Difference between revisions of "Part:BBa K592011"

Revision as of 09:47, 21 October 2019

cjBlue, green chromoprotein

This chromoprotein from the Cnidopus japonicus sea anemone, cjBlue, naturally exhibits dark green color when expressed. Compared to some other chromoproteins, such as amilCP (BBa_K592009), amilGFP (BBa_K592010), spisPink (BBa_K1033932), asPink (BBa_K1033933) and aeBlue (BBa_K864401), the color development is slower. The color is readily observed in both LB or on agar plates after 24-48 hours of incubation. The sequence is codon optimized for expression in E coli.

Usage and Biology

This part is useful as a reporter.

iGEM11_Uppsala-Sweden: Expression of green chromoprotein. Escherichia coli constitutively expressing cjBlue (BBa_K592011).

iGEM12_Uppsala_University: The Uppsala chromoprotein collection and RFP. The image shows pellets of E coli expressing chromoproteins eforRed BBa_K592012, RFP BBa_E1010, cjBlue BBa_K592011, aeBlue BBa_K864401, amilGFP BBa_K592010 and amilCP BBa_K592009.

iGEM17_SJTU_BioX_Shanghai:

J23119+Target3+cjBlue BBa_K2285014
J23119+Target1+cjBlue BBa_K2285018

An improved part has been constructed. Since this part is a coding sequence, we added a RBS which is on the upstream of sfGFP BBa_K515005 and terminator BBa_B1006 by PCR. What's more, our device have a stem-loop structure(called Target) following the constitutive promoter J23119, to achieve further control of cjBlue expression.

References

[http://www.jbc.org/content/281/49/37813.abstract] Chan MC, Karasawa S, Mizuno H, Bosanac I, Ho D, Privé GG, Miyawaki A, Ikura M. (2006) Structural characterization of a blue chromoprotein and its yellow mutant from the sea anemone Cnidopus japonicus. J. Biol. Chem. 281(49). 37813-9

Sequence and Features

Structure and SWISS MODEL Homology Report

(The following information has been contributed by SVCE_Chennai 2016)

The following sections give a detailed information on this chromoprotein done by its in silico analysis.

3D Structure

Cartoon representation of cjBlue chromoprotein.

The best match during structure prediction.

Ligands -None

Plot 1

Plot 2

Plot 3

Plot 1

The area built by the circles colored in different shades of grey in the plot on the left hand side represent the QMEAN scores of the reference structures from the PDB. The model's QMEAN score is compared to the scores obtain for experimental structures of similar size (model size +/- 10%) and a Z-score is calculated. A Z-score (or standard score) is a score which is normalised to mean 0 and standard deviation 1. Thus the QMEAN Z-score directly indicates how many standard deviations the model's QMEAN score differs from expected values for experimental structures. In analogy, Z-scores are calculated for all four statistical potential terms as well as the agreement terms being part of the QMEAN score.

Plot 2

The plot in the middle shows the density plot (based on the QMEAN score) of all reference models used in the Z-score calculation. The location of the query model with respect to the background distribution is marked in red. This plot basically is a "projection" of the first plot for the given protein size. The number of reference models used in the calculation is shown at the bottom of the plot.

Plot 3

The analysis of these Z-scores of the individual terms can help identifying the geometrical features responsible for an observed large negative QMEAN Z-score. Models of low quality are expected to have strongly negative Z-scores for QMEAN but also for most of the contributing terms. Large negative values correspond to red regions in the color gradient. "Good structures" are expected to have all sliders in the light red to blue region.”

The QMEAN6 score is a composite score consisting of a linear combination of 6 terms (estimated model reliability between 0-1). The pseudo-energies of the contributing terms are given below together with their Z-scores with respect to scores obtained for high-resolution experimental structures of similar size solved by X-ray crystallography:

Ramachandran Plot- SWISS MODEL Workspace

(The following information has been contributed by SVCE_Chennai 2016)

File:Ramaplot cjblue.pdf

A score of 94% in the most favoured regions as seen in the Ramachandran plot shows that the above predicted model of cjBlue, green chromoprotein is a very good prediction.

ProtParam Results

(The following information has been contributed by SVCE_Chennai 2016)

Number of amino acids: 232
Molecular weight: 26173.80
Theoretical pI: 7.53
Total number of negatively charged residues (Asp + Glu): 24
Total number of positively charged residues (Arg + Lys): 25

Atomic composition:

Carbon (C) - 1151 Hydrogen (H) - 1776 Nitrogen (N) - 314 Oxygen (O) - 347 Sulfur (S) - 19

Formula: C₁₁₅₁H₁₇₇₆N₃₁₄O₃₄₇S₁₉
Total number of atoms: 3607

Extinction coefficients:

Extinction coefficients are in units of M^-1 cm^-1, at 280 nm measured in water.

Ext. coefficient : 28015
Abs 0.1% (=1 g/l): 1.070, assuming all pairs of Cys residues form cystines

Ext. coefficient : 27390
Abs 0.1% (=1 g/l): 1.046, 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 29.86.
This classifies the protein as stable.

Aliphatic index: 58.84

Grand average of hydropathicity (GRAVY): -0.558

===Newcastle 2019 Characterisation===