Difference between revisions of "Part:BBa K1998011"

Revision as of 07:31, 30 October 2017

Ferredoxin-FNR

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
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

Overview

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 in vivo 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 in vitro [5].

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

Fig 1. A gel consisting of EcorI/PstI double digests for the Ferredoxin-FNR part. The expected bands for Ferredoxin-FNR (1672 bp) was observed.

Improved Characterisation

The Macquarie iGEM team of 2017 has further characterise the expression and function of the proteins in this part. The part was first induced and expressed in E. coli then purified on a Source Q column. Following the confirmation of protein expression in the collected fractions via SDS-PAGE gel a cytochrome C enzymatic assay was performed to measure the activity of the FNR enzyme.

Fig 2. Showing the cytochrome C enzymatic reduction measured at 550 nm (right Y axis) plotted against the graph of the observed fractions from the chromatography performed measured at 410 nm (left Y axis).

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

@@ Line 29: / Line 29: @@
 ===Improved Characterisation===
 The Macquarie iGEM team of 2017 has further characterise the expression and function of the proteins in this part.
+The part was first induced and expressed in <i>E. coli</i> then purified on a Source Q column. Following the confirmation of protein expression in the collected fractions via SDS-PAGE gel a cytochrome C enzymatic assay was performed to measure the activity of the FNR enzyme.
 <html><center><img src="https://static.igem.org/mediawiki/parts/e/ee/Cytc%29.png" alt="HydrogenProduction" height="45%"width="70%"></center></html>
 <b>Fig 2.</b> Showing the cytochrome C enzymatic reduction measured at 550 nm (right Y axis) plotted against the graph of the observed fractions from the chromatography performed measured at 410 nm (left Y axis).