实验室操作手册(20130328更新).doc

目录：按ctrl点击目录项进入所需章节欢迎大家积极更新纠正并注明贡献者，愿实验室知识准确传承，平台越来越好。Cracking初筛重组子3Pseudomonas aeruginosa Transformation4electroporation of Pseudomonas aeruginosa5多糖的检测6Swimming，swarming，twiching三种运动能力的检测7绿浓菌素和弹性蛋白酶产量的检测（未庆）9Rapid biofilm assay11cyclic-di-GMP的HPLC检测（王世伟）12Biofilm Antibiotic Resistance Assay13Congo Red agar plate14Electrophoretic Mobility Shift Assay (EMSA)15ELISA protocol for Psl bound to plates17Biparental Spot Mating（启动子加lacZ）18Pellicle assay19Sytox Green染色观察菌落（朱斌）20-半乳糖苷酶活性21流式细胞仪（朱斌）23大肠杆菌感受态细胞的制备CaCl2法24常用培养基25Matlab计算生物量（王世伟）26Mating1. 将pEX18质粒转入E.coli SM10，选择质粒marker（Ap或Gm）。将P.aeruginosa在LBNS平板上培养。2. 第一天：分别在LB和LBNS中过夜培养含接合质粒的大肠杆菌和PAO1-UW。3. 第二天：在无抗LBNS上点PAO1-UW培养液7ul，吹干后再在其上点等体积的大肠培养液并吹干。无抗板放入37恒温培养箱培养过夜培养。4. 第三天：将板上的菌苔刮下，划线涂布在如下培养基中37过夜培养：LBNS+lrg25（三氯生）+Gm100/ Cb300 (三氯生可以杀死E.coli)。5. 第四天：把上一步得到的单菌落在下述平板上点板： LBNS+5%sucrose。（SacB基因在sucrose诱导下使不发生同源双交换的菌死亡留下质粒与基因组同源双交换，产生两种可能结果:1.回复成野生型；2.产生敲除）6. 第五-六天：涂布单菌落于LBNS，LBNS+Cb300，37过夜培养，寻找对Cb敏感（丢失pEX）的菌株。7. 第七天：转接目的单菌落于LBNS，LBNS+Cb300，37过夜培养。8. 选取目的单菌落，提取基因组。 9. PCR鉴定目的基因是否插入。Cracking初筛重组子用无菌牙签挑取单菌落逐一接种于含相应抗生素的LB平板上，37培养1216h。用无菌牙签挑取少量培养物与10uL无菌水混匀，加入10uL2Crackingbuffer(0.2MNaOH，0.5%SDS，20%蔗糖)，剧烈振荡35min，将裂解液全部上样进行琼脂糖凝胶电泳。电泳结果中虽然含有染色体带，但不影响质粒条带的观察，很容易将重组质粒与载体质粒区分开，可大大节省工作量。对于10kb以下的高拷贝质粒可以检测出0.81kb以上的插入片段，并能一次筛选大量转化子。该法对于大质粒也适用，但若插入片段太小，则由于重组质粒与载体间的大小差别太小而不易将它们区分开。Pseudomonas aeruginosa TransformationCompetent cells preparation:1. Prepare o/n culture.2. Inoculate 1ml o/n culture to 50ml LBNS.3. Shaking 37C until OD600 reach 0.4.4. Pellet cells at 7000g and suspend its in 10ml cold 150mM MgCl2.5. Incubate on ice for 30min.6. Pellet cells and resuspend in 1.0ml 150mM MgCl2. Transformation:1. Mix 100ul competent cells with 50ng-1ug plasmid DNA/up to 100ug chromosome DNA.2. Incubate on ice for 1 hr.3. Heat shock cells at 42C for 3 min, then place on ice for 5 min.4. Add 1ml LBNS and incubate at 37C for 30 min.5. Pellet cells and resuspend in 200ul LBNS.6. Plate 100ul onto the antibiotic plate.7. Incubate at 37C overnight.electroporation of Pseudomonas aeruginosa1. P. aeruginosa PA103 was grown in L-broth with vigorous shaking at 37 until early log phase (ABS540 -0.3-0.5). 2. The cells were harvested by centrifugation at 7000 g for 10 min at 40C,washed in the same volume of 300mM sucrose, 3. re-centrifuged, washed in 0.5 volume of wash medium and fiallyresuspended in 0.01 volumes of 300mM sucrose (-4 x 1011 cfu/ml). （The efficiency of electroporation was approximately 103-fold lower when either water or 1mM HEPES buffer was used as the wash medium, due to celllysis. ）4. The cell suspension was chilled on ice for 30 min prior to electroporation. 