QPIX 論證報告

1、Qpix系列微生物篩選系統(tǒng)介紹基因有限公司 市場部 李磊lilei2009/12/30目錄一、系統(tǒng)概述31. 技術(shù)背景32. 篩選原理43. 功能強大的軟件44. Qpix克隆挑取過程5二、技術(shù)特點51. 高通量的微生物篩選系統(tǒng)52. 用于多種微生物類型53 數(shù)據(jù)追蹤功能（Data Tracking）6三、系統(tǒng)應(yīng)用-基本功能61克隆自動挑?。–olony Picking）：62膜芯片（Macroarraying）：73復制（Replicating）：84. 重排（Re-arraying）8四、系統(tǒng)應(yīng)用-拓展應(yīng)用81蛋白質(zhì)工程(推薦型號：QPExpression)92. 蛋白表達，轉(zhuǎn)化和亞克隆

2、(推薦型號：QPExpression)93. 大規(guī)模DNA測序(推薦型號：QPExpression或Qpix2)94. 克隆管理（推薦型號：Qpix2 XT）105. 克隆管理-文庫篩選（推薦型號：Qpix2 XT加點膜模塊）10四、參考用戶111. 國際用戶112. 國內(nèi)用戶11五、發(fā)表文獻及相關(guān)121. 部分發(fā)表文獻12六、儀器性能：131. 各型號儀器性能132. 篩選功能:143. 挑樣針清潔144. 系統(tǒng)環(huán)境控制145. 條碼讀取器146. 選配功能157. 儀器規(guī)格15一、系統(tǒng)概述1. 技術(shù)背景 QPix系列是目前全世界最好的多功能文庫管理系統(tǒng)，是基因組學研究領(lǐng)域的第一品牌，目前全

3、球總裝機量在600臺以上. Genetix公司的挑克隆系統(tǒng)為全球基因組學研究立下了赫赫功勞，它廣泛分布在全世界著名的各大基因組學研究中心。在上世紀90年代，大規(guī)模的測序工作迫切的需要一款性能可靠的自動化微生物克隆挑選系統(tǒng)。Genetix公司開發(fā)了Qbot系統(tǒng)，它的獨特的技術(shù)以及可靠的性能得到了廣大用戶的認可。隨著科學技術(shù)的發(fā)展，人們對于微生物克隆篩選系統(tǒng)的需求不僅僅局限于高通量，而更多的要求儀器能夠更加智能。Qpix系列的出現(xiàn)，很好的滿足了當前科研人員對于該儀器的要求。該系統(tǒng)可以進行動植物基因組文庫克隆挑取，EST克隆或者全長cDNA文庫克隆的高速全自動挑取，以及文庫的復制，膜芯片的制作等工作

4、，大大加快了基因組研究的進程。該系統(tǒng)保證功能并最大程度的節(jié)省空間。高智能、界面友好的軟件系統(tǒng)使其能夠輕松高效地勝任您的每一項需要。Qpix系列共有三個型號，其區(qū)別如下：2. 篩選原理 Qpix系列是一款多功能微生物篩選系統(tǒng)，通過白光對菌落成像后，軟件自動根據(jù)菌落的大小，圓度，相鄰程度等信息對所有菌落進行分析，并根據(jù)自定義參數(shù)自動推薦符合要求的菌落，96道德高精度的挑取裝置可以快速的將菌落挑取并轉(zhuǎn)移到培養(yǎng)基中。3. 功能強大的軟件軟件可以自動根據(jù)菌落的大小，圓度和灰度值等信息對其進行評估（圖 1），并可根據(jù)用戶自定義來判定哪些是符合用戶要求的克隆并將之挑選出來放入準備好的96孔板進行后續(xù)培養(yǎng)。

