QPIX 论证报告

1、Qpix系列微生物筛选系统介绍基因有限公司 市场部 李磊lilei2009/12/30目录一、系统概述31. 技术背景32. 筛选原理43. 功能强大的软件44. Qpix克隆挑取过程5二、技术特点51. 高通量的微生物筛选系统52. 用于多种微生物类型53 数据追踪功能（Data Tracking）6三、系统应用-基本功能61克隆自动挑取（Colony Picking）：62膜芯片（Macroarraying）：73复制（Replicating）：84. 重排（Re-arraying）8四、系统应用-拓展应用81蛋白质工程(推荐型号：QPExpression)92. 蛋白表达，转化和亚克隆

2、(推荐型号：QPExpression)93. 大规模DNA测序(推荐型号：QPExpression或Qpix2)94. 克隆管理（推荐型号：Qpix2 XT）105. 克隆管理-文库筛选（推荐型号：Qpix2 XT加点膜模块）10四、参考用户111. 国际用户112. 国内用户11五、发表文献及相关121. 部分发表文献12六、仪器性能：131. 各型号仪器性能132. 筛选功能:143. 挑样针清洁144. 系统环境控制145. 条码读取器146. 选配功能157. 仪器规格15一、系统概述1. 技术背景 QPix系列是目前全世界最好的多功能文库管理系统，是基因组学研究领域的第一品牌，目前全

3、球总装机量在600台以上. Genetix公司的挑克隆系统为全球基因组学研究立下了赫赫功劳，它广泛分布在全世界著名的各大基因组学研究中心。在上世纪90年代，大规模的测序工作迫切的需要一款性能可靠的自动化微生物克隆挑选系统。Genetix公司开发了Qbot系统，它的独特的技术以及可靠的性能得到了广大用户的认可。随着科学技术的发展，人们对于微生物克隆筛选系统的需求不仅仅局限于高通量，而更多的要求仪器能够更加智能。Qpix系列的出现，很好的满足了当前科研人员对于该仪器的要求。该系统可以进行动植物基因组文库克隆挑取，EST克隆或者全长cDNA文库克隆的高速全自动挑取，以及文库的复制，膜芯片的制作等工作

4、，大大加快了基因组研究的进程。该系统保证功能并最大程度的节省空间。高智能、界面友好的软件系统使其能够轻松高效地胜任您的每一项需要。Qpix系列共有三个型号，其区别如下：2. 筛选原理 Qpix系列是一款多功能微生物筛选系统，通过白光对菌落成像后，软件自动根据菌落的大小，圆度，相邻程度等信息对所有菌落进行分析，并根据自定义参数自动推荐符合要求的菌落，96道德高精度的挑取装置可以快速的将菌落挑取并转移到培养基中。3. 功能强大的软件软件可以自动根据菌落的大小，圆度和灰度值等信息对其进行评估（图 1），并可根据用户自定义来判定哪些是符合用户要求的克隆并将之挑选出来放入准备好的96孔板进行后续培养。

5、系统自动记录克隆挑选前后图像，并与相关数据整合链接，可回顾整个挑选过程。 图 1软件中的数据显示4. Qpix克隆挑取过程图 2 96道独立控制的挑取针Qpix首先根据用户设定的标准（例如菌落大小，圆度，灰度值等）创建一个具有高度选择性的挑取列表，之后通过一个96通道，每个通道可独立控制的克隆挑取头（图 2）自动从琼脂培养基中沾取目的菌落，待全部克隆挑选完成之后，将之转入96孔板，之后进行后续培养。使用Qpix，用户可在1个小时内挑选约4600个克隆。Qpix系列配备的功能完备的软件系统具有追踪功能（Qpix2除外），它可以记录目标培养板和源培养板信息。相关的克隆数据以及图像可被储存并根据用户

6、需要导出。每次克隆挑选结束后，克隆挑取针可在机器内置的清洗平台上进行清洗、消毒和干燥。清洗过程通常是先用浸泡在80%的酒精中的刷子清洁针的外部可能残留的培养基等杂质，之后用灭菌水冲洗挑样针，最后再用卤素灯干燥装置去除针上可能残留的酒精等液体溶剂，从而有效避免样品间的交叉污染。二、技术特点 1. 高通量的微生物筛选系统 Qpix系列通过白光成像识别微生物菌落克隆，并依靠高精度挑取装置将符合特定标准的克隆从培养基中挑选出来。和传统方法相比，挑选出来的细胞具有更高的准确性。有了Qpix，一个人可以在一小时的时间内挑选约约4600个克隆，并且挑取的过程完全由机器自动完成；而采用传统方法，同样的时间不仅