5. 40ul aliquots of cell suspension were mixed with 5ul of DNA solution and transfenred to chilled 0.2 cm gap cuvettes (Bio-Rad). 6. Following delivery of the pulse, the cells were mixed with 3ml L-broth and shaken for 2h at 37C. Prelimiary experiments indicated that, in contrast to E.coli (2), electroporated P. aeruginosa cells could be held on ice for up to 10 min before addition of the outgrowthmedium with only a 10% decrease in transformation efficiency. The cells were diluted in L-broth and plated on L-agar supplemented with carbenicillin at 200 pg/ml. Transfonnation efficiency was calculated at CFU/tg of plasmid DNA. Figure 1 shows the effect of field strength on transfonnation efficiency at an exponential decayconstant of 5 msec. Maximum efficiency was reached at 8 lV/cm. The data in Fig. 2 shows the number of transformants obtained as a ftmction of the DNA concentration, indicating that up to 104 transformants can beobtained with picogram quantities of plasmid DNA. Unlike E. coli, P. aeruginosa cells could not be electroporated with high efficiency after being frozen.多糖的检测1.1.2.1 MIC检测(参照AB bioMerieux说明书) 根据说明要求倒取一定厚度的培养基平板，用过夜的菌液配取麦氏浊度0.5的菌液用灭菌的棉签沾取菌液在平板上连续均匀涂布，晾干用无菌镊子夹取E-Test药敏试纸条贴在平板中央（正面朝上），若有气泡，可用镊子按压，但不可移动试条37培养过夜，观察记录结果，并按照全国临床检验操作标准中对于药敏试验的规定分析耐药情况1.1.2.2 Psl多糖提取8, 33 测定过夜培养的菌液的OD600值，收集3个 OD的菌液。 13000r/min离心5min,弃上清。100L0.5mol/L的EDTA，重悬菌泥沸水煮5min,离心，取上清加入适量蛋白酶K处理（0.5mg/mL），60温浴1h后80处理30min,每个样品三个重复。1.1.2.3 Immuno-blotting 检测Psl多糖8, 33取适当大小的硝酸纤维素膜，点样5L，晾干。TBST润洗3次，以10%脱脂牛奶封闭1h，TBST润洗27min，11min。加一抗（1:25000）处理1hTBST润洗如前，加二抗（1:7500）1hTBST润洗27min，11min。用碱性磷酸酶buffer润洗一下显色。用Bio-Rad凝胶成像系统照相，后用Quantity One软件进行灰度值分析。1.1.2.4 标准曲线的绘制以提纯过的Psl多糖作为标准品，根据以上试验方法。以灰度值和Psl多糖浓度用绘制做出标准曲线，并根据标准曲线分析Psl具体产量。1.1.2.5 统计结果并分析 以Excel表格自带分析软件，分析实验结果。Swimming，swarming，twiching三种运动能力的检测2.1.1.2试剂与培养基细菌用胰蛋白胨 英国Oxoid公司酵母提取物 英国Oxoid公司营养肉汤 英国Oxoid公司琼脂粉 中国惠兴生化制剂有限公司Bacto Agar BD公司TTC为 Sigma公司Swimming培养基：细菌用胰蛋白胨: 10g/l酵母提取物： 5g/l琼脂粉: 0.15%加蒸馏水定容至1L，121湿热灭菌20min。需要测定swimming能力之前，加热凝固的培养基，融化后冷却至50左右倒平板，平板的厚度约为4mm左右，冷却凝固后备用。由于培养基中琼脂粉较少，属于软平板，所以移动平板时需要柔和平稳移动，不可倾斜和倒置。Swarmming培养基：营养肉汤： 8g/lD-葡萄糖： 5g/lBacto Agar： 0.5%加蒸馏水定容至1L，115湿热灭菌30min。需要测定swarming能力之前，加热凝固的培养基，融化后冷却至50左右倒平板，平板的厚度约为3mm左右，冷却凝固，干燥过夜。培养基属于半固体，平板不可倒置。（需晾干一些，平板不能叠加放置，防止水分不同）Twitching 培养基：细菌用胰蛋白胨: 10g/l酵母提取物： 5g/l琼脂粉: 1.0%加蒸馏水定容至1L，121湿热灭菌20min。