5、系統(tǒng)自動記錄克隆挑選前后圖像，并與相關(guān)數(shù)據(jù)整合鏈接，可回顧整個挑選過程。 圖 1軟件中的數(shù)據(jù)顯示4. Qpix克隆挑取過程圖 2 96道獨立控制的挑取針Qpix首先根據(jù)用戶設(shè)定的標準（例如菌落大小，圓度，灰度值等）創(chuàng)建一個具有高度選擇性的挑取列表，之后通過一個96通道，每個通道可獨立控制的克隆挑取頭（圖 2）自動從瓊脂培養(yǎng)基中沾取目的菌落，待全部克隆挑選完成之后，將之轉(zhuǎn)入96孔板，之后進行后續(xù)培養(yǎng)。使用Qpix，用戶可在1個小時內(nèi)挑選約4600個克隆。Qpix系列配備的功能完備的軟件系統(tǒng)具有追蹤功能（Qpix2除外），它可以記錄目標培養(yǎng)板和源培養(yǎng)板信息。相關(guān)的克隆數(shù)據(jù)以及圖像可被儲存并根據(jù)用戶

6、需要導出。每次克隆挑選結(jié)束后，克隆挑取針可在機器內(nèi)置的清洗平臺上進行清洗、消毒和干燥。清洗過程通常是先用浸泡在80%的酒精中的刷子清潔針的外部可能殘留的培養(yǎng)基等雜質(zhì)，之后用滅菌水沖洗挑樣針，最后再用鹵素燈干燥裝置去除針上可能殘留的酒精等液體溶劑，從而有效避免樣品間的交叉污染。二、技術(shù)特點 1. 高通量的微生物篩選系統(tǒng) Qpix系列通過白光成像識別微生物菌落克隆，并依靠高精度挑取裝置將符合特定標準的克隆從培養(yǎng)基中挑選出來。和傳統(tǒng)方法相比，挑選出來的細胞具有更高的準確性。有了Qpix，一個人可以在一小時的時間內(nèi)挑選約約4600個克隆，并且挑取的過程完全由機器自動完成；而采用傳統(tǒng)方法，同樣的時間不僅

7、挑選的克隆數(shù)目很少，而且重復性和準確性也較差（如下表）。傳統(tǒng)方法Clonepix FL篩選標準人工主管判斷儀器客觀分析挑選時間（10000個克?。? man weeks小于3小時位置精度人工操作10um重復性低99.5% 2. 用于多種微生物類型圖 3 針對不同微生物的挑樣針Qpix系列針對不同的微生物，可以配備不同的挑樣針（圖 3），以保證微生物挑選后的存活率。適用于大腸桿菌、噬菌體文庫、鏈霉菌、酵母等。3 數(shù)據(jù)追蹤功能（Data Tracking）Qpix（Qpix2除外）系列該系統(tǒng)還標配了自動條碼閱讀器，可閱讀符合21CFRPart11要求的編碼，并記錄目標培養(yǎng)板和源培養(yǎng)板信息。三、系統(tǒng)

8、應(yīng)用-基本功能 Qpix系列的主要功能有：1克隆自動挑?。–olony Picking）： · 在線CCD實時監(jiān)測分析· 根據(jù)克隆大小,顏色,形狀來自動選擇挑取符合條件的克隆斑· 96針高速運行，挑4600個克隆/小時· 1×222mm×222mmQTray、1×150mm培養(yǎng)皿、5×90mm培養(yǎng)皿或15×96/384微孔板· 可以進行大腸桿菌,酵母,噬菌體文庫的自動挑取克隆挑取全過程如下所示： 克隆自動挑取 克隆轉(zhuǎn)移至96孔培養(yǎng)板96根針清洗 96根針滅菌 2膜芯片（Macroarraying

9、）：u 可將培養(yǎng)的克隆點樣至膜上,進行高通量的DNA篩選u 22cm×22cm膜上可點55,296個樣,也可以點樣至8x12cm膜u XY軸上達到1m高精確解析度，Z軸由伺服馬達控制u 同樣適合點DNA膜芯片陣列3復制（Replicating）：可以進行96孔板克隆和384孔板克隆的相互復制轉(zhuǎn)移1個384孔板到4個96孔板4個96孔板到1個384孔板1個96孔板到4個96孔板4. 重排（Re-arraying） 通過屏幕選取特定的點或DNA序列進行再排列，自動轉(zhuǎn)移到指定位置四、系統(tǒng)應(yīng)用-拓展應(yīng)用1蛋白質(zhì)工程(推薦型號：QPExpression)節(jié)約時間 · 自動吸取樣品并涂

