amilCP, blue chromoprotein

This chromoprotein from the coral Acropora millepora, amilCP, naturally exhibits strong color when expressed. The protein has an absorbance maximum at 588 nm giving it a blue/purple color visible to the naked eye, thereby requiring no instruments to observe. The strong color is readily observed in both LB or agar culture, in less than 24 hours of incubation.

This part has been adaptated to B. subtilis with a LVA tail. by the UPMC-Paris Team in 2016. (see part BBa K2180019)

Usage and Biology

This part is useful as a reporter. In the pictures below it's built into BBa_K592015.

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). Also see the green chromoprotein 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.

References

[http://www.ncbi.nlm.nih.gov/pubmed/18648549] Alieva, N. O., et al. 2008. Diversity and evolution of coral fluorescent proteins. PLoS One 3:e2680.

[1] Levy, Appelbaum, Leggat, Gothlif, Hayward, Miller, & Hoegh-Guldberg. (2007). Light-responsive cryptochromes from a simple multicellular animal, the coral Acropora millepora. Science (New York, N.Y.), 318(5849), 467-70. GenBank: AY646075.1

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]

CONTRIBUTION: Uppsala 2018

Authors iGEM Uppsala 2018, Matilda Brink, Elin Ramstrom.

The original native AmilCP sequence, Part:BBa_K592009 was codon optimized for E.coli K12 with a codon optimization tool provided by Integrated DNA Technologies (IDT). The codon optimized basic AmilCP part is attached to a constitutive promotor, RBS and a double terminator. Part:BBa_K2669003

Purpose

The Uppsala 2018 team have performed a codon optimization on the original native AmilCP basic part in order to get a chromoprotein expression that can be maintained for a longer period of time trough several generations of growth, hence making the expression of the blue chromoprotein more stable.

Method

In order to conduct a stability assay through liquid experiment, the codon optimized basic part and the original native AmilCP part was attached to a constitutive promotor, RBS and a double terminator separately. The two Biobrick parts were ordered as gBlocks from IDT inserted in pUCIDT (Amp).

Both vectors were transformed into DH5-aplha E.coli cells through single tube transformation and yielded phenotypically blue colonies for cells containing plasmid with original part and plasmid containing codon optimized part. Stability assay was perfored according to protocol [2].

Results

Figure 3: Results after 10 generations of growth. The upper row represents 10 different culture pellets of the cells with the plasmid containing the codon optimized sequence of AmilCP. The lower row represents 10 different culture pellets with the original native AmilCP (part:BBa_K592009) sequence incorporated in the plasmid.

Already after 10 generations of growth it was clearly visible that the color intensity was better distributed in the cells containing the plasmid with the codon optimized AmilCP sequence. The result confirms that the plasmid containing codon optimized part generates better maintenance of protein expression than the plasmid containing the original native part. The improved stability of expression makes this Biobrick part a good candidate as a reliable reporter or biosensor.

IMPROVEMENT REFERENCE: IONIS_PARIS 2017

The part used to improve BBa_K592009 (Group: iGEM11_Uppsala-Sweden) is : BBa_K2282006 (Group: iGEM17_IONIS-PARIS). https://parts.igem.org/Part:BBa_K2282006

IMPROVEMENT REFERENCE: Austin_UTexas 2017

The original blue chromoprotein part (BBa_K592009) was codon-optimized in order to increase its overall stability as a color reporter. The modified blue chromoprotein part is: BBa_K2253002 (Group: iGEM17_Austin_UTexas). Designed by Surta Dave.

IMPROVEMENT REFERENCE: DTU-Denmark 2017

A 6x his-tag was added to this part, to the C-terminal end of the expressed amilCP. The new part was submitted as BBa_K2355002 (Group: iGEM17_DTU-Denmark), Designed by:Philip Sørensen.

IMPROVEMENT REFERENCE: BNU-China 2017

The part was added to the end of a silk protein from honeybee. The new part is : BBa_K2220028(Group: iGEM17_BNU-China), Designed by: Jiawei Xing.

IMPROVEMENT REFERENCE: IISER Pune India 2017

The part was assembled with B0034 RBS and B0015 double terminator. The new part is : BBa_K2258003(Group: iGEM17_IISER Pune India), Designed by: Jyothish S.

IMPROVEMENT REFERENCE: Peshawar 2017

One of the most used chromoprotein used in iGEM is AmilCP blue, BBa_K592009. Because our modelling team was doing homology models for other proteins and AmilCP doesn't have a homology model on it's page, we decided to do one for AmilCP blue.

Click <a href="http://2017.igem.org/Team:Peshawar/Model" target="_blank"> here</a>to check out our modeling page!

Contribution: Valencia UPV iGEM 2018

Authors: Carolina Ropero, Adrián Requena Gutiérrez Part BBa_K2656018 is the amil Chromoprotein coding sequence BBa_K592009 compatible with both BioBricks and [http://2018.igem.org/Team:Valencia_UPV/GB3 Golden Braid 3.0] assembly methods.

In order to carry out a correct characterization of the protein and to be able to use it to make measurements of the different transcriptional units that we have assembled with it, we obtained the absorbance spectrum in the conditions of our equipment. To do so, we assembled composite part BBa_K2656113. By using this [http://2018.igem.org/Team:Valencia_UPV/Modeling#spectra protocol] with the parameters of Table 1, Figure 1 was obtained.

Parameter	Value
Number of samples	3
Wavelength measurement range (nm)	[300-700]
Table 1. Parameters used to obtain the spectum

Figure 1. amilCP absorbance spectrum

abs	588 nm
color	Blue