Part:BBa_E0040

green fluorescent protein derived from jellyfish Aequeora victoria wild-type GFP (SwissProt: P42212

GFP (mut3b) [note that this part does not have a barcode]

Usage and Biology

Untagged version of gfp from Repressilator reporter. See the design page for more source information.

The original citation for GFPmut3b is as follows:

Cormack, B.P., Valdivia, R.H., and S. Falkow. FACS-optimized mutants of green fluorescent protein (GFP). Gene 173: 33-38 (1996).

Here's the link: http://www.sciencedirect.com/science/article/pii/0378111995006850

Fluorescence wavelengths

Cormack et al.Cormack report the following excitation and emission data for GFPmut3 -

• Excitation max - 501nm
• Emission max - 511nm

Latency

Cormack et al.Cormack report detectable fluorescence within 8 mins. Please add maturation time data for E0040 here.

References

<biblio>

1. Cormack pmid=10659856

</biblio>

Part Characteristics in Cell-Free Chassis

Parameter	Value and Description
Calibration	A conversion factor of 79.429 from Au to concentraion in nM
Half-life	33 hours in the cell-free chassis, with a degradation constant of 0.0210 (in hours)

Purificaton

GFPmut3b can be purified for calibration after the addition of a his-tag. The detailed [http://2007.igem.org/Imperial/Wet_Lab/Protocols/Prot1.6 protocols]and [http://2007.igem.org/Imperial/Wet_Lab/Results/Res1.6 results]for the purification can be found.

Calibration

The fluorescence of purified GFPmut3B was calibrated in the cell-free chassis. The derived [http://2007.igem.org/Imperial/Wet_Lab/Results/Res1.3 calilbration curve]allows the determination of the concentration of GFPmut3b in the cell-free chassis. [http://2007.igem.org/Imperial/Wet_Lab/Protocols/Prot1.3 Detailed protocols]for generating the calibration curve are available. Other calibration curves for are also available on the results page.

Calibration curve of concentration of GFP against fluorescence. The conversion factor is 79.429.

Degradation

The degradation of GFPmut3B in the cell-free chassis was also characterized. Purified GFPmut3B was allowed to degrade in the cell-free chassis and the fluorescence was measured over time. [http://2007.igem.org/Imperial/Wet_Lab/Protocols/Prot1.4 Detailed protocols]and [http://2007.igem.org/Imperial/Wet_Lab/Results/Res1.4 results]are attached.

Degradation curve of different GFP concentrations.

Semi-log plot of GFP degradation at different GFP concentrations

From the semi-log plot, the degradation constant (in minutes) was derived to be 0.0003501, which is equivalent to GFPmut3b having a half-life of 33 hours in the cell-free chassis.

Expression of GFP-mut3b in cell lysate

Cell-free GFP-mut3b synthesis was analyzed in self-made E.Coli lysate from strain BL21(DE3). Fluorescence was measured at 37°C for five hours on a plate reader. For details on how the lysate and the energy solution were made and which components went into the final reaction volume of 10uL, check out our [http://2017.igem.org/Team:EPFL/Protocols protocols]. Shown are three repeats with a negative control as well as a shaded error graph (control was subtracted) summarizing the result. GFP-mut3b expression yields high signals in lysate and thus is a good choice of reporter while working on a cell-free chassis. Saturation occurs after about five hours.

Three repeats and control of GFP-mut3b expression in cell lysate.

Shaded error graph of GFP-mut3b expression in cell lysate (control was subtracted).

Improvements

• Chlamydomonas reinhardtii chloroplast optimised: BBa_K2148009
• Yeast- and FACS optimized GFP: BBa_K194001
• Yeast- and FACS optimized, fast degradable GFP: BBa_K194002
• RFC[1000] compatible GFP: BBa_K2294444

Protein data table for BioBrick BBa_E0040 automatically created by the BioBrick-AutoAnnotator version 1.0
Nucleotide sequence in RFC 10: (underlined part encodes the protein)  ATGCGTAAA ... CTATACAAATAATAA  ORF from nucleotide position 1 to 714 (excluding stop-codon)
Amino acid sequence: (RFC 25 scars in shown in bold, other sequence features underlined; both given below) 1  101  201  MRKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFF KDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHY LSTQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYK*
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) 9 (3.8%) Arg (R) 7 (2.9%) Asn (N) 13 (5.5%) Asp (D) 18 (7.6%) Cys (C) 2 (0.8%) Gln (Q) 8 (3.4%) Glu (E) 16 (6.7%) Gly (G) 23 (9.7%) His (H) 10 (4.2%) Ile (I) 12 (5.0%) Leu (L) 19 (8.0%) Lys (K) 20 (8.4%) Met (M) 6 (2.5%) Phe (F) 13 (5.5%) Pro (P) 10 (4.2%) Ser (S) 8 (3.4%) Thr (T) 15 (6.3%) Trp (W) 1 (0.4%) Tyr (Y) 11 (4.6%) Val (V) 17 (7.1%)
Amino acid counting Total number: 238 Positively charged (Arg+Lys): 27 (11.3%) Negatively charged (Asp+Glu): 34 (14.3%) Aromatic (Phe+His+Try+Tyr): 35 (14.7%)	Biochemical parameters Atomic composition: C1207H1853N321O362S8 Molecular mass [Da]: 26909.4 Theoretical pI: 5.80 Extinction coefficient at 280 nm [M-1 cm-1]: 21890 / 22015 (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): good (0.72) good (0.76) good (0.75) excellent (0.82) good (0.77) good (0.68)
Alignments (obtained from PredictProtein.org) SwissProt: P42212 (98% identity on 238 AAs), Q9U6Y3 (25% identity on 217 AAs) TrEML: B6UPG7 (99% identity on 238 AAs), B9U1C3 (98% identity on 238 AAs), J8VIQ3 (98% identity on 238 AAs), Q71RY9 (98% identity on 238 AAs), Q8GHE2 (98% identity on 238 AAs) PDB: 1gfl (98% identity on 229 AAs), 3vht (98% identity on 228 AAs)
Predictions (obtained from PredictProtein.org)
Subcellular Localization (reliability in brackets) Archaea: secreted (99%) Bacteria: cytosol (62%) Eukarya: cytosol (36%)	Gene Ontology (reliability in brackets) Molecular Function Ontology: - Biological Process Ontology: GO:0018298 (45%), GO:0008218 (32%)
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.

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
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 644