Abstract
BackgroundCoral 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.ResultsHere, 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.ConclusionAvailability 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.
Highlights
Coral reefs are colored by eukaryotic chromoproteins (CPs) that are homologous to green fluorescent protein
Recoding of CPs for expression as markers in E. coli All CP genes (Table 1), except the native amilGFP and amilCP genes, were codon optimized for plasmid-based expression in E. coli by proprietary computational programs and synthesized commercially without illegal BioBrick restriction sites
The genes encoding amilGFP and amilCP were amplified by the polymerase chain reaction (PCR) from the plasmids pGEM-T-11 and pGEM-T-14 [6] and illegal BioBrick sites were removed by mutagenesis
Summary
Coral reefs are colored by eukaryotic chromoproteins (CPs) that are homologous to green fluorescent protein. Coral reefs are colored by fluorescent proteins (FPs) and chromoproteins (CPs) that constitute a homologous eukaryotic protein family with the jellyfish green FP (GFP) [1, 2] These GFP homologs are small proteins each encoded by a single gene, comprise a relatively high percentage of soluble proteins in expressed tissues, and form their chromophore without needing cofactors or substrates other than oxygen. Bacteriophage have been engineered to cause bioluminescence of pathogenic bacteria in food, and bacteria have been engineered to fluoresce upon detection of spoiled meat gas [11], trinitrotoluene (TNT) products [12] or arsenic [13] Adaptation of these biosensors to non-fluorescent detection for use in supermarkets or the field beckons, but it is unclear which CP genes might be suitable or how best to assay them quantitatively
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