETCHIIIETEGKIPFWEGQSYGVIPP
GTKINSKGKEVPHGTRLYSIASSRYGDDFDGQTASLCVRRAVYVDPETGKEDPAKKGLCSNFLCDATPGTEISMTGPTGKVLLLPADANAPLICVATGTGIAPFRS
FWRRCFIENVPSYKFTGLFWLFMGVANSDAKLYDEELQAIAKAYPGQFRLDYALSREQNNRKGGKMYIQDKVEEYADEIFDLLDNGAHMYFCGLKGMMPGIQD
MLERVAKEKGLNYEEWVEGLKHKNQWHVEVY
Hyd1
Mass: 53.13 kDa
Sequence:
MSALVLKPCAAVSIRGSSCRARQVAPRAPLAASTVRVALATLEAPARRLGNVACAAAAPAAEAPLSHVQQALAELAKPKDDPTRKHVCVQVAPAVRVAIAETLGLAPGATT
PKQLAEGLRRLGFDEVFDTLFGADLTIMEEGSELLHRLTEHLEAHPHSDEPLPMFTSCCPGWIAMLEKSYPDLIPYVSSCKSPQMMLAAMVKSYLAEKKGIAPKDMVMV
SIMPCTRKQSEADRDWFCVDADPTLRQLDHVITTVELGNIFKERGINLAELPEGEWDNPMGVGSGAGVLFGTTGGVMEAALRTAYELFTGTPLPRLSLSEVRGMDGIKET
NITMVPAPGSKFEELLKHRAAARAEAAAHGTPGPLAWDGGAGFTSEDGRGGITLRVAVANGLGNAKKLITKMQAGEAKYDFVEIMACPAGCVGGGGQPRSTDKAITQKR
QAALYNLDEKSTLRRSHENPSIRELYDTYLGEPLGHKAHELLHTHYVAGGVEEKDEKK
HydEF
Mass: 121.95 kDa
Sequence:
MAHSLSAHSRQAGDRKLGAGAASSRPSCPSRRIVRVAAHASASKATPDVPVDDLPPAHARAAVAAANRRARAMASAEAAAETLGDFLGLGKGGLSP
GATANLDREQVLGVLEAVWRRGDLNLERALYSHANAVTNKYCGGGVYYRGLVEFSNICQNDCSYCGIRNNQKEVWRYTMPVEEVVEVAKWALENGI
RNIMLQGGELKTEQRLAYLEACVRAIREETTQLDLEMRARAASTTTAEAAASAQADAEAKRGEPELGVVVSLSVGELPMEQYERLFRAGARRYLIRIET
SNPDLYAALHPEPMSWHARVECLRNLKKAGYMLGTGVMVGLPGQTLHDLAGDVMFFRDIKADMIGMGPFITQPGTPATDKWTALYPNANKNSHMK
SMFDLTTAMNALVRITMGNVNISATTALQAIIPTGREIALERGANVVMPILTPTQYRESYQLYEGKPCITDTAVQCRRCLDMRLHSVGKTSAAGVWGDPA
SFLHPIVGVPVPHDLSSPALAAAASADFHEVGAGPWNPIRLERLVEVPDRYPDPDNHGRKKAGAGKGGKAHDSHDDGDHDDHHHHHGAAPAGAAA
GKGTGAAAIGGGAGASRQRVAGAAAASARLCAGARRAGRVVASPLRPAAACRGVAVKAAAAAAGEDAGAGTSGVGSNIVTSPGIASTTAHGVPRINI
GVFGVMNAGKSTLVNALAQQEACIVDSTPGTTADVKTVLLELHALGPAKLLDTAGLDEVGGLGDKKRRKALNTLKECDVAVLVVDTDTAAAAIKSGRLA
EALEWESKVMEQAHKYNVSPVLLLNVKSRGLPEAQAASMLEAVAGMLDPSKQIPRMSLDLASTPLHERSTITSAFVKEGAVRSSRYGAPLPGCLPRW
SLGRNARLLMVIPMDAETPGGRLLRPQAQVMEEAIRHWATVLSVRLDLDAARGKLGPEACEMERQRFDGVIAMMERNDGPTLVVTDSQAIDVVHPW
TLDRSSGRPLVPITTFSIAMAYQQNGGRLDPFVEGLEALETLQDGDRVLISEACNHNRITSACNDIGMVQIPNKLEAALGGKKLQIEHAFGREFPELESG
GMDGLKLAIHCGGCMIDAQKMQQRMKDLHEAGVPVTNYGVFFSWAAWPDALRRALEPWGVEPPVGTPATPAAAPATAASGV
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. & PETERS, J. W. 2011. Insights into [FeFe]-hydrogenase structure, mechanism, and maturation. Structure, 19, 1038-1052.