Exploring the impact of terminators on transgene expression in Chlamydomonas reinhardtii with a synthetic biology approach
Exploring the impact of terminators on transgene expression in Chlamydomonas reinhardtii with a synthetic biology approach
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用合成生物学方法探索终止子对莱茵衣藻转基因表达的影响
DOI：
10.1101/2021.08.04.455025
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发表时间：
2021
期刊：
影响因子：
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通讯作者：
Geisler K
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作者：
Geisler K
莱茵衣藻具有许多有吸引力的特征，可用作基础研究的模式生物和生物技术平台。尽管如此，尽管已经开发了许多分子工具和资源，但其成功的工程设计仍面临挑战，特别是获得可重复和高水平的转基因表达。在这里，我们描述了一种合成生物学方法，使用标准化部件筛选数百个独立的转化体，以探索可能影响转基因表达的不同参数。我们专注于终止子，并使用标准化工作流程和定量输出，测试了代表三种不同大小类别的天然终止子的 9 种不同元素，以确定它们支持 GFP 报告基因高水平表达的能力。我们发现最佳大小反映了 C 中元素的中值大小。莱茵哈特基因组。不同启动子、不同宿主菌株和不同转基因的终止子部分的行为相似。该方法适用于其他遗传元件的系统测试，有助于比较以确定最佳的转基因设计。
Chlamydomonas reinhardtiihas many attractive features for use as a model organism for both fundamental studies and as a biotechnological platform. Nonetheless, despite the many molecular tools and resources that have been developed, there are challenges for its successful engineering, in particular to obtain reproducible and high levels of transgene expression. Here we describe a synthetic biology approach to screen several hundred independent transformants using standardised parts to explore different parameters that might affect transgene expression. We focused on terminators and, using a standardised workflow and quantitative outputs, tested 9 different elements representing three different size classes of native terminators to determine their ability to support high level expression of a GFP reporter gene. We found that the optimal size reflected the median size of element found in theC. reinhardtiigenome. The behaviour of the terminator parts was similar with different promoters, in different host strains and with different transgenes. This approach is applicable to the systematic testing of other genetic elements, facilitating comparison to determine optimal transgene design.