| Rapid Genome Engineering of Pseudomonas Assisted by Fluorescent Markers and Tractable Curing of Plasmids
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Author:
Date:
2021-02-20
[Abstract] Precise genome engineering has become a commonplace technique for metabolic engineering. Also, insertion, deletion and alteration of genes and other functional DNA sequences are essential for understanding and engineering cells. Several techniques have been developed to this end (e.g., CRISPR/Cas-assisted methods, homologous recombination, or λ Red recombineering), yet most of them rely on the use of auxiliary plasmids, which have to be cured after the editing procedure. Temperature-sensitive replicons, counter-selectable markers or repeated passaging of plasmid-bearing cells have been traditionally employed to circumvent this hurdle. While these protocols work reasonably well in some bacteria, they are not applicable for other species or are time consuming and laborious. Here, we present ...
[摘要] [摘要]精确的基因组工程已成为代谢工程的一种普遍技术。同样，基因和其他功能性DNA序列的插入，缺失和改变对于理解和改造细胞也是必不可少的。几种技术已经发展到该端部（例如，CRISPR / CAS-辅助方法，同源重组，或 λ 红色重组），但其中大多数依赖于辅助质粒的使用，必须在编辑程序后将其固化。传统上已采用对温度敏感的复制子，反向选择标记或带有质粒的细胞的重复传代来规避这一障碍。尽管这些协议在某些细菌中可以很好地发挥作用，但它们不适用于其他物种，或者既费时又费力。在这里，我们提出了快速和通用的荧光假单胞菌荧光标记辅助基因组编辑协议，然后通过用户控制的质粒复制干净固化辅助质粒。一种荧光标记有助于鉴定基因组编辑的菌落，而第二种报道分子能够检测无质粒的细菌克隆。该协议不仅是用于假单胞菌物种的最快方法，而且可以轻松地适应任何类型的基因组修饰，包括序列删除，插入和替换。
图形概要：
带有可治愈质粒的假单胞菌的快速基因组工程
[背景]靶向，精确的基因组操纵技术已经大大推进了微生物工程领域。这样的方法不仅允许评估基因型与表型的关系，而且使微生物细胞工厂的复杂工程化成为可能。近年来，CRISPR / Cas9方法为真核生物的精确基因组工程铺平了道路。在细菌中，CRISPR / ...
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| Rapid Isolation and Purification of Secreted Bacteriocins from Streptococcus mutans and Other Lactic Acid Bacteria
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Author:
Date:
2020-11-20
[Abstract] Bacteriocins are small ribosomally synthesized antimicrobial peptides produced by some microorganisms including lactic acid bacteria (LAB), a group of Gram-positive bacteria (cocci, rods) expressing high tolerance for low pH. Bacteriocins kill bacteria rapidly and are biologically active at very low concentrations. Bacteriocins produced by LAB are primarily active against closely related bacterial species. Many bacteriocins have been investigated with respect to their potential use in promoting human, plant, and animal health, and as food biopreservatives. Bacteriocins produced by LAB are particularly interesting since several LAB have been granted GRAS (Generally Recognized as Safe) status. Because it is not always possible to extract active bacteriocins secreted from cells grown in ...
[摘要] [摘要]细菌素是由一些核糖体合成的抗菌肽微生物，包括乳酸菌（LAB），一组革兰氏阳性菌（球菌，棒）表现出对低pH的高耐受性。细菌素可迅速杀死细菌，并在极高的温度下具有生物活性。低浓度。LAB生产的细菌素主要对紧密相关的细菌具有活性种类。已经研究了许多细菌素在促进细菌生长方面的潜在用途。对人类，植物和动物健康，以及作为食品生物防腐剂。LAB生产的细菌素是特别有趣的是，由于一些实验室已获得GRAS（通常被认为是安全的）状态。因为并非总是可能提取液体中生长的细胞分泌的活性细菌素介质，我们开发了一种使用半固体的简单而廉价的肽提取程序营养丰富的琼脂培养基。我们在此提出一种详细的程序，以快速提取出从口腔物种分泌的生物活性细菌素肽变异链球菌，多产的菌种的生产及其在其他实验室提取细菌素的潜在应用（例如，链球菌，乳球菌，肠球菌）。我们还提出了一种简单的检测方法纯化的细胞外肽提取物的细菌素活性测定
[背景]自然界中的大多数细菌并不是独立存在，而是存在于复杂的多物种中生物膜群落（López等，2010）。两者之间存在大量的身体和营养相互作用细菌有助于生物膜的生长和存活。细菌素的产生和分泌细胞外空间为生产者与其他竞争者赋予了独特的生态优势存在于同一生态位中的细菌（Donia and ...
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| Analysis of Gram-negative Bacteria Peptidoglycan by Ultra-performance Liquid Chromatography
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Author:
Date:
2020-10-05
[Abstract] Bacteria are surrounded by a protective peptidoglycan cell wall. Provided that this structure and the enzymes involved are the preferred target for our most successful antibiotics, determining its structural and chemical complexity is of the highest interest. Traditionally, high-performance liquid chromatography (HPLC) analyses have been performed, but these methods are very time consuming in terms of sample preparation and chromatographic separation. Here we describe an optimized method for preparation of Gram-negative bacteria peptidoglycan and its subsequent analysis by ultra-performance liquid chromatography (UPLC). The use of UPLC in peptidoglycan analyses provides a dramatic reduction of the sample volume and hands-on time required and, furthermore, permits in-line mass spectrometry ...
[摘要] [摘要]细菌被保护性肽聚糖细胞壁包围。如果这种结构和涉及的酶是我们最成功的抗生素的首选靶标，那么确定其结构和化学复杂性便是最重要的。传统上，已经进行了高效液相色谱（HPLC）分析，但是这些方法在样品制备和色谱分离方面非常耗时。在这里我们描述了一种优化的制备方法 革兰氏阴性细菌肽聚糖及其随后的超高效液相色谱（UPLC）分析。在肽聚糖分析中使用UPLC可以大大减少所需的样品量和动手时间，此外，还可以对U PLC解析的多肽进行在线质谱（MS），从而有助于对其进行鉴定。这种方法提高了我们执行高通量分析以更好地了解细胞壁生物学的能力。
[背景]细菌由肽聚糖（PG）的细胞壁所包围，除了到结构的作用，传达细胞形状和保护细菌免受外部损害，作为抗生物，化学屏障iCal和物理应力。murein囊或PG是细胞壁的主要成分。革兰氏阴性细菌在周质空间中呈现单层（Gan等，2008），而它构成了一个厚的网状结构，在革兰氏阳性细菌中具有多个堆积和交联的层（Pasquina-Lemonche等，2020）。Ť他细胞壁是交联的聚糖链的三维网状结构包围所述电池主体（Glauner等人，1988; Typas ...
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