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Effects of codon bias on heterologous expression of fluorescent proteins in Xylella fastidiosa

Tiffany Lowe-Power: University of California Berkeley

<div>The xylem pathogenic bacterium <i>Xylella fastidiosa</i> is characterized by slow growth, a small genome, and low codon usage bias in protein-coding genes. In most organisms, genes have bias in codon usage that reflects availability of cognate tRNAs charged with amino acids. Codon usage mismatch between a genome and a heterologous gene can reduce protein expression. Although a constitutive GFP-expressing <i>X.</i><i> fastidiosa </i>strain was created by expressing the <i>gfp</i> gene from a strong ribosomal promoter, previous attempts to create moderately-expressed fluorescent biosensors in <i>X. fastidiosa</i> have been unsuccessful. Therefore, we hypothesize that a codon-harmonized fluorescent protein expresses better in <i>X. fastidiosa</i>. To test this hypothesis<span>, we adapted the fluorescent protein mNeonGreen<i>. </i>After normalization to <i>X. fastidiosa</i> codon usage the gene was synthesized <span class="m_319172630067195215gmail-msoDel"><del datetime="2017-12-07T09:52"> </del></span>and the codon-harmonized and wildtype <i>mNeonGreen</i> alleles were fused to the <i>hxfA</i> promoter and transformed into <i>X. fastidiosa </i>Temecula1 to generate biosensors that are specific for the <i>X. fastidiosa </i>quorum sensing signal diffusible signal factor (DSF). The effect of codon normalization on protein expression and fluorescence output in bacteria will be discussed. </span></div>