7、挑选的克隆数目很少，而且重复性和准确性也较差（如下表）。传统方法Clonepix FL筛选标准人工主管判断仪器客观分析挑选时间（10000个克隆）2 man weeks小于3小时位置精度人工操作10um重复性低99.5% 2. 用于多种微生物类型图 3 针对不同微生物的挑样针Qpix系列针对不同的微生物，可以配备不同的挑样针（图 3），以保证微生物挑选后的存活率。适用于大肠杆菌、噬菌体文库、链霉菌、酵母等。3 数据追踪功能（Data Tracking）Qpix（Qpix2除外）系列该系统还标配了自动条码阅读器，可阅读符合21CFRPart11要求的编码，并记录目标培养板和源培养板信息。三、系统

8、应用-基本功能 Qpix系列的主要功能有：1克隆自动挑取（Colony Picking）： · 在线CCD实时监测分析· 根据克隆大小,颜色,形状来自动选择挑取符合条件的克隆斑· 96针高速运行，挑4600个克隆/小时· 1×222mm×222mmQTray、1×150mm培养皿、5×90mm培养皿或15×96/384微孔板· 可以进行大肠杆菌,酵母,噬菌体文库的自动挑取克隆挑取全过程如下所示： 克隆自动挑取 克隆转移至96孔培养板96根针清洗 96根针灭菌 2膜芯片（Macroarraying

9、）：u 可将培养的克隆点样至膜上,进行高通量的DNA筛选u 22cm×22cm膜上可点55,296个样,也可以点样至8x12cm膜u XY轴上达到1m高精确解析度，Z轴由伺服马达控制u 同样适合点DNA膜芯片阵列3复制（Replicating）：可以进行96孔板克隆和384孔板克隆的相互复制转移1个384孔板到4个96孔板4个96孔板到1个384孔板1个96孔板到4个96孔板4. 重排（Re-arraying） 通过屏幕选取特定的点或DNA序列进行再排列，自动转移到指定位置四、系统应用-拓展应用1蛋白质工程(推荐型号：QPExpression)节约时间 · 自动吸取样品并涂

10、布于Qtray上 · 客观选择 根据预设的参数对克隆进行鉴别和筛选· 同时操作对照样品减少下游的工作量· 自动挑取每天可以挑取30000个克隆 · 精确、温和挑取 仅挑取单个克隆，最小化误操作· 确保选择的是最好的克隆2. 蛋白表达，转化和亚克隆 (推荐型号：QPExpression) 在蛋白表达、转化和亚克隆过程中，成功的筛选到好的克隆需要保持其完整性，即保持其始终为单克隆，并且在处理大量的样本的时候不出现操作失误以及污染等情况。在复杂的多步骤，多次操作中保证克隆完整性· 自动监控克隆挑取和涂布过程 · 精确的克隆选择和挑

11、取提供检测和追踪蛋白表达水平· 适用于可溶蛋白，不溶蛋白以及包涵体· 将克隆挑取至96孔板，并且可以复制到多个96孔板QPExpression 提高操作精确性，减少手工误操作的几率· 将所有的转化产物转移到 QTrays· 过夜培养后进行克隆挑取· 根据预设的数目将克隆精确挑取至深孔板不会出现重复挑取或遗漏挑取的情况· Cherry-pick感兴趣的克隆· 挑样针的灭菌步骤保证不发生交叉污染3. 大规模DNA测序(推荐型号：QPExpression或Qpix2)人类基因组计划中很多大型测序中心都在使用Qpix系列。 提高效率

12、和可靠性Increase productivity and reliability· 减少手工的误操作· 减少DNA延滞效应· 数据跟踪o 可以根据挑取日志核对测序结果o 在测序前跟踪克隆生长的深孔板信息 4. 克隆管理（推荐型号：Qpix2 XT） Qpix2 XT以其高通量，高准确度的克隆挑取、复制/重排，有效的筛选以及数据跟踪功能，提供了一个经济而且行之有效的DNA文库建立和管理的系统。经济的DNA 文库构建于管理· 高通量，高准确度的克隆挑取· 精确的复制/重排 · 高效的文库筛选 建立BAC和DNA文库· 以100

