Difference between revisions of "Part:BBa K2664002"
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===Overview===
 
===Overview===
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The ferredoxin and ferredoxin reductase enzyme which are encoded by this part makes up the ferredoxin reductase operon for testing the hydrogen production pathway of our project. Oxidised ferredoxin is reduced by ferredoxin reductase using NADPH. The reduced ferredoxin supplies electrons to hydrogenase to allow the testing for hydrogen production with the hydrogenase operon <i>in vivo</i> in the absence of PSII. In the presence of PSII we plan to enable the direct transfer of electrons to the hydrogenase from PSII, similar to the proof of concept for PSII-hydrogenase coupling demonstrated <i>in vitro</i> [5].
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FNR and FDX genes were included in the Macquarie iGEM 2018 complete Chlorophyll Biosynthesis Plasmid (https://parts.igem.org/Part:BBa_K2664008) based upon reports that they may aid chlorophyll biosynthesis (Herbst et al. 2018). The part is similar to the FER part (https://parts.igem.org/Part:BBa_K1998011) from the 2017 iGEM project used in hydrogen production. The order of the genes has been changed and the lac promoter was changed to a higher expression Trc promoter.
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(added trc promoter 2018 for higher expression)
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<html><center><img src="https://static.igem.org/mediawiki/parts/b/b5/HydrogenPathwayUpdated2016.jpeg" alt="HydrogenProduction" height="50%"width="75%"></center></html>
 
  
 
===Biology & Literature===
 
===Biology & Literature===

Revision as of 12:27, 17 October 2018


trc-FNR-Ferredoxin-Double-terminator

Ferredoxin Reductase-Ferredoxin with high expression Trc promoter and a double terminator.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Overview

FNR and FDX genes were included in the Macquarie iGEM 2018 complete Chlorophyll Biosynthesis Plasmid (https://parts.igem.org/Part:BBa_K2664008) based upon reports that they may aid chlorophyll biosynthesis (Herbst et al. 2018). The part is similar to the FER part (https://parts.igem.org/Part:BBa_K1998011) from the 2017 iGEM project used in hydrogen production. The order of the genes has been changed and the lac promoter was changed to a higher expression Trc promoter.

Biology & Literature

Ferredoxin-NADP reductase (FNR) has a mass of 36kDa and shows ferredoxin-dependent cytochrome c reduction activity [1]. This enzyme facilitates electron transfer from ferredoxin to NADP [2]. In vivo studies showed that this protein is N-trimethylated on 3 different lysines – K135, K83 and K89). This modification is thought to be involved in regulation and structural modulation since the methylated protein is smaller and more compact. Studies comparing FNR protein from Chlamydomonas reinhardtii with recombinant protein expressed in E. coli showed that the latter had a higher Km value for NADPH [3].

PETF plays a role in transfer of electrons between photosystem I and FNR [4]. It has been shown to enhance heat stress tolerance when it is overexpressed in Chlamydomonas reinhardtii by downregulating the levels of reactive oxygen species.

Part Verification

HydrogenProduction

Figure 1. Agarose gel (1%) electrophoresis with GelRed of single (E) and double (E+P) digests of submitted basic parts: trc-ChlH (4198bp), trc-FNR-fdx (1495bp) and trc-POR (1125bp).

Protein information

Ferredoxin
Mass: 13.0 kDa
Sequence:
MAMRSTFAARVGAKPAVRGARPASRMSCMAYKVTLKTPSGDKTIECPADTYILDAAEEAGLDLPYSCRAGACSSCAGKVAAGTVDQSDQSFLDDAQMGNGFV LTCVAYPTSDCTIQTHQEEALY

Ferredoxin NADP+ reductase (FNR)
Mass: 38.27 kDa
Sequence:
MQTVRAPAASGVATRVAGRRMCRPVAATKASTAVTTDMSKRTVPTKLEEGEMPLNTYSNKAPFKAKVRSVEKITGPKATGETCHIIIETEGKIPFWEGQSYGVIPP GTKINSKGKEVPHGTRLYSIASSRYGDDFDGQTASLCVRRAVYVDPETGKEDPAKKGLCSNFLCDATPGTEISMTGPTGKVLLLPADANAPLICVATGTGIAPFRS FWRRCFIENVPSYKFTGLFWLFMGVANSDAKLYDEELQAIAKAYPGQFRLDYALSREQNNRKGGKMYIQDKVEEYADEIFDLLDNGAHMYFCGLKGMMPGIQD MLERVAKEKGLNYEEWVEGLKHKNQWHVEVY

References

[1] Decottignies, P., Lemarechal, P., Jacquot, J., Schmitter, J. and Gadal, P. (1995). Primary Structure and Post-translational Modification of Ferredoxin-NADP Reductase from Chlamydomonas reinhardtii. Archives of Biochemistry and Biophysics, 316(1), pp.249-259.

[2] Rogers, W., Hodges, M., Decottignies, P., Schmitter, J., Gadal, P. and Jacquot, J. (1992). Isolation of a cDNA fragment coding for Chlamydomonas reinhardtii ferredoxin and expression of the recombinant protein in Escherichia coli. FEBS Letters, 310(3), pp.240-245.

[3] Decottignies, P., Flesch, V., Gérard-Hirne, C. and Le Maréchal, P. (2003). Role of positively charged residues in Chlamydomonas reinhardtii ferredoxin-NADP+-reductase. Plant Physiology and Biochemistry, 41(6-7), pp.637-642.

[4] Lin, Y., Pan, K., Hung, C., Huang, H., Chen, C., Feng, T. and Huang, L. (2013). Overexpression of Ferredoxin, PETF, Enhances Tolerance to Heat Stress in Chlamydomonas reinhardtii. International Journal of Molecular Sciences, 14(10), pp.20913-20929.

[5] Mersch D., Lee C-Y., Zhang J.Z., BrinkertK., Fontecilla-Camps J.C., Rutherford A.W., and Reisner E. (2015) Wiring of Photosystem II to Hydrogenase for Photoelectrochemical Water Splitting J. Am. Chem. Soc., 137 (26), pp 8541–8549