10、布于Qtray上 · 客觀選擇 根據(jù)預(yù)設(shè)的參數(shù)對克隆進行鑒別和篩選· 同時操作對照樣品減少下游的工作量· 自動挑取每天可以挑取30000個克隆 · 精確、溫和挑取 僅挑取單個克隆，最小化誤操作· 確保選擇的是最好的克隆2. 蛋白表達，轉(zhuǎn)化和亞克隆 (推薦型號：QPExpression) 在蛋白表達、轉(zhuǎn)化和亞克隆過程中，成功的篩選到好的克隆需要保持其完整性，即保持其始終為單克隆，并且在處理大量的樣本的時候不出現(xiàn)操作失誤以及污染等情況。在復雜的多步驟，多次操作中保證克隆完整性· 自動監(jiān)控克隆挑取和涂布過程 · 精確的克隆選擇和挑

11、取提供檢測和追蹤蛋白表達水平· 適用于可溶蛋白，不溶蛋白以及包涵體· 將克隆挑取至96孔板，并且可以復制到多個96孔板QPExpression 提高操作精確性，減少手工誤操作的幾率· 將所有的轉(zhuǎn)化產(chǎn)物轉(zhuǎn)移到 QTrays· 過夜培養(yǎng)后進行克隆挑取· 根據(jù)預(yù)設(shè)的數(shù)目將克隆精確挑取至深孔板不會出現(xiàn)重復挑取或遺漏挑取的情況· Cherry-pick感興趣的克隆· 挑樣針的滅菌步驟保證不發(fā)生交叉污染3. 大規(guī)模DNA測序(推薦型號：QPExpression或Qpix2)人類基因組計劃中很多大型測序中心都在使用Qpix系列。 提高效率

12、和可靠性Increase productivity and reliability· 減少手工的誤操作· 減少DNA延滯效應(yīng)· 數(shù)據(jù)跟蹤o 可以根據(jù)挑取日志核對測序結(jié)果o 在測序前跟蹤克隆生長的深孔板信息 4. 克隆管理（推薦型號：Qpix2 XT） Qpix2 XT以其高通量，高準確度的克隆挑取、復制/重排，有效的篩選以及數(shù)據(jù)跟蹤功能，提供了一個經(jīng)濟而且行之有效的DNA文庫建立和管理的系統(tǒng)。經(jīng)濟的DNA 文庫構(gòu)建于管理· 高通量，高準確度的克隆挑取· 精確的復制/重排 · 高效的文庫篩選 建立BAC和DNA文庫· 以100

13、%的精確性復制達70 x 384孔板或70 x 96孔板· 減少DNA交叉污染 · Cherry pick功能精確選擇克隆 圖 4 Cherry Picking示意圖5. 克隆管理-文庫篩選（推薦型號：Qpix2 XT加點膜模塊） 傳統(tǒng)的文庫篩選是一個費時又費力的工作，并且很容易出錯。配備了點膜模塊的QPix2 XT提供了有效的文庫篩選方案。取代傳統(tǒng)的費時費力的文庫篩選技術(shù)· 96- or 384-點樣針將每一個樣品點到尼龍膜上o 可以同時放置6張膜，每張膜可達57,600個點· 源96- or 384孔板儲存于孔板堆棧中o 孔板堆?？煞胖?0 個標準孔

14、板或70個淺孔板經(jīng)濟型的篩選· 從包含100000個克隆的文庫中篩選鑒別10個陽性克隆只需要四張尼龍膜6. 噬菌斑應(yīng)用 （推薦型號：Qpix2 XT或QP Expression） 很多大型實驗室都推薦使用QPix解決方案，如MorphoSys公司。提高生產(chǎn)效率· 高通量篩選· 根據(jù)實驗靈活性應(yīng)用減少污染· 儀器滅菌能夠有效減少污染 客觀圖像分析及克隆挑選· 根據(jù)克隆大小，形狀，顏色等信息挑選單克隆 針對不同微生物設(shè)計的挑樣針最大程度保證樣品的存活率四、參考用戶1. 國際用戶全球總裝機量在600臺以上。2. 國內(nèi)用戶部分國內(nèi)用戶：1. 中國農(nóng)科院

15、作物所2. 武漢大學3. 華大基因4. 北京諾和諾德醫(yī)藥研究中心5. 五、發(fā)表文獻及相關(guān) 1. 部分發(fā)表文獻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