No abstract

The integration of genes into the nuclear genome of Chlamydomonas reinhardtii is mediated by Non-Homologous-End-Joining, thus resulting in unpredicted insertion locations. This phenomenon defines 'the position-effect', which is used to explain the variation of expression levels between different clones transformed with the same DNA fragment. Likewise, nuclear transgenes often undergo epigenetic silencing that reduces their expression; hence, nuclear transformations require high-throughput screening methods to isolate clones that express the foreign gene at a desirable level. Here, we show that the number of integration sites of heterologous genes results in higher mRNA levels. By transforming both a synthetic ferredoxin-hydrogenase fusion enzyme and a Gaussia-Luciferase reporter protein, we were able to obtain 33 positive clones that exhibit a wide range of synthetic expression. We then performed a droplet-digital polymerasechain-reaction for these lines to measure their transgene DNA copy-number and mRNA levels. Surprisingly, most clones contain two integration sites of the synthetic gene (45.5%), whilst 33.3% contain one, 18.1% include three and 3.1% encompass four. Remarkably, we observed a positive correlation between the raw DNA copy-number values to the mRNA levels, suggesting a general effect of which transcription of transgenes is partially modulated by their number of copies in the genome. However, our data indicate that only clones harboring at least three copies of the target amplicon show a significant increment in mRNA levels of the reporter transgene. Lastly, we measured protein activity for each of the reporter genes to elucidate the effect of copynumber variation on heterologous expression.

Motivation Regulation of the amount of protein that is synthesized from genes has proved to be a serious challenge in terms of analysis and prediction, and in terms of engineering and optimization, due to the large diversity in expression machinery across species. Results To address this challenge, we developed a methodology and a software tool (ChimeraUGEM) for predicting gene expression as well as adapting the coding sequence of a target gene to any host organism. We demonstrate these methods by predicting protein levels in seven organisms, in seven human tissues, and by increasing in vivo the expression of a synthetic gene up to 26-fold in the single-cell green alga Chlamydomonas reinhardtii. The underlying model is designed to capture sequence patterns and regulatory signals with minimal prior knowledge on the host organism and can be applied to a multitude of species and applications. Availability and implementation Source code (MATLAB, C) and binaries are freely available for download for non-commercial use at http://www.cs.tau.ac.il/~tamirtul/ChimeraUGEM/, and supported on macOS, Linux and Windows. Supplementary information Supplementary data are available at Bioinformatics online.