| Easy and Efficient Permeabilization of Cyanobacteria for in vivo Enzyme Assays Using B-PER
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Author:
Date:
2018-01-05
[Abstract] Cyanobacteria are photosynthetic bacteria that thrive in diverse ecosystems and play major roles in the global carbon cycle. The abilities of cyanobacteria to fix atmospheric CO2 and to allocate the fixed carbons to chemicals and biofuels have attracted growing attentions as sustainable microbial cell factories. A better understanding of activities of enzymes involved in the central carbon metabolism might lead to increased product yields. Currently, cell-free lysates are widely used for the determination of intracellular enzyme activities. However, due to thick cell walls in cyanobacteria, lysis of cyanobacterial cells is inefficient and often laborious. The present protocol describes an easy and efficient method to permeabilize cyanobacterial cells, without lysing them, and ...
[摘要] 蓝细菌是光合细菌，在不同的生态系统中繁衍，在全球碳循环中发挥重要作用。 蓝藻固定大气CO 2和将固定碳分配到化学品和生物燃料的能力作为可持续的微生物细胞工厂已经引起越来越多的关注。 更好地了解参与中央碳代谢的酶的活性可能导致产物产量增加。 目前，无细胞裂解物被广泛用于细胞内酶活性的测定。 然而，由于蓝细菌细胞壁较厚，蓝细菌细胞的裂解效率低下且费力。 目前的方案描述了一种简单而有效的方法来渗透蓝藻细胞，而不溶解它们，并直接使用透化细胞来测定体内代谢酶活性。
【背景】我们之前已经报道了使用B-PER TM试剂（Thermo Fisher Science）（Rasmussen等人，2016）简单，有效且可扩展的蓝细菌的透化。 B-PER TM TM试剂含有溶解在Tris-HCl缓冲液中的未公开的温和洗涤剂，通常用于裂解细菌细胞如大肠杆菌（Escherichia coli）。偶然地，我们发现B-PER TM TM试剂渗透蓝细菌细胞而不是溶解它们，可能是因为厚的蓝细菌细胞壁（Hoiczyk和Hansel，2000）赋予了试剂中使用的去污剂的抗性。在生物技术感兴趣的蓝细菌中进行通透化。聚球蓝细菌PCC 7002（以下简称“Synechococcus”7002）和“Synechocystis”sp。 PCC ...
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| In Gel Kinase Assay
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Author:
Date:
2017-03-05
[Abstract] Proper spatiotemporal regulation of protein phosphorylation in cells and tissues is required for normal development and homeostasis. We present the protocol ‘In Gel Kinase Assay’, which is useful for protein kinase activity measurements from crude protein extracts. We have successfully used ‘In Gel Kinase Assay’ protocol to show that the Arabidopsis thaliana sextuple mutant in the PYRABACTIN RESISTANCE1/PYR1-LIKE/REGULATORY COMPONENTS OF ABA RECEPTORS (PYR/PYL/RCAR-ABA receptors; line pyr/pyl112458) is impaired in ABA-mediated activation of SnRK2.2, SnRK2.3 and OST1/SnRK2.6, as much as the triple mutant snrk2.2/2.3/2.6 (Gonzalez-Guzman et al., 2012).
[摘要] 正常发育和体内平衡需要细胞和组织中蛋白质磷酸化的适时时空调节。我们提出方案“凝胶激酶测定”，其可用于粗蛋白质提取物的蛋白激酶活性测量。我们已经成功地使用“凝胶激酶测定”方案来证明在ABA受体（PYR / PYL / RCAR-ABA受体）的PYRABACTIN RESISTANCE1 / PYR1-样/调节组分中的拟南芥线条pyr / pyl112458 ）在ABA介导的SnRK2.2，SnRK2.3和OST1 / SnRK2.6的活化中受损，多达三重突变体snrk2.2 / 2.3 / 2.6 （Gonzalez-Guzman等人，2012）。
背景 植物激素脱落酸（ABA）是涉及植物生长发育以及植物对非生物和生物胁迫的反应的关键信号。 ABA感知和信号通路由ABA受体（PYR / PYL / RCAR-ABA受体）的PYRABACTIN RESISTANCE1 / PYR1-调节组分，PP2C磷酸酶和SnRK2s激酶组成（在Antoni等人， ，，2011）。模块受体-ABA-磷酸酶通过调节ABA激活的SnRK2而以配体依赖的方式控制磷酸化信号级联。反过来，SnRK2s激酶使细胞核和细胞质中的无数效应物从转录因子（例如，ABFs）到离子通道（例如）磷酸化， ...
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