需要测定Twitching能力之前，加热凝固的培养基，融化后冷却至50左右添加1%的TTC（2，3，5氯化三苯基四氮唑）50，振荡混匀后倒平板，平板的厚度约为12mm,需要比较薄，冷却凝固，4保存备用。培养基属于高浓度琼脂固体培养基，培养时可以倒置。（需晾干一些，平板不能叠加放置，防止水分不同）2.1.2.1 游动（Swimming）能力检测51将菌种接种到 LBNS 琼脂平板上，37培养过夜。取单菌落接种到含有0.15%琼脂粉的游动平板表面，每个平板可接种4个菌，每个菌三个重复。将游动平板在 37下培养过夜，之后测量每个平板上细菌游动所形成的半透明区域的直径，记录并进行统计。以PAO1菌株作为正对照，失去鞭毛的P18作为阴性对照。2.2.2颤触运动（twitching）能力检测50, 51将菌种接种从-80到 LBNS 琼脂平板上，37培养过夜。取单菌落穿刺接种到含有 1% 琼脂平板底部，每个板上接种4个，每菌三个重复。细菌在琼脂与塑料板交界面的生长即为颤触运动。在培养基内添加1%的TTC,细菌运动范围就会变成红色。测量每个平板红色区域的直径，记录并进行统计。以PAO1菌株作为正对照，失去菌毛的P77作为阴性对照。2.1.2.3丛集运动（swarming）能力检测52, 53将菌种从-80接种到 LBNS 琼脂平板上，37培养过夜。挑取单菌落接种于LBNS液体培养基，37震荡培养过夜。取1l菌液接种于营养肉汤琼脂平板中央，每个菌做3个重复。将平板在 37下培养过夜。测量每个平板细菌运动区域的直径，记录并进行统计。2.1.2.4 鼠李糖脂分泌检测41在检测丛集运动的同时，在菌体运动范围的外围会出现一个透明的圈，有文献报道，为鼠李糖脂分泌的范围，鼠李糖脂是swarming所必须的一种外在条件。在记录swarming数据的同时记录鼠李糖脂分泌范围，定性的了解鼠李糖脂的分泌水平。2.1.2.5统计学处理实验数据用均数标准差（xs)表示，最后以PAO1运动能力为标准分为三个等级，运动能力与PAO1运动能力近似的，用+表示，没有明显运动迹象的为-,显著高于PAO1的用+表示。绿浓菌素和弹性蛋白酶产量的检测（未庆）液体培养时 色素提取测定(如图3-1所示)51从-80的菌种活化，接种于LBNS固体培养基上，之字形划线法得到单菌落。挑取单菌落，接种于5ml液体LBNS培养基中，37200r/min振荡培养48h。3000rpm离心2min,把上清液转移至一个15ml离心管中。加入3mlCHCl3，充分振荡。移除上清，加入1ml0.2M的HCl，混匀，得到一种粉红至深红的液体。测粉红色的反应液的OD520值，记录结果并分析。固体培养时绿浓菌素检测方法（未师兄）glycerol alanine minimal (GA) medium：(1% v/v glycerol, 6g L-alanine, 2 g MgSO4, 0.1 g K2HPO4, 0.018 g FeSO4) for 24 h.Briefly, the agar was collected in a Falcon tube and 10 ml chloroform was added per 12.5 g agar medium. The phenazine pigment was extracted during 2 h incubation at 37 uC, after which 2 ml 0.5 M HCl was added and the mixture shaken vigorously. The pink top layer was removed and its absorbance was measured at 520 nm.Pigment production medium: 4.0 g alanine; 8.0 g leucine, 10 g glycerol; 4.0 g K2HPO4; 2.0 g MgSO4 .7H2O, 0.01 g FeSO4.7H20, final pH 7.0.Glycerol alanine minimal (GA) medium: 1% v/v glycerol, 6 g L-alanine, 2 g MgSO4, 0.1 g K2HPO4, 0.018 g FeSO4.Grow at 37C for 24 h.液体提取示意图3.1.2.2 弹性蛋白酶的测定64-66 从-80的菌种活化，接种于LBNS固体培养基上，之字形划线法得到单菌落。 挑取单菌落，接种于3ml液体LBNS培养基中，37200r/min振荡培养过夜。 在脱脂牛奶培养基上点1l菌液，37培养过夜。 测定弹性蛋白酶分解蛋白所产生的透明圈的直径，记录结果并分析。3.1.2.3 群体感应信号分子检测62如图3-1和图3-2所示。群体感应信号分子检测如下：从-80的菌种活化，接种于适宜固体培养基上，之字形划线法适宜温度（指示菌株30，其他菌株37）得到单菌落。如下图3-2所示，挑取检测菌株在平板边缘划一条直线（一个平板最多三条），而在挑取指示菌株于上述直线中央垂直于该直线划一条直线。30摄氏度培养2d左右，观察结果并记录。图3-2 短链信号分子检测Fig 3-2 Short chain QS signal molecules assay 图3-3 长链信号分子检测Fig 3-3 Long chain QS signal molecules assay图注：1为M23检测结果,与PAO1近似。为“+”；2为M19检测结果与 指示菌本底颜色近似，为“”；3上为PAO1检测结果，下为指示菌株NTL4在添加X-Gal培养基中显色结果。Rapid biofilm assay(Otool et al. Mol. Microbiol. 