Part:BBa_E1010

**highly** engineered mutant of red fluorescent protein from Discosoma striata (coral)

monomeric RFP: Red Fluorescent Protein. Excitation peak: 584 nm Emission peak: 607 nm

Usage and Biology

Robert E. Campbell started with Discosoma RFP (DsRed) and evolved a faster folding, monomeric variant. See paper listed in source. Codon optimized for expression in bacteria (?? DE)

iGEM11_Uppsala-Sweden: Expression of chromoproteins. The images above show E coli constitutively expressing amilCP BBa_K592009 (blue), amilGFP BBa_K592010 (yellow) and RFP BBa_E1010 (red).

Peking iGEM 2016 has fused this part with triple spytag. The fused protein is participate in Peking’s polymer network. By adding this protein, the whole polymer network become visible in most conditions. If you want to learn more about Peking’s polymer network and the role of mRFP in this network, please click here https://parts.igem.org/Part:BBa_K1989004".

Contribution

Group: Hong Kong-CUHK iGEM 2017
Author: Yuet Ching Lin
Summary: We measured the fluorescent signal of mRFP in buffers with different pH.
Documentation:
Charaterization of mRFP pH stabillity:

We grew C41 bacteria with parts BBa_J61002 in 2XYT for 24 hours. After purifying the mRFP by Ion Exchange Chromatography and Hydrophobic Interaction Chromatography, we measured the fluoresece (ex ,em ) of purified mRFP, which is diluted to 10µg/100µl (total 200µl) in triplicates, into different buffers (ranges from pH2 to pH12; Volume of mRFP:buffer = 1:1.8). To facilitate reproducibility of the experiment, we correlated the relative fluorescent intensity to an absolute fluorophore concentration by referring it to a standard curve of the fluorophores(Rhodamine) using the interlab study protocol. The result shows that the stability drops dramatically in pH condition below 6 and relatively stable in pH 6-10.

Fig.1 Vary pH attributed to different fluorescent intensity of RFP.

Table 1 Plate reader setting of fluorescent measurement

Measurement Type	Fluorescence
Microplate name	COSTAR 96
Scan mode	orbital averaging
Scan diameter [nm]	3
Excitation	550-20
Emission	605-40
Dichronic filter	auto 572.5
Gain	500
Focal height [nm]	9

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Parts table Protein data table for BioBrick BBa_E1010 automatically created by the BioBrick-AutoAnnotator version 1.0 Nucleotide sequence in RFC 10: (underlined part encodes the protein)  ATGGCTTCC ... ACCGGTGCTTAATAACGCTGATAGTGCTAGTGTAGATCGC  ORF from nucleotide position 1 to 675 (excluding stop-codon) Amino acid sequence: (RFC 25 scars in shown in bold, other sequence features underlined; both given below) 1  101  201  MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKMRLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLD ITSHNEDYTIVEQYERAEGRHSTGA* Sequence features: (with their position in the amino acid sequence, see the list of supported features) None of the supported features appeared in the sequence Amino acid composition: Ala (A) 12 (5.3%) Arg (R) 9 (4.0%) Asn (N) 4 (1.8%) Asp (D) 14 (6.2%) Cys (C) 0 (0.0%) Gln (Q) 8 (3.6%) Glu (E) 22 (9.8%) Gly (G) 23 (10.2%) His (H) 5 (2.2%) Ile (I) 9 (4.0%) Leu (L) 12 (5.3%) Lys (K) 22 (9.8%) Met (M) 9 (4.0%) Phe (F) 10 (4.4%) Pro (P) 12 (5.3%) Ser (S) 12 (5.3%) Thr (T) 14 (6.2%) Trp (W) 3 (1.3%) Tyr (Y) 11 (4.9%) Val (V) 14 (6.2%) Amino acid counting Total number: 225 Positively charged (Arg+Lys): 31 (13.8%) Negatively charged (Asp+Glu): 36 (16.0%) Aromatic (Phe+His+Try+Tyr): 29 (12.9%) Biochemical parameters Atomic composition: C1135H1749N299O347S9 Molecular mass [Da]: 25423.7 Theoretical pI: 5.65 Extinction coefficient at 280 nm [M-1 cm-1]: 32890 / 32890 (all Cys red/ox) Plot for hydrophobicity, charge, predicted secondary structure, solvent accessability, transmembrane helices and disulfid bridges  Codon usage Organism: E. coli B. subtilis S. cerevisiae A. thaliana P. patens Mammals Codon quality (CAI): excellent (0.84) good (0.72) good (0.68) good (0.74) good (0.78) good (0.71) Alignments (obtained from PredictProtein.org) SwissProt: Q9U6Y8 (86% identity on 221 AAs), P83690 (63% identity on 215 AAs) TrEML: Q5S3G8 (95% identity on 225 AAs), D0VWW2 (94% identity on 220 AAs) PDB: 2h5q (94% identity on 216 AAs), 2qlg (94% identity on 215 AAs) Predictions (obtained from PredictProtein.org) Subcellular Localization (reliability in brackets) Archaea: secreted (100%) Bacteria: cytosol (52%) Eukarya: cytosol (20%) Gene Ontology (reliability in brackets) Molecular Function Ontology: - Biological Process Ontology: GO:0018298 (40%), GO:0008218 (27%)   Predicted features: Disulfid bridges:  - Transmembrane helices:  - The BioBrick-AutoAnnotator was created by TU-Munich 2013 iGEM team. For more information please see the documentation. If you have any questions, comments or suggestions, please leave us a comment. } Contribution2 Group: iGEM17_CSMU_NCHU_Taiwan 2017 Author: SHAO-CHI LO Summary: We add a His Tag at the end. Therefore, a fusion protein with this part may have red color and the ability to be purified easily. For more, we use the IDT Codon Optimization Tool(http://sg.idtdna.com/CodonOpt) to optimize the sequence and make it more suit in E.coli K12 Documentation: 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 INCOMPATIBLE WITH RFC[25] Illegal AgeI site found at 606 1000 COMPATIBLE WITH RFC[1000]