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. 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. 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.

Two of the many goals of synthetic biology are synthesizing large biochemical systems and simplifying their assembly. While several genes have been assembled together by modular idempotent cloning, it is unclear if such simplified strategies scale to very large constructs for expression and purification of whole pathways. Here we synthesize from oligodeoxyribonucleotides a completely de-novo-designed, 58-kb multigene DNA. This BioBrick plasmid insert encodes 30 of the 31 translation factors of the PURE translation system, each His-tagged and in separate transcription cistrons. Dividing the insert between three high-copy expression plasmids enables the bulk purification of the aminoacyl-tRNA synthetases and translation factors necessary for affordable, scalable reconstitution of an in vitro transcription and translation system, PURE 3.0.