28:449)1. Culture P.aer O/N in LBNS.2. The following day inoculate 100ml o/n culture in 5ml LBNS or Jensens media.3. Shaking at 37C for 3h until OD600 reach 0.5.4. Add OD600=0.5 culture 100ml (or equal amount cells for culture had lower/higher OD) into a well of 96 well microtiter dish made of PVC (Falcon 3911).5. Incubate at 30 for 30min.6. Remove non attached bacteri a by dumping out media/cells and rinse the wells for 3-4 times by dunking the plate in a tub of water, then dumping the water out of the wells. Pad plate on paper towels to dry out the excess dye. 7. Add 120ml of 0.1% solution of crystal violet.8. Incubate at 30 for 30min. 9. Rinse the wells for 3-4 times as described in step 6.10. Add 200ml of 95% ethanol. Try to solubilize all the CV on the wall.11. Remove 125ml from each well to a fresh polystyrene microtiter dish (Costar) and determine the A560 in a microplate reader.cyclic-di-GMP的HPLC检测（王世伟）一 样品制备：Cells were grown o/n in LBNS and transfer it to Jenson(1:50) to an OD600 of 0.02 in 125 ml baffled shake flasks containing 15ml of VBMM.When cells reached an OD600 of 0.4-0.6 , 30 ml was removed, centrifuged for 8 min at 8000g, and the supernatant was removed.The cell pellet was re-suspended in 200 ul 0.6 M perchloric acid(高氯酸). Samples were incubated on ice for 30 min, and cell debris was removed by microcentrifugation at 4 for 8 min at 8000g. Supernatants(200ul) were removed and samples were neutralized by addition of 20 ul 2.5 M K2CO3.The resulting precipitate was removed by centrifugation at 4 for 5 min at 15000 g.These neutralized supernatants were stored at -80 until analysed by LC-MS.二 HPLC检测：HPLC was performed on a 250*4 mm RP-18 column(merck) at 40, detection at 252 nm; on a Merck-Hitachi liquid chromatograph, Run were carrede out in 0.15 M NaH2PO4, pH 5.2,at 1 ml min-1 ,using a multi-step gradient of acetonitrile (steps are isocratic, unless otherwise stated); 1 min(0-2% linear gradient), 1-12 minutes (2%) : 12-15 minutes(2-6% linear gradient), 15-20minutes(6%), 20-25 minutes(6-0% linear gradient), Biofilm Antibiotic Resistance AssayMaterial: Microtiter dish Jensens Media 50% arabinose 0.85% Nacl saline Sterile beaker (2)Procedure:1. Inoculate 1ul culture into a Microtiter dish well containing 99ul Jensens media W/O arabinose.2. Incubate 24h at 30C.3. Remove non-attached cells by clumping out the media and rinsing wells once in the saline.4. Add 100ul/well Jensens media with antibiotic and corresponding amount arabinose.5. Incubate 24h at 30C.6. Removed media and rinse wells once in saline.7. Add 100ul Jensens media into each well.8. Incubate 