Geisler Katrin, Scaife Mark A, Mordaka Paweł M, Holzer Andre, Tomsett Eleanor V, Mehrshahi Payam, Mendoza Ochoa Gonzalo I, Smith Alison G
Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge CB2 3EA, UK.
Mara Renewables Corporation, Dartmouth, NS B2Y 4T6, Canada.
Life (Basel). 2021 Sep 14;11(9):964. doi: 10.3390/life11090964.
has 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 the genome. 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.
作为基础研究的模式生物和生物技术平台，它具有许多吸引人的特性。然而，尽管已经开发了许多分子工具和资源，但对其进行成功改造仍面临挑战，特别是要获得可重复的高水平转基因表达。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转化体，以探索可能影响转基因表达的不同参数。我们聚焦于终止子，并使用标准化流程和定量输出，测试了代表三种不同大小类别的天然终止子的9种不同元件，以确定它们支持绿色荧光蛋白（GFP）报告基因高水平表达的能力。我们发现，最佳大小反映了基因组中元件的中位数大小。终止子部件在不同启动子、不同宿主菌株和不同转基因情况下的行为相似。这种方法适用于对其他遗传元件的系统测试，便于进行比较以确定最佳转基因设计。