13、%的精确性复制达70 x 384孔板或70 x 96孔板· 减少DNA交叉污染 · Cherry pick功能精确选择克隆 图 4 Cherry Picking示意图5. 克隆管理-文库筛选（推荐型号：Qpix2 XT加点膜模块） 传统的文库筛选是一个费时又费力的工作，并且很容易出错。配备了点膜模块的QPix2 XT提供了有效的文库筛选方案。取代传统的费时费力的文库筛选技术· 96- or 384-点样针将每一个样品点到尼龙膜上o 可以同时放置6张膜，每张膜可达57,600个点· 源96- or 384孔板储存于孔板堆栈中o 孔板堆栈可放置50 个标准孔

14、板或70个浅孔板经济型的筛选· 从包含100000个克隆的文库中筛选鉴别10个阳性克隆只需要四张尼龙膜6. 噬菌斑应用 （推荐型号：Qpix2 XT或QP Expression） 很多大型实验室都推荐使用QPix解决方案，如MorphoSys公司。提高生产效率· 高通量筛选· 根据实验灵活性应用减少污染· 仪器灭菌能够有效减少污染 客观图像分析及克隆挑选· 根据克隆大小，形状，颜色等信息挑选单克隆 针对不同微生物设计的挑样针最大程度保证样品的存活率四、参考用户1. 国际用户全球总装机量在600台以上。2. 国内用户部分国内用户：1. 中国农科院

15、作物所2. 武汉大学3. 华大基因4. 北京诺和诺德医药研究中心5. 五、发表文献及相关 1. 部分发表文献Distinct sensory pathways in Vibrio cholerae El Tor and classical biotypes modulate cyclic dimeric GMP levels to control biofilm formation.J Bacteriol. 2009 Jan;191(1):169-77. Epub 2008 Oct 24.Hammer BK, Bassler BL.Quorum sensing (QS), or cell-ce

16、ll communication in bacteria, is achieved through the production and subsequent response to the accumulation of extracellular signal molecules called autoinducers (AIs). To identify AI-regulated target genes in Vibrio cholerae El Tor (V. cholerae(El), the strain responsible for the current cholera p

17、andemic, luciferase expression was assayed in an AI(-) strain carrying a random lux transcriptional reporter library in the presence and absence of exogenously added AIs. Twenty-three genes were identified and shown to require the QS transcription factor, HapR, for their regulation. Several of the Q

18、S-dependent target genes, annotated as encoding hypothetical proteins, in fact encode HD-GYP proteins, phosphodiesterases that degrade the intracellular second messenger cyclic dimeric GMP (c-di-GMP), which is important for controlling biofilm formation. Indeed, overexpression of a representative QS

19、-activated HD-GYP protein in V. cholerae(El) reduced the intracellular concentration of c-di-GMP, which in turn decreased exopolysaccharide production and biofilm formation. The V. cholerae classical biotype (V. cholerae(Cl), which caused previous cholera pandemics and is HapR(-), controls c-di-GMP

20、levels and biofilm formation by the VieA signaling pathway. We show that the VieA pathway is dispensable for biofilm formation in V. cholerae(El) but that restoring HapR in V. cholerae(Cl) reestablishes QS-dependent repression of exopolysaccharide production. Thus, different pandemic strains of V. c

21、holerae modulate c-di-GMP levels and control biofilm formation in response to distinct sensory pathways.Automated panning and screening procedure on microplates for antibody generation from phage display libraries.J Biomol Screen. 2009 Mar;14(3):282-93. Epub 2009 Feb 17.Turunen L, Takkinen K, Sö

22、;derlund H, Pulli T.Antibody phage display technology is well established and widely used for selecting specific antibodies against desired targets. Using conventional manual methods, it is laborious to perform multiple selections with different antigens simultaneously. Furthermore, manual screening

23、 of the positive clones requires much effort. The authors describe optimized and automated procedures of these processes using a magnetic bead processor for the selection and a robotic station for the screening step. Both steps are performed in a 96-well microplate format. In addition, adopting the

24、antibody phage display technology to automated platform polyethylene glycol precipitation of the enriched phage pool was unnecessary. For screening, an enzyme-linked immunosorbent assay protocol suitable for a robotic station was developed. This system was set up using human gamma-globulin as a mode

25、l antigen to select antibodies from a VTT naive human single-chain antibody (scFv) library. In total, 161 gamma-globulin-selected clones were screened, and according to fingerprinting analysis, 9 of the 13 analyzed clones were different. The system was further tested using testosterone bovine serum

26、albumin (BSA) and beta-estradiol-BSA as antigens with the same library. In total, 1536 clones were screened from 4 rounds of selection with both antigens, and 29 different testosterone-BSA and 23 beta-estradiol-BSA binding clones were found and verified by sequencing. This automated antibody phage d

