Coding
GFP

Part:BBa_E0040

Designed by: jcbraff   Group: Antiquity   (2004-09-30)

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. report the following excitation and emission data for GFPmut3 -

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

Latency

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

References


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 protocolsand resultsfor the purification can be found.

Calibration

The fluorescence of purified GFPmut3B was calibrated in the cell-free chassis. The derived calilbration curveallows the determination of the concentration of GFPmut3b in the cell-free chassis. Detailed protocolsfor 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. Detailed protocolsand resultsare 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.

Improvements

Yeast- and FACS optimized GFP: BBa_K194001 Yeast- and FACS optimized, fast degradable GFP: BBa_K194002

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)

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. coliB. subtilisS. cerevisiaeA. thalianaP. patensMammals
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: -
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[edit]
Categories
//chassis/prokaryote/bsubtilis
//chassis/prokaryote/ecoli
//function/reporter
//function/reporter/fluorescence
Parameters
colorGreen
directionForward
emission
emit511
excitation
excite501
kegg
lum
proteinGFPmut3b
swisspro
tagNone