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Coding

Part:BBa_K1351021

Designed by: Anna Sommer   Group: iGEM14_LMU-Munich   (2014-09-30)

Monomeric Red Fluorescent Protein from Discosoma striata

Mutant of the Biobrick BBa_E1010. In order to make it compatible with the Freiburg-Standard RFC25, two AgeI-Restriction sites were deleted.

monomeric RFP: Red Fluorescent Protein.

Excitation peak: 584 nm Emission peak: 607 nm


Usage and Biology

This part was created in order to enhance the Bacillus BioBrick Box by adding different fluorescent proteins to have more reporters available in B. subtilis. Since the Biobrick BBa_E1010 is already evaluated very well and shows a very bright color in E. coli, the rather light coloring in our experimental setup may be due to the used expression vector pBS0K-Pspac. This vector is optimized for the use in B. subtilis and shows a rather weak and heterogenous expression in E. coli.

LMU14 K1351021 RFP.jpg


Picture 1: BBa_K1351021 in the B. subtilis expression vector pBS0K-Pspac, cloned into E. coli. The rather weak color is due to the vector, which shows a rather weak an heterogenous expression. On the right picture, the left eppendorf tube contains a sample of E. coli containing the Biobrick BBa_K1351021, the right eppendorf tube contains E. coli with just the empty vector.

Design

This part was generated in a modified version of RFC25, where a strong Shine Dalgarno Sequence (SD) is included, and has the following prefix and suffix:

prefix with EcoRI, NotI, XbaI, SD and NgoMIV: GAATTCGCGGCCGCTTCTAGAGTAAGGAGGAGCCGGC
suffix with AgeI, SpeI, NotI and PstI: ACCGGTTAATACTAGTAGCGGCCGCTGCAG

Sites of restriction enzymes generating compatible overhangs have the same color:

EcoRI and PstI in blue, NotI in green, XbaI and SpeI in red, NgoMIV and AgeI in orange. Shine-Dalgarno sequence and stop codons are underlined.

This part is used in the 2014 LMU-Munich iGEM project BaKillus.


BioBrick AutoAnnotator

Protein data table for BioBrick BBa_ automatically created by the BioBrick-AutoAnnotator version 1.0
Nucleotide sequence in RFC 25, so ATGGCCGGC and ACCGGT were added (in italics) to the 5' and 3' ends: (underlined part encodes the protein)
 ATGGCCGGCATGGCTTCC ... ACCTGTGCTACCGGT
 ORF from nucleotide position -8 to 681 (excluding stop-codon)
Amino acid sequence: (RFC 25 scars in shown in bold, other sequence features underlined; both given below)

101 
201 
MAGMASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVM
NFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKMRLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDI
KLDITSHNEDYTIVEQYERAEGRHSTCATG*
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)13 (5.7%)
Arg (R)9 (3.9%)
Asn (N)4 (1.7%)
Asp (D)14 (6.1%)
Cys (C)1 (0.4%)
Gln (Q)8 (3.5%)
Glu (E)22 (9.6%)
Gly (G)24 (10.4%)
His (H)5 (2.2%)
Ile (I)9 (3.9%)
Leu (L)12 (5.2%)
Lys (K)22 (9.6%)
Met (M)10 (4.3%)
Phe (F)10 (4.3%)
Pro (P)12 (5.2%)
Ser (S)12 (5.2%)
Thr (T)15 (6.5%)
Trp (W)3 (1.3%)
Tyr (Y)11 (4.8%)
Val (V)14 (6.1%)
Amino acid counting
Total number:230
Positively charged (Arg+Lys):31 (13.5%)
Negatively charged (Asp+Glu):36 (15.7%)
Aromatic (Phe+His+Try+Tyr):29 (12.6%)
Biochemical parameters
Atomic composition:C1152H1778N304O353S11
Molecular mass [Da]:25887.3
Theoretical pI:5.65
Extinction coefficient at 280 nm [M-1 cm-1]:32890 / 32953 (all Cys red/ox)
Plot for hydrophobicity, charge, predicted secondary structure, solvent accessability, transmembrane helices and disulfid bridges 
Codon usage
Organism:E. coliB. subtilisS. cerevisiaeA. thalianaP. patensMammals
Codon quality (CAI):excellent (0.84)good (0.72)good (0.68)good (0.74)good (0.79)good (0.72)
Alignments (obtained from PredictProtein.org)
   There were no alignments for this protein in the data base. The BLAST search was initialized and should be ready in a few hours.
Predictions (obtained from PredictProtein.org)
   There were no predictions for this protein in the data base. The prediction was initialized and should be ready in a few hours.
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.


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]


[edit]
Categories
//cds/reporter/rfp
//chassis/prokaryote/bsubtilis
//function/reporter/fluorescence
Parameters
colorRed