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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RNA Capping by Transcription Initiation with Non-canonical Initiating Nucleotides (NCINs): Determination of Relative Efficiencies of Transcription Initiation with NCINs and NTPs
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Author:
Date:
2017-06-20
[Abstract] It recently has been established that adenine-containing cofactors, including nicotinamide adenine dinucleotide (NAD+), reduced nicotinamide adenine dinucleotide (NADH), and 3’-desphospho-coenzyme A (dpCoA), can serve as ‘non-canonical initiating nucleotides’ (NCINs) for transcription initiation by bacterial and eukaryotic cellular RNA polymerases (RNAPs) and that the efficiency of the reaction is determined by promoter sequence (Bird et al., 2016). Here we describe a protocol to quantify the relative efficiencies of transcription initiation using an NCIN vs. transcription initiation using a nucleoside triphosphate (NTP) for a given promoter sequence.
[摘要] 最近已经确定,含有腺嘌呤的辅因子,包括烟酰胺腺嘌呤二核苷酸(NAD +),还原型烟酰胺腺嘌呤二核苷酸(NADH)和3'-脱磷酸辅酶A(dpCoA)可以作为“非规范起始核苷酸” NCIN),用于通过细菌和真核细胞RNA聚合酶(RNAP)进行转录起始,并且通过启动子序列确定反应的效率(Bird等,2016)。 在这里,我们描述了使用NCIN与使用三磷酸核苷(NTP)对于给定启动子序列的转录起始来定量转录起始的相对效率的方案。 【背景】在细菌,古细菌和真核生物中的转录由序列,结构和机制保守的多亚基RNA聚合酶(RNAPs)进行(Ebright,2000; Lane和Darst,2010)。为了启动转录,RNAP与一个或多个引发因子一起结合称为“启动子”的特异性DNA序列,并解开启动子DNA以形成含有未解链“转录泡”的RNAP启动子开放复合物(RPo)(图1A; Ruff等人,2015)。 RNAP然后通过扩增(“剔除”)或收缩(“抗锯齿”)转录起始点来选择转录起始位点,以将转录起始位点的核苷酸置于RNAP活性中心起始位点(“i位点”)和扩增位点'i + 1位点')结合i位点的互补起始核苷酸底物和“i + 1”位点的互补延伸底物,并催化磷酸二酯键形成产生初始RNA产物(Winkelman等, 2016)。 在标准的从头转录启动中,起始底物是核苷三磷酸(NTP),通常为ATP或GTP(Nickels ...
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