Part:BBa_K2664001
Trc POR
POR part with high expression Trc promoter.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 3
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Overview
Usage and Biology
A crucial regulatory step in the biosynthesis of chlorophyll in higher plants and algae is the reduction of protochlorophyllide to chlorophyllide. POR is unique in its formation as it directly utilizes light for catalysis. POR is a major protein in the membrane of plants, without it, the chloroplasts would remain inactive. The protochlorophyllide absorbs light, excites the molecule and is reduced by NADPH to form chlorophyllide. The illumination of POR and its conversion of protochlorophyllide to chlorophyllide transforms etiolated membranes to active chloroplasts, which in turn provides energy to the plant once chlorophyll is produced. The absorption of light by the co-enzyme complex developed from the interaction of POR and NADPH induces hydrogen transfer resulting in the formation of chlorophyllide and NADP+ [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1133702/].
Figure 1: The reaction mechanism between POR and NADPH in the active site. [http://www.sciencedirect.com/science/article/pii/S136013851000155X#]
Light-dependent protochlorophyllidereductase - Light-dependent protochlorophyllidereductase, chloroplast precursor; Converts protochlorophyllide to chlorophyllide using NADPH and light as the reductant; Chlamydomonas mutant known as pc-1 has a two-nucleotide deletion within the fourth and fifth codons of this gene giving rise to a premature termination [PMID: 8616232; identical to U36752]
Protein Expression - Increasing transcription of the POR gene 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), we were confident our improved plasmid would be more efficient at producing chlorophyll.
Part Verification
Biobrick Design:
Source Genbank accession: [http://www.ncbi.nlm.nih.gov/nuccore/NW_001843471.1?report=genbank&from=693790&to=696964 NW_001843471]
Source Uniprot reference: [http://www.uniprot.org/uniprot/A8HPJ2 A8HPJ2]
cDNA gene sequence from Chlamydomonas reinhardtii was sourced from NCBI database, chloroplast targeting sequence was removed. EcoRI/XbaI/SpeI/PstI restriction sites were removed via codon adjustment, biobrick prefix and RBS were added to start of gene, biobrick suffix added to end of gene. Part was synthesised with new high expression Trc promoter in 2018.
Biobrick construction: Gibson assembly of 2 synthesised DNA fragments into BB vector.
Number of amino acids: 397
Molecular weight: 41871.0
Theoretical pI: 9.48
Amino acid composition:
Ala (A) 61 15.4%
Arg (R) 17 4.3%
Asn (N) 16 4.0%
Asp (D) 16 4.0%
Cys (C) 6 1.5%
Gln (Q) 14 3.5%
Glu (E) 13 3.3%
Gly (G) 27 6.8%
His (H) 7 1.8%
Ile (I) 12 3.0%
Leu (L) 36 9.1%
Lys (K) 25 6.3%
Met (M) 10 2.5%
Phe (F) 15 3.8%
Pro (P) 22 5.5%
Ser (S) 37 9.3%
Thr (T) 24 6.0%
Trp (W) 4 1.0%
Tyr (Y) 7 1.8%
Val (V) 28 7.1%
Pyl (O) 0 0.0%
Sec (U) 0 0.0%
(B) 0 0.0% (Z) 0 0.0% (X) 0 0.0%
Total number of negatively charged residues (Asp + Glu): 29
Total number of positively charged residues (Arg + Lys): 42
Atomic composition:
Carbon C 1849 Hydrogen H 2965 Nitrogen N 521 Oxygen O 554 Sulfur S 16
Formula: C1849H2965N521O554S16 Total number of atoms: 5905
Extinction coefficients:
Extinction coefficients are in units of M-1 cm-1, at 280 nm measured in water.
Ext. coefficient 32805 Abs 0.1% (=1 g/l) 0.783, assuming all pairs of Cys residues form cystines
Ext. coefficient 32430
Abs 0.1% (=1 g/l) 0.775, assuming all Cys residues are reduced
Estimated half-life:
The N-terminal of the sequence considered is M (Met).
The estimated half-life is: 30 hours (mammalian reticulocytes, in vitro).
>20 hours (yeast, in vivo).
>10 hours (Escherichia coli, in vivo).
Instability index:
The instability index (II) is computed to be 36.61 This classifies the protein as stable.
Aliphatic index: 82.97
Grand average of hydropathicity (GRAVY): -0.035
Figure 2: This model is based on template from pbd 1HU4. This enzyme has 85% sequence similarity with CTH1. [http://modbase.compbio.ucsf.edu/modbase-cgi/model_details.cgi?searchmode=default&displaymode=moddetail&seq_id=&model_id=93ba682ed61b336603a63529a6c3e0bc&queryfile=1413329888_4558]
Source
Chlamydomonas reinhardtii
Reference:
Darrah, P. M., et al. (1990). "Cloning and sequencing of protochlorophyllide reductase." Biochem J 265(3): 789-798.
Fujita, Y. (1996). "Protochlorophyllide reduction: a key step in the greening of plants." Plant Cell Physiol 37(4): 411-421.
Griffiths, W. T. (1975). "Characterization of the terminal stages of chlorophyll (ide) synthesis in etioplast membrane preparations." Biochem J 152(3): 623-635.
Reinbothe, C., et al. (2010). "Chlorophyll biosynthesis: spotlight on protochlorophyllide reduction." Trends in Plant Science 15(11): 614-624.
Sperling, U., et al. (1997). "Overexpression of light-dependent PORA or PORB in plants depleted of endogenous POR by far-red light enhances seedling survival in white light and protects against photooxidative damage." The Plant Journal 12(3): 649-658.
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.
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