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J Biol Eng. 2018 May 10;12:8. doi: 10.1186/s13036-018-0100-0. eCollection 2018.

Engineering a palette of eukaryotic chromoproteins for bacterial synthetic biology.

Author information

1
1Department of Cell and Molecular Biology, Uppsala University, Uppsala, Sweden.
2
2iGEM Uppsala, Uppsala University, Uppsala, Sweden.
3
3Biology Education Centre at Uppsala University, Uppsala, Sweden.
4
4Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden.
5
5Science for Life Laboratory, Uppsala University, Uppsala, Sweden.

Abstract

Background:

Coral reefs are colored by eukaryotic chromoproteins (CPs) that are homologous to green fluorescent protein. CPs differ from fluorescent proteins (FPs) by intensely absorbing visible light to give strong colors in ambient light. This endows CPs with certain advantages over FPs, such as instrument-free detection uncomplicated by ultra-violet light damage or background fluorescence, efficient Förster resonance energy transfer (FRET) quenching, and photoacoustic imaging. Thus, CPs have found utility as genetic markers and in teaching, and are attractive for potential cell biosensor applications in the field. Most near-term applications of CPs require expression in a different domain of life: bacteria. However, it is unclear which of the eukaryotic CP genes might be suitable and how best to assay them.

Results:

Here, taking advantage of codon optimization programs in 12 cases, we engineered 14 CP sequences (meffRed, eforRed, asPink, spisPink, scOrange, fwYellow, amilGFP, amajLime, cjBlue, meffBlue, aeBlue, amilCP, tsPurple and gfasPurple) into a palette of Escherichia coli BioBrick plasmids. BioBricks comply with synthetic biology's most widely used, simplified, cloning standard. Differences in color intensities, maturation times and fitness costs of expression were compared under the same conditions, and visible readout of gene expression was quantitated. A surprisingly large variation in cellular fitness costs was found, resulting in loss of color in some overnight liquid cultures of certain high-copy-plasmid-borne CPs, and cautioning the use of multiple CPs as markers in competition assays. We solved these two problems by integrating pairs of these genes into the chromosome and by engineering versions of the same CP with very different colors.

Conclusion:

Availability of 14 engineered CP genes compared in E. coli, together with chromosomal mutants suitable for competition assays, should simplify and expand CP study and applications. There was no single plasmid-borne CP that combined all of the most desirable features of intense color, fast maturation and low fitness cost, so this study should help direct future engineering efforts.

KEYWORDS:

BioBrick; Chromoprotein; Coral; Escherichia coli; Fitness cost; Fluorescent protein; Genetic marker; Integration; Reporter gene; iGEM

Conflict of interest statement

Not applicableThe authors declare that they have no competing interests.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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