Engineering a palette of eukaryotic chromoproteins for bacterial synthetic biology.

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.

Effects of GABA agonists on body temperature regulation in GABA(B(1))-/- mice.

1. Activation of GABA(B) receptors evokes hypothermia in wildtype (GABA(B(1))+/+) but not in GABA(B) receptor knockout (GABA(B(1))-/-) mice. The aim of the present study was to determine the hypothermic and behavioural effects of the putative GABA(B) receptor agonist gamma-hydroxybutyrate (GHB), and of the GABA(A) receptor agonist muscimol. In addition, basal body temperature was determined in GABA(B(1))+/+, GABA(B(1))+/- and GABA(B(1))-/- mice. 2. GABA(B(1))-/- mice were generated by homologous recombination in embryonic stem cells. Correct gene targeting was assessed by Southern blotting, PCR and Western blotting. GABA(B) receptor-binding sites were quantified with radioligand binding. Measurement of body temperature was done using subcutaneous temperature-sensitive chips, and behavioural changes after drug administration were scored according to a semiquantitative scale. 3. GABA(B(1))-/- mice had a short lifespan, probably caused by generalised seizure activity. No histopathological or blood chemistry changes were seen, but the expression of GABA(B(2)) receptor protein was below the detection limit in brains from GABA(B(1))-/- mice, in the absence of changes in mRNA levels. 4. GABA(B) receptor-binding sites were absent in brain membranes from GABA(B(1))-/- mice. 5. GABA(B(1))-/- mice were hypothermic by approximately 1 degrees C compared to GABA(B(1))+/+ and GABA(B(1))+/- mice. 6. Injection of baclofen (9.6 mg kg-1) produced a large reduction in body temperature and behavioural effects in GABA(B(1))+/+ and in GABA(B(1))+/- mice, but GABA(B(1))-/- mice were unaffected. The same pattern was seen after administration of GHB (400 mg kg-1). The GABA(A) receptor agonist muscimol (2 mg kg-1), on the other hand, produced a more pronounced hypothermia in GABA(B(1))-/-mice. In GABA(B(1))+/+ and GABA(B(1))+/- mice, muscimol induced sedation and reduced locomotor activity. However, when given to GABA(B(1))-/- mice, muscimol triggered periods of intense jumping and wild running. 7. It is concluded that hypothermia should be added to the characteristics of the GABAB(1)-/-phenotype. Using this model, GHB was shown to be a selective GABAB receptor agonist. In addition, GABAB(1)-/- mice are hypersensitive to GABAA receptor stimulation, indicating that GABAB tone normally balances GABAA-mediated effects.

Effects of repeated administration of baclofen to rats on GABAB receptor binding sites and subunit expression in the brain.

Repeated stimulation of the GABAB receptor with baclofen frequently produces tolerance, the underlying mechanisms of which are poorly understood. The purpose of the present work was to determine whether repeated administration of baclofen to rats is accompanied by changes in cerebral GABAB receptor binding sites, mRNA for the subunits GABAB(1) and GABAB(2), and protein levels for these subunits. Rats were injected with placebo or baclofen (20 micromol/kg subcutaneously) once daily for 14 days. Decreases in body temperature were measured as an index of pharmacological effects of baclofen. Binding of radiolabeled GABA to GABAB receptors was quantitated in brain membranes, mRNA levels were determined using quantitative real-time PCR, and GABAB receptor protein levels were assessed with Western blot analysis. Baclofen caused a decline in temperature amounting to approximately 2.5 degrees C after the first dose. This effect was partly lost after the fifth and abolished after the seventh injection. Despite the complete development of tolerance, there were no significant alterations in GABAB receptor binding sites (number or affinity) or mRNA levels for the subtypes GABAB(1a), GABAB(1b), or GABAB(2). Receptor protein levels were also unchanged. It is concluded that baclofen induces tolerance through mechanisms other than down-regulation of GABAB receptor transcription or translation.