24h at 30C.9. Take viable count by plating 5ul culture from each well on a LBNS plate.10. Count the number of colonies grown on the plates.Congo Red agar plate2% (vol/vol) Congo red solution into media with 1% agar (kolter) or 1.6% agar (Parsek). Congo red solution: 2mg/ml Congo red 1mg/ml Coomassie brilliant blue 70% (vol/vol) ethanolCongo red assaySamples 1, 5, or 10ul of OD600 =0.025 culture are plated and grown at room temperature to assess colony morphology (kolter).In liquid media, 40ug/ml Congo red will be used.Electrophoretic Mobility Shift Assay (EMSA) Materials-5X Binding Buffer (20 mM Tris-HCl, pH 8.0; 200 mM NaCl; 20 mM MgCl2; 20% glycerol); store at 4C-Source of nonspecific competitor DNA (we use Poly d(I-C) at 1.5 g/l from Amersham)-Sterile water-Source of protein (can be pure protein or from whole-cell extracts)-Labeled DNA-Running buffer: 0.5X TBE Pre-running the gel-Pre-run a small gel (8 x 10 cm) for 30 minutes at 200V in 4C cold room in 0.5X TBE. DNA-Binding Reactions-Prepare ten standard 10-ml DNA binding reactions for small gels (8 x 10 cm). -Protein Sources: The negative control reaction contains no protein (NP). The “Vector Control” reaction utilizes a whole cell extract made from E. coli that contains the protein expression vector only. Samples 1-8 utilize increasing amounts of a whole cell extract (5 g/l) containing wildtype AlgZ; this is not a source of pure protein. The “1/10” designation means a 1:10 dilution of the stock protein extract. (-) ControlVector Alone123456785X Binding Buffer2 ml2 ml2 ml2 ml2 ml2 ml2 ml2 ml2 ml2 mlPoly d(I-C) (1.5g/l)0.5 ml0.5 ml0.5 ml0.5 ml0.5 ml0.5 ml0.5 ml0.5 ml0.5 ml0.5 mldH2O6.5 ml6 ml6 ml5.5 ml5.2 ml4.5 ml3.5 ml2.5 ml2.1 ml6 mlProtein-0.5 ml0.5 ml(1/10)1 ml(1/10)1.3 ml(1/10)2 ml(1/10)3 ml(1/10)4 ml(1/10)4.4 ml(1/10)0.5 mlLabeled DNA1 ml1 ml1 ml1 ml1 ml1 ml1 ml1 ml1 ml1 ml10 ml10 ml10 ml10 ml10 ml10 ml10 ml10 ml10 ml10 ml-Incubate DNA-binding reactions at room temperature for 10 minutes.-Load samples-Run gel for 30-45 minutes (depending on the size of your DNA fragment) at 200V in 4C cold room in 0.5X TBE running buffer-Dry gel for 20 minutes at 80C in vacuum gel dryer-Expose to film or phosphorimaging screen Results - V 1 2 3 4 5 6 7 8ELISA protocol for Psl bound to plates1. Prepare samples. If rapid EPS prep, spin down equal ODs of culture and resuspend in 100L 0.5M EDTA. Boil 5 min at 100C. Remove 100 L supernatant and to this add 2.5L 20mg/mL proteinase K. Incubate 1h at 60C, then inactivate 30