Coding

Part:BBa_K1998011

Designed by: Shauna Winchester   Group: iGEM16_Macquarie_Australia   (2016-10-13)
Revision as of 23:59, 1 November 2017 by Ari edmonds (Talk | contribs) (→‎Improved Characterisation)


Ferredoxin-FNR

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Unknown
  • 12
    INCOMPATIBLE WITH RFC[12]
    Unknown
  • 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].

HydrogenProduction

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 characterised the expression and function of the proteins in this part (http://2017.igem.org/Team:Macquarie_Australia/Improve). We first induced operon expression with 1 mM IPTG in Escherichia coli DH5α. The cells were pelleted by centrifugation at 14,000xg, resuspended in 1xPBS, then lysed by 2 rounds of French Press. Following this, anionic exchange chromatography was performed on the lysate using a Source Q column. Pronounced colour change can be observed in the fractionated proteins, as shown in Figure 1. The yellow colour changes are due to the FNR proteins tightly bound co-factor FAD (Flavin Adenine Dinucleotide) which absorbs at 400 and 450nm. The yellow-brown colour change is due to FDX iron-sulfur cluster which absorbs at 410nm.

HydrogenProduction

Figure 1. Showing the 1 mL fractions produced from the anionic exchange chromatography. Samples were labelled A, B and C with 12 fractions taken from each. The fractions pictured (A10-12, B12-6, B1 and C1) contained purified protein and are presented in order collected. The darker yellow colour corresponds to presence of expressed Ferredoxin (FDX) and FNR. B10 was the most yellow fraction, indicating highest concentration of FNR.


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. The observed activity shown in green in Figure 2 shows the FNR enzyme oxidising the NADPH to NADP+ while reducing the Ferredoxin. The electrons from the NADPH are then transported to the cytochrome C protein by the Ferredoxin protein. The reduction of cytochrome C is responsible for the observed change in absorbance.


CYTox + Ferrodoxin-NADPH -Ferredoxin NADP Reductase→ CYTred + Ferredoxin-NADP+


The fractions collected at 19 mLs and at 23 mLs were shown to be the most active in reduction of cytochrome C, as can be observed in Figure 2. This is due to the higher concentration of FNR at 19 mLs and the higher concentration of FDX at 23 mLs.

We were able to not only express and purify the Ferredoxin and FNR proteins, but we also were able to prove that the proteins were capable of the oxidation of NADPH and reduction with the use of Ferredoxin.

HydrogenProduction
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). Each assay was performed using a master mix of 5 µM Ferredoxin, 250 µM Cytochrome C and 0.2 µM NADPH. 5 µL of each fraction was added to the master mix and the change in absorbance was observed using a spectrometer at 550 nm.

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

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