Fluorescent proteins are essential reporters in cell and molecular biology. Here, we found that red-fluorescent proteins possess an alternative translation initiation site that produces a short functional protein isoform in both prokaryotes and eukaryotes. The short isoform creates significant background fluorescence that biases the outcome of expression studies. In this study, we identified the short protein isoform, traced its origin, and determined the extent of the issue within the family of red fluorescent protein. Our analysis showed that the short isoform defect of the red fluorescent protein family may affect the interpretation of many published studies. We provided a re-engineered mCherry variant that lacks background expression as an improved tool for imaging and protein expression studies.

In this study, we provide a universal approach to Gene Expression Engineering (GeneEE) for creating artificial expression systems. GeneEE leads to the generation of artificial 5ʹ regulatory sequences (ARES) consisting of promoters and 5ʹ untranslated regions. The ARES lead to successful recruitment of RNA polymerase, related sigma factors, and ribosomal proteins that result in a wide range of expression levels. We also demonstrate that by engaging native transcription regulators, GeneEE can be used to generate inducible promoters. To showcase the universality of the approach, we demonstrate that 200-nucleotide-long DNA with random composition can be used to generate functional expression systems in six bacterial species, Escherichia coli, Pseudomonas putida, Corynebacterium glutamicum, Thermus thermophilus, Streptomyces albus, Streptomyces lividans; and the eukaryote yeast Saccharomyces cerevisiae.

Fluorescent proteins are essential reporters in cell biology and molecular biology. Here, we reveal that red-fluorescent proteins possess an alternative translation initiation site that produces a short functional protein isoform. The short isoform creates significant background fluorescence that biases the outcome of expression studies. Our investigation identifies the short protein isoform, traces its origin, and determines the extent of the issue within the family of red fluorescent protein. Our analysis shows that the short isoform defect of the red fluorescent protein family may affect the interpretation of many published studies. Finally, we provide a re-engineered mCherry variant that lacks background expression as an improved tool for imaging and protein expression studies.

The chloroplast is a promising platform for biotechnological innovation due to its compact translation machinery. Nucleotide modifications within a minimal set of tRNAs modulate codon–anticodon interactions that are crucial for translation efficiency. However, a comprehensive assessment of these modifications does not presently exist in chloroplasts. Here, we synthesize all available information concerning tRNA modifications in the chloroplast and assign translation efficiency for each modified anticodon–codon pair. In addition, we perform a bioinformatics analysis that links enzymes to tRNA modifications and aminoacylation in the chloroplast of Chlamydomonas reinhardtii. This work provides the first comprehensive analysis of codon and anticodon interactions of chloroplasts and its implication for translation efficiency.

The development of antifouling coating for sensor is desirable because the biofilm can shorten sensor’s life and cause inaccurate reading. In this study, a facile one-pot reaction was used to synthesized ZnO–graphene oxide (GO) (ZnO–GO) nanocomposites. Different amount of ZnO–GO was incorporated in the polydimethylsiloxane (PDMS) matrix respectively though a simple solution mixing method, in order to create PDMS/ZnO–GO nanocomposite (PZGO). The coating was obtained directly by spin coating of PZGO/tetrahydrofuran suspension. The hydrophobicity, surface roughness (Ra), surface free-energy (SFE) and nanoscale structure were investigated as antifouling factors. Antifouling tests were performed using two marine microorganisms, the cyanobacterium Synechococcus sp. Strain PCC 7002 and the diatom Phaeodactylum tricornutum. PZGO0.2 (mass ratio of ZnO–GO to PDMS: 0.2 wt%) displayed excellent antifouling property with 8.5% of Synechococcus sp. Strain PCC 7002 biofilm coverage, while PZGO0.1 (mass ratio of ZnO–GO to PDMS: 0.1 wt%) showed 2.4% P. tricornutum biofilm coverage. The antifouling property of the synthesized PZGO nanocomposite can be attributed to its high Ra and hydrophobicity which was caused by the good dispersion of ZnO–GO in PDMS matrix. This study suggests a potential of PZGO nanocomposite for sensor’s antifouling coating, which could contribute to improve sensor’s durability relating to biofouling in future. Graphic Abstract