Upon starvation, the soil bacterium Bacillus subtilis forms an intracellular, metabolically inactive endospore. Its core contains the DNA, encased by three protein layers protecting it against a multitude of environmental threats. The outermost layer, the crust, harbors great potential as a protein-displaying platform: a gene of interest can be translationally fused to a crust protein gene, resulting in endospores displaying the desired protein on their surface. To unlock this potential in a standardized fashion, we designed a suite of 12 vectors (Sporovectors), based on the BioBrick cloning standard. With these vectors, proteins can easily be fused N- or C-terminally to the six crust proteins CotV, CotW, CotX, CotY, CotZ, and CgeA under the control of the strongest crust gene promoter P_cotYZ. All Sporovectors were evaluated with GFP and two different laccases. On the basis of our data, CotY and CotZ represent the best anchor proteins. But there are significant differences in activity and functional stability between the two tested laccases. Our vector suite is a powerful tool to generate and evaluate a vast variety of functionalized endospores. It allows quickly identifying the best anchor and fusion site for the protein of interest. Our findings demonstrate that the crust of B. subtilis endospores is an inexpensive and easy platform for displaying different proteins of interest.

中文翻译：

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孢子虫：枯草芽孢杆菌内生孢子壳作为蛋白质展示平台的利用。

饥饿时，土壤细菌枯草芽孢杆菌会形成细胞内的，代谢不活跃的内生孢子。它的核心包含被三层蛋白质包裹的DNA，可以保护其免受多种环境威胁。最外层的外壳具有作为蛋白质展示平台的巨大潜力：可以将感兴趣的基因与外壳蛋白基因翻译融合，从而使内生孢子在其表面上展示所需的蛋白质。为了以标准化的方式释放这种潜力，我们根据BioBrick克隆标准设计了12种载体（Sporovectors）套件。使用这些载体，可以在最强的外壳基因启动子P _cotYZ的控制下，将蛋白质轻松地在N端或C端与六种外壳蛋白CotV，CotW，CotX，CotY，CotZ和CgeA_融合。。用GFP和两种不同的漆酶评估所有孢子载体。根据我们的数据，CotY和CotZ代表最好的锚蛋白。但是两种测试的漆酶在活性和功能稳定性方面存在显着差异。我们的载体套件是生成和评估各种功能化内生孢子的强大工具。它可以快速确定目标蛋白的最佳锚定和融合位点。我们的发现表明，枯草芽孢杆菌内生孢子的壳是展示不同目的蛋白的廉价且容易的平台。