Difference between revisions of "Part:BBa S05396"
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__NOTOC__ | __NOTOC__ | ||
<partinfo>BBa_S05396 short</partinfo> | <partinfo>BBa_S05396 short</partinfo> | ||
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<!-- Add more about the biology of this part here | <!-- Add more about the biology of this part here | ||
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<partinfo>BBa_S05396 SequenceAndFeatures</partinfo> | <partinfo>BBa_S05396 SequenceAndFeatures</partinfo> | ||
+ | ===Overview=== | ||
+ | |||
+ | Fer/Hyd was created as a construction intermediate to create the final Hydrogen Gas Producing Gene Cluster (HGPGC) composite part (BBa_K2300001). Fer contains the <i>Chlamydomonas reinhardtii</i> genes encoding Ferredoxin and Ferredoxin NADP+ reductase. Hyd refers to the Hyd1 hydrogenase protein also from <i>C. reinhardtii</i>. | ||
+ | |||
+ | All genes within this plasmid are sequences obtained from <i>C. reinhardtii</i> and codon optimised to be expressed in <i>Escherichia coli</i>. | ||
+ | |||
+ | ===Biology & Literature=== | ||
+ | The process by which hydrogen gas is created in our <i>E. coli</i> requires each individual part in the HGPGC (BBa_K2300001). This construction intermediate was required in the stepwise assembly of the composite part. 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). | ||
+ | |||
+ | This construction intermediate is missing the hydrogenase maturation enzymes HydEFG (BBa_K2300000). Without this the H-cluster which is the catalytic site cannot be inserted successfully into the Hyd1 protein (Mulder et al., 2010). | ||
+ | |||
+ | ===Part Verification=== | ||
+ | The entire Hydrogen Gas Producing Gene Cluster (BBa_K2300001) was sequenced and confirmed once it had been ligated together. This included the Fer/Hyd1 construction intermediate 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. | ||
+ | |||
+ | <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> | ||
+ | <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. | ||
+ | |||
+ | 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=== | ||
+ | |||
+ | <!-- Uncomment this to enable Functional Parameter display | ||
+ | ===Functional Parameters=== | ||
+ | <partinfo>BBa_K2300001 parameters</partinfo> | ||
+ | <!-- --> | ||
+ | Ferredoxin (FDX)<br> | ||
+ | Mass: 13.0 kDa<br> | ||
+ | Sequence: <br> | ||
+ | MAMRSTFAARVGAKPAVRGARPASRMSCMAYKVTLKTPSGDKTIECPADTYILDAAEEAGLDLPYSCRAGACSSCAGKVAAGTVDQSDQSFLDDAQMGNGFV | ||
+ | LTCVAYPTSDCTIQTHQEEALY | ||
+ | <br> | ||
+ | <br> | ||
+ | Ferredoxin NADP+ Reductase (FNR)<br> | ||
+ | Mass: 38.27 kDa<br> | ||
+ | Sequence: <br> | ||
+ | MQTVRAPAASGVATRVAGRRMCRPVAATKASTAVTTDMSKRTVPTKLEEGEMPLNTYSNKAPFKAKVRSVEKITGPKATGETCHIIIETEGKIPFWEGQSYGVIPP | ||
+ | GTKINSKGKEVPHGTRLYSIASSRYGDDFDGQTASLCVRRAVYVDPETGKEDPAKKGLCSNFLCDATPGTEISMTGPTGKVLLLPADANAPLICVATGTGIAPFRS | ||
+ | FWRRCFIENVPSYKFTGLFWLFMGVANSDAKLYDEELQAIAKAYPGQFRLDYALSREQNNRKGGKMYIQDKVEEYADEIFDLLDNGAHMYFCGLKGMMPGIQD | ||
+ | MLERVAKEKGLNYEEWVEGLKHKNQWHVEVY | ||
+ | <br> | ||
+ | <br> | ||
+ | Hyd1<br> | ||
+ | Mass: 53.13 kDa<br> | ||
+ | Sequence: <br> | ||
+ | MSALVLKPCAAVSIRGSSCRARQVAPRAPLAASTVRVALATLEAPARRLGNVACAAAAPAAEAPLSHVQQALAELAKPKDDPTRKHVCVQVAPAVRVAIAETLGLAPGATT | ||
+ | PKQLAEGLRRLGFDEVFDTLFGADLTIMEEGSELLHRLTEHLEAHPHSDEPLPMFTSCCPGWIAMLEKSYPDLIPYVSSCKSPQMMLAAMVKSYLAEKKGIAPKDMVMV | ||
+ | SIMPCTRKQSEADRDWFCVDADPTLRQLDHVITTVELGNIFKERGINLAELPEGEWDNPMGVGSGAGVLFGTTGGVMEAALRTAYELFTGTPLPRLSLSEVRGMDGIKET | ||
+ | NITMVPAPGSKFEELLKHRAAARAEAAAHGTPGPLAWDGGAGFTSEDGRGGITLRVAVANGLGNAKKLITKMQAGEAKYDFVEIMACPAGCVGGGGQPRSTDKAITQKR | ||
+ | QAALYNLDEKSTLRRSHENPSIRELYDTYLGEPLGHKAHELLHTHYVAGGVEEKDEKK | ||
+ | <br> | ||
+ | |||
+ | ===References=== | ||
+ | |||
+ | Decottignies, P., Lemarechal, P., Jacquot, J.-P., Schmitter, J.-M. & Gadal, P. 1995. Primary structure and post-translational modification of ferredoxin-NADP reductase from <i>Chlamydomonas reinhardtii</i>. <i>Archives of Biochemistry and Biophysics</i>, 316, 249-259. | ||
+ | |||
+ | |||
+ | 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. <i>Nature</i>, 465(7295), pp.248-251. | ||
<!-- Uncomment this to enable Functional Parameter display | <!-- Uncomment this to enable Functional Parameter display |
Latest revision as of 00:05, 2 November 2017
FDX/FNR/Hyd1
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1792
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Overview
Fer/Hyd was created as a construction intermediate to create the final Hydrogen Gas Producing Gene Cluster (HGPGC) composite part (BBa_K2300001). Fer contains the Chlamydomonas reinhardtii genes encoding Ferredoxin and Ferredoxin NADP+ reductase. Hyd refers to the Hyd1 hydrogenase protein also from C. reinhardtii.
All genes within this plasmid are sequences obtained from C. reinhardtii and codon optimised to be expressed in Escherichia coli.
Biology & Literature
The process by which hydrogen gas is created in our E. coli requires each individual part in the HGPGC (BBa_K2300001). This construction intermediate was required in the stepwise assembly of the composite part. 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).
This construction intermediate is missing the hydrogenase maturation enzymes HydEFG (BBa_K2300000). Without this the H-cluster which is the catalytic site cannot be inserted successfully into the Hyd1 protein (Mulder et al., 2010).
Part Verification
The entire Hydrogen Gas Producing Gene Cluster (BBa_K2300001) was sequenced and confirmed once it had been ligated together. This included the Fer/Hyd1 construction intermediate 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.
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
Ferredoxin (FDX)
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
References
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.
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.