Although some strategies have been reported for the elimination of stop and redundant codons during the chemical synthesis of degenerate oligonucleotides, incorporating an expensive cocktail of 20 trimer-phosphoramidites is currently a commonly employed and straightforward approach. As an alternative option, we describe here a cheaper strategy based on standard monomer-phosphoramidites and a simplified resin-splitting procedure. The accurate division of the resin, containing the growing oligonucleotide, into four columns represents the key step in this approach. The synthesis of the degenerate codon NDT in column 1, loaded with 60% of the resin, produces 12 codons, while a degenerate codon VMA in column 2, loaded with 30% of the resin, produces 6 codons. Codons ATG and TGG, independently synthesized in columns 3 and 4, respectively, and loaded with 5% each, completes the 20 different codons. The experimental frequency of each mutant codon in the library was assessed by randomizing 12 contiguous codons that encode for amino acids located in the chromophore region of the enhanced red fluorescent protein mKate-S158A. Furthermore, randomization of three contiguous codons that encode for the amino acids Phe62, Met63, and Tyr64, which are equivalent to Phe64, Ser65, and Tyr66 in GFP, gave rise to some red and golden yellow fluorescent mutants displaying interesting phenotypes and spectroscopic properties. The absorption and emission spectra of two of these mutants also suggested that the complete maturation of the red and golden yellow chromophores in mKate proceeds via the formation of a green-type chromophore and a cyan-type chromophore, respectively.

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In this study we cloned a chitinase gene (SmchiC), from Serratia marcescens isolated from the corpse of a Diatraea magnifactella lepidopteran, which is an important sugarcane pest. The chitinase gene SmchiC amplified from the S. marcescens genome was cloned into the transformation vector p2X35SChiC and used to transform tobacco (Nicotiana tabacum L. cv Petit Havana SR1). The resistance of these transgenic plants to the necrotrophic fungus Botrytis cinerea and to the pest Spodoptera frugiperda was evaluated: both the activity of chitinase as well as the resistance against B. cinerea and S. frugiperda was significantly higher in transgenic plants compared to the wild-type.

In vitro mutagenesis methods have revolutionized biological research and the biotechnology industry. In this study, we describe a mutagenesis method based on synthesizing a gene using a complete set of forward and reverse spiked oligonucleotides that have been modified to introduce a low ratio of mutant nucleotides at each position. This novel mutagenesis scheme named “Spiked Genes” yields a library of clones with an enhanced mutation distribution due to its unbiased nucleotide incorporation. Using the far-red fluorescent protein emKate as a model, we demonstrated that Spiked Genes yields richer libraries than those obtained via enzymatic methods. We obtained a library without bias toward any nucleotide or base pair and with even mutations, transitions, and transversion frequencies. Compared with enzymatic methods, the proposed synthetic approach for the creation of gene libraries represents an improved strategy for screening protein variants and does not require a starting template.

Recombinant protein expression is one of the key issues in protein engineering and biotechnology. Among the different models for assessing protein production and structure-function studies, green fluorescent protein (GFP) is one of the preferred models because of its importance as a reporter in cellular and molecular studies. In this research we analyze the effect of codon deletions near the amino terminus of different GFP proteins on fluorescence. Our study includes Gly4 deletions in the enhanced GFP (EGFP), the red-shifted GFP and the red-shifted EGFP. The Gly4 deletion mutants and their corresponding wild-type counterparts were transcribed under the control of the T7 or Trc promoters and their expression patterns were analyzed. Different fluorescent outcomes were observed depending on the type of fluorescent gene versions. In silico analysis of the RNA secondary structures near the ribosome binding site revealed a direct relationship between their minimum free energy and GFP production. Integrative analysis of these results, including SDS-PAGE analysis, led us to conclude that the fluorescence improvement of cells expressing different versions of GFPs with Gly4 deleted is due to an enhancement of the accessibility of the ribosome binding site by reducing the stability of the RNA secondary structures at their mRNA leader regions.