27、isplay procedure increases the throughput of generating wide panels of target-binding antibody candidates and allows the selection and screening of antibodies against several different targets in parallel with high efficiency.Functional and comparative metagenomic analysis of bile salt hydrolase act

28、ivity in the human gut microbiomeProc Natl Acad Sci U S A., Sep 2008; 105: 13580 - 13585.Brian V. Jones, Máire Begley, Colin Hill, Cormac G. M. ahan, and Julian R. Marchesi Abstract: Bile salt hydrolases (BSHs) catalyze the “gateway” reaction in a wider pathway of bile acid modification by

29、 the gut microbiota. Because bile acids function as signaling molecules regulating their own biosynthesis, lipid absorption, cholesterol homeostasis, and local mucosal defenses in the intestine, microbial BSH activity has the potential to greatly influence host physiology. However, the function, dis

30、tribution, and abundance of BSH enzymes in the gut community are unknown. Here, we show that BSH activity is a conserved microbial adaptation to the human gut environment with a high level of redundancy in this ecosystem. Through metagenomic analyses we identified functional BSH in all major bacteri

31、al divisions and archaeal species in the gut and demonstrate that BSH is enriched in the human gut microbiome. Phylogenetic analysis illustrates that selective pressure in the form of conjugated bile acid has driven the evolution of members of the Ntn_CGH-like family of proteins toward BSH activity

32、in gut-associated species. Furthermore, we demonstrate that BSH mediates bile tolerance in vitro and enhances survival in the murine gut in vivo. Overall, we demonstrate the use of function-driven metagenomics to identify functional anchors in complex microbial communities, and dissect the gut micro

33、biome according to activities relevant to survival in the mammalian gastrointestinal tract.The genome sequence of Bifidobacterium longum subsp. infantis reveals adaptations for milk utilization within the infant microbiomeProc Natl Acad Sci U S A. 2008 Dec 2;105(48):18964-9. Epub 2008 Nov 24.Sela DA

34、, Chapman J, Adeuya A, Kim JH, Chen F, Whitehead TR, Lapidus A, Rokhsar DS, Lebrilla CB, German JB, Price NP, Richardson PM, Mills DA. Following birth, the breast-fed infant gastrointestinal tract is rapidly colonized by a microbial consortium often dominated by bifidobacteria. Accordingly, the

35、 complete genome sequence of Bifidobacterium longum subsp. infantis ATCC15697 reflects a competitive nutrient-utilization strategy targeting milk-borne molecules which lack a nutritive value to the neonate. Several chromosomal loci reflect potential adaptation to the infant host including a 43 kbp c

36、luster encoding catabolic genes, extracellular solute binding proteins and permeases predicted to be active on milk oligosaccharides. An examination of in vivo metabolism has detected the hallmarks of milk oligosaccharide utilization via the central fermentative pathway using metabolomic and proteom

37、ic approaches. Finally, conservation of gene clusters in multiple isolates corroborates the genomic mechanism underlying milk utilization for this infant-associated phylotype.Factor Xa active site substrate specificity with substrate phage display and computational molecular modeling.J Biol Chem. 20

38、08 May 2;283(18):12343-53. Epub 2008 Feb 22.Hsu HJ, Tsai KC, Sun YK, Chang HJ, Huang YJ, Yu HM, Lin CH, Mao SS, Yang AS.Abstract:Structural origin of substrate-enzyme recognition remains incompletely understood. In the model enzyme system of serine protease, canonical anti-parallel beta-structure su

39、bstrate-enzyme complex is the predominant hypothesis for the substrate-enzyme interaction at the atomic level. We used factor Xa (fXa), a key serine protease of the coagulation system, as a model enzyme to test the canonical conformation hypothesis. More than 160 fXa-cleavable substrate phage varian

40、ts were experimentally selected from three designed substrate phage display libraries. These substrate phage variants were sequenced and their specificities to the model enzyme were quantified with quantitative enzyme-linked immunosorbent assay for substrate phage-enzyme reaction kinetics. At least

41、three substrate-enzyme recognition modes emerged from the experimental data as necessary to account for the sequence-dependent specificity of the model enzyme. Computational molecular models were constructed, with both energetics and pharmacophore criteria, for the substrate-enzyme complexes of several of the representative substrate peptide sequences. In contrast to the canonical conformation hypothesis, the binding modes of the substrates to the model enzyme varied according to the substrate peptide sequenc