Tracking of Engineered Bacteria In Vivo Using Nonstandard Amino Acid Incorporation,ACS Synthetic Biology

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Tracking of Engineered Bacteria In Vivo Using Nonstandard Amino Acid Incorporation
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2018-05-23 00:00:00 , DOI: 10.1021/acssynbio.8b00135
Pichet Praveschotinunt _{1,

2} , Noémie-Manuelle Dorval Courchesne _{1,

3} , Ilona den Hartog _{1,

4,

5} , Chaochen Lu _{1,

6,

7,

8} , Jessica J Kim ₉ , Peter Q Nguyen ₁ , Neel S Joshi _{1,

2}

Affiliation

The rapidly growing field of microbiome research presents a need for better methods of monitoring gut microbes in vivo with high spatial and temporal resolution. We report a method of tracking microbes in vivo within the gastrointestinal tract by programming them to incorporate nonstandard amino acids (NSAA) and labeling them via click chemistry. Using established machinery constituting an orthogonal translation system (OTS), we engineered Escherichia coli to incorporate p-azido-l-phenylalanine (pAzF) in place of the UAG (amber) stop codon. We also introduced a mutant gene encoding for a cell surface protein (CsgA) that was altered to contain an in-frame UAG codon. After pAzF incorporation and extracellular display, the engineered strains could be covalently labeled via copper-free click reaction with a Cy5 dye conjugated to the dibenzocyclooctyl (DBCO) group. We confirmed the functionality of the labeling strategy in vivo using a murine model. Labeling of the engineered strain could be observed using oral administration of the dye to mice several days after colonization of the gastrointestinal tract. This work sets the foundation for the development of in vivo tracking microbial strategies that may be compatible with noninvasive imaging modalities and are capable of longitudinal spatiotemporal monitoring of specific microbial populations.

中文翻译：

使用非标准氨基酸掺入追踪体内工程细菌

快速发展的微生物组研究领域需要更好的方法来以高空间和时间分辨率监测体内肠道微生物。我们报告了一种跟踪胃肠道内体内微生物的方法，方法是对微生物进行编程，使其掺入非标准氨基酸（NSAA）并通过点击化学对其进行标记。使用构成正交翻译系统（OTS）的已建立的机器，我们对大肠杆菌进行了改造，以掺入对叠氮基-L-苯丙氨酸（pAzF）来代替UAG（琥珀）终止密码子。我们还引入了编码细胞表面蛋白（CsgA）的突变基因，该基因被改变为包含框内 UAG 密码子。pAzF 掺入和细胞外展示后，工程菌株可以通过无铜点击反应与与二苯并环辛基 (DBCO) 基团缀合的 Cy5 染料进行共价标记。我们使用小鼠模型证实了体内标记策略的功能。在胃肠道定植几天后，通过给小鼠口服染料可以观察到工程菌株的标记。这项工作为开发体内追踪微生物策略奠定了基础，该策略可能与非侵入性成像方式兼容，并且能够对特定微生物种群进行纵向时空监测。

更新日期：2018-05-23

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