Control of ? C31 integrase-mediated site-specific recombination by protein trans splicing
Control of ? C31 integrase-mediated site-specific recombination by protein trans splicing
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DOI：
10.1101/540872
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发表时间：
2019
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影响因子：
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通讯作者：
Olorunniji F
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文献类型：
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作者：
Olorunniji F
丝氨酸整合酶正在成为合成生物学的核心工具，并在生物技术和基因组工程中得到应用。我们设计了一种基于分裂内含肽丝氨酸整合酶的系统，具有在体内蛋白质水平调节位点特异性重组事件的潜力。 φC31整合酶被分成两个外显肽结构域，内含肽序列（NpuDnaEN和SspDnaEC）连接到两个末端以进行融合。这两种成分在大肠杆菌中表达，然后进行翻译后蛋白质反式剪接，生成功能齐全的 phiC31 整合酶。我们证明蛋白质剪接对于重组活性是必需的；内含肽结构域的缺失或关键内含肽残基的突变会导致重组失活。我们使用可逆启动子报告系统来证明分裂内含肽调节的位点特异性重组系统在构建可逆基因开关中的潜在应用。我们使用相同的分裂内含子来控制分裂整合酶重组方向性因子融合（Integrase-RDF）的重构，该融合有效地催化了反向attRxattL重组。这证明了分别使用整合酶和整合酶-RDF 融合来调节正向和反向反应的分裂内含肽的潜力。分裂内含肽整合酶是一种潜在的多功能、可调节的组件，用于构建合成遗传电路和设备。
Serine integrases are emerging as core tools in synthetic biology and have applications in biotechnology and genome engineering. We have designed a split-intein serine integrase-based system with potential for regulation of site-specific recombination events at the protein levelin vivo. The ϕC31 integrase was split into two extein domains, and intein sequences (NpuDnaENandSspDnaEC) were attached to the two termini to be fused. Expression of these two components followed by post-translational proteintrans-splicing inEscherichia coligenerated a fully functional ϕC31 integrase. We showed that protein splicing is necessary for recombination activity; deletion of intein domains or mutation of key intein residues inactivated recombination. We used an invertible promoter reporter system to demonstrate a potential application of the split intein-regulated site-specific recombination system in building reversible genetic switches. We used the same split inteins to control the reconstitution of a split Integrase-Recombination Directionality Factor fusion (Integrase-RDF) that efficiently catalysed the reverseattRxattLrecombination. This demonstrates the potential for split-intein regulation of the forward and reverse reactions using the integrase and the integrase-RDF fusion, respectively. The split-intein integrase is a potentially versatile, regulatable component for building synthetic genetic circuits and devices.