Difference between revisions of "Part:BBa K2664002"

(→‎References)
(→‎Part Verification)
 
Line 28: Line 28:
  
 
<html><center><img src="https://static.igem.org/mediawiki/parts/d/da/Basic_parts_2018_igem.jpeg" alt="HydrogenProduction" height="45%"width="70%"></center></html>
 
<html><center><img src="https://static.igem.org/mediawiki/parts/d/da/Basic_parts_2018_igem.jpeg" alt="HydrogenProduction" height="45%"width="70%"></center></html>
−
<center><b>Figure 1.</b>  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).</center>
+
<center><b>Figure 1.</b>  Agarose gel (1%) electrophoresis with GelRed of single (E) and double (E+P) digests of submitted trc-FNR-fdx (1495bp), as shown in Lanes 4 and 5, respectively.</center>
  
 
===Protein information===
 
===Protein information===

Latest revision as of 01:28, 18 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 [1]. Increasing transcription of the FNR and FDX genes would improve the efficiency of chlorophyll production. As such in 2018, we decided not to use many of the parts assembled by previous Macquarie teams. We created the second iteration of our design, in which a trc protomoter was placed before the genes. Modelling this promoter swap based on gene expression data obtained by Tegel, Ottosson, and Hober (2011) [2], we were confident our improved plasmid would be more efficient at producing chlorophyll. 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 [3]. This enzyme facilitates electron transfer from ferredoxin to NADP [4]. 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 [5].

PETF plays a role in transfer of electrons between photosystem I and FNR [6]. 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 trc-FNR-fdx (1495bp), as shown in Lanes 4 and 5, respectively.

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] Herbst, J., Girke, A., Hajirezaei, M.R., Hanke, G. and Grimm, B., 2018. Potential roles of YCF 54 and ferredoxin‐NADPH reductase for magnesium protoporphyrin monomethylester cyclase. The Plant Journal, 94(3), pp.485-496.

[2] Tegel, H., Ottosson, J. and Hober, S., 2011. Enhancing the protein production levels in Escherichia coli with a strong promoter. The FEBS journal, 278(5), pp.729-739.

[3] 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.

[4] 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.

[5] 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.

[6] 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.