Difference between revisions of "Part:BBa K2300001"
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===Biology & Literature=== | ===Biology & Literature=== | ||
| − | The process by which hydrogen gas is created in our E. coli requires each individual part in this HGPGC. The FNR enzyme first oxidises NADPH to NADP+ while reducing the Ferredoxin protein (both produced from BBa_K1998011). The Ferredoxin protein then donates the electron to the Hyd1 hydrogenase enzyme which using 2 protons gained from either the NADPH or the breakdown of glucose creates H2 (Decottignies et al., 1995). To do this Hyd1 requires the H-cluster which is the site of catalysis to be inserted into the protein. This is done by the maturation enzymes HydEF and HydG (Mulder et al., 2010). These maturation enzymes were assembled into a composite part by our team previously (BBa_K2300000). | + | The process by which hydrogen gas is created in our <i>E. coli</i> requires each individual part in this HGPGC. The FNR enzyme first oxidises NADPH to NADP+ while reducing the Ferredoxin protein (both produced from BBa_K1998011). The Ferredoxin protein then donates the electron to the Hyd1 hydrogenase enzyme (BBa_K1998009), which using 2 protons gained from either the NADPH, or the breakdown of glucose creates H2 (Decottignies et al., 1995). To do this Hyd1 requires the H-cluster which is the site of catalysis to be inserted into the protein. This is done by the maturation enzymes HydEF and HydG (Mulder et al., 2010). These maturation enzymes were assembled into a composite part by our team previously (BBa_K2300000). |
| − | One of the main problems with producing hydrogen gas naturally in C. reinhardtii | + | One of the main problems with producing hydrogen gas naturally in <i>C. reinhardtii</i> for industry, is that the Hyd1 hydrogenase is catalytically inactivated by oxygen which is produces naturally in the algae (Esquível et al., 2011). We are seeking to solve this through producing hydrogen gas in <i>E. coli</i> which is a facultative anaerobe. As all the genes are produced naturally in <i>C. reinhardtii</i> we chose to attempt to mimic its natural state as closely as possible in our system to ensure a greater efficiency. |
| − | A similar experiment was carried out by Agapakis et al. (2010) producing hydrogen via co-expression of an [FeFe] hydrogenase with ferredoxin and pyruvate-ferredoxin oxidoreductase (PFOR) in E. coli. Through testing combinations of hydrogenases, ferredoxins and PFOR from different sources they found the highest levels of hydrogen production were seen with the PFOR from Desulfovibrio africanus co-expressed with the hydrogenase and ferredoxin from Clostridium acetobutylicum. However, this model limited the theoretical yield to two moles hydrogen per one mole of glucose. They did not co-express the C. reinhardtii hydrogenase with the C. reinhardtii FNR as we have. Theoretical yield for hydrogen gas production from glucose is twelve molecules of hydrogen per glucose, however natural and genetically-modified microorganisms to date cannot produce hydrogen with a yield of more than four (Zhang, 2015). | + | A similar experiment was carried out by Agapakis et al. (2010) producing hydrogen via co-expression of an [FeFe] hydrogenase with ferredoxin and pyruvate-ferredoxin oxidoreductase (PFOR) in <i>E. coli</i>. Through testing combinations of hydrogenases, ferredoxins and PFOR from different sources they found the highest levels of hydrogen production were seen with the PFOR from <i>Desulfovibrio africanus</i> co-expressed with the hydrogenase and ferredoxin from <i>Clostridium acetobutylicum</i>. However, this model limited the theoretical yield to two moles hydrogen per one mole of glucose. They did not co-express the <i>C. reinhardtii</i> hydrogenase with the <i>C. reinhardtii</i> FNR as we have. Theoretical yield for hydrogen gas production from glucose is twelve molecules of hydrogen per glucose, however natural and genetically-modified microorganisms to date cannot produce hydrogen with a yield of more than four (Zhang, 2015). |
===Part Verification=== | ===Part Verification=== | ||
Revision as of 12:22, 31 October 2017
Hydrogen Gas Producing Gene Cluster
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Unknown
- 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
Overview
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.
Biology & Literature
The process by which hydrogen gas is created in our E. coli requires each individual part in this HGPGC. The FNR enzyme first oxidises NADPH to NADP+ while reducing the Ferredoxin protein (both produced from BBa_K1998011). The Ferredoxin protein then donates the electron to the Hyd1 hydrogenase enzyme (BBa_K1998009), which using 2 protons gained from either the NADPH, or the breakdown of glucose creates H2 (Decottignies et al., 1995). To do this Hyd1 requires the H-cluster which is the site of catalysis to be inserted into the protein. This is done by the maturation enzymes HydEF and HydG (Mulder et al., 2010). These maturation enzymes were assembled into a composite part by our team previously (BBa_K2300000).
One of the main problems with producing hydrogen gas naturally in C. reinhardtii for industry, is that the Hyd1 hydrogenase is catalytically inactivated by oxygen which is produces naturally in the algae (Esquível et al., 2011). We are seeking to solve this through producing hydrogen gas in E. coli which is a facultative anaerobe. As all the genes are produced naturally in C. reinhardtii we chose to attempt to mimic its natural state as closely as possible in our system to ensure a greater efficiency.
A similar experiment was carried out by Agapakis et al. (2010) producing hydrogen via co-expression of an [FeFe] hydrogenase with ferredoxin and pyruvate-ferredoxin oxidoreductase (PFOR) in E. coli. Through testing combinations of hydrogenases, ferredoxins and PFOR from different sources they found the highest levels of hydrogen production were seen with the PFOR from Desulfovibrio africanus co-expressed with the hydrogenase and ferredoxin from Clostridium acetobutylicum. However, this model limited the theoretical yield to two moles hydrogen per one mole of glucose. They did not co-express the C. reinhardtii hydrogenase with the C. reinhardtii FNR as we have. Theoretical yield for hydrogen gas production from glucose is twelve molecules of hydrogen per glucose, however natural and genetically-modified microorganisms to date cannot produce hydrogen with a yield of more than four (Zhang, 2015).
Part Verification
The entire Hydrogen Gas Producing Gene Cluster was sequenced and confirmed once it had been ligated together.
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.
Part Validation
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
Agapakis, C.M., Ducat, D.C., Boyle, P.M., Wintermute, E.H., Way, J.C. and Silver, P.A., 2010. Insulation of a synthetic hydrogen metabolism circuit in bacteria. Journal of Biological Engineering, 4(1), p.3.
DECOTTIGNIES, P., LEMARECHAL, P., JACQUOT, J.-P., SCHMITTER, J.-M. & GADAL, P. 1995. Primary structure and post-translational modification of ferredoxin-NADP reductase from Chlamydomonas reinhardtii. Archives of Biochemistry and Biophysics, 316, 249-259.
ESQUÍVEL, M. G., AMARO, H. M., PINTO, T. S., FEVEREIRO, P. S. & MALCATA, F. X. 2011. Efficient H 2 production via Chlamydomonas reinhardtii. Trends in Biotechnology, 29, 595-600.
Mulder, D.W., Boyd, E.S., Sarma, R., Lange, R.K., Endrizzi, J.A., Broderick, J.B. and Peters, J.W., 2010. Stepwise [FeFe]-hydrogenase H-cluster assembly revealed in the structure of HydA [Dgr] EFG. Nature, 465(7295), pp.248-251.
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.
Zhang, Y.H.P., 2015. Production of biofuels and biochemicals by in vitro synthetic biosystems: opportunities and challenges. Biotechnology Advances, 33(7), pp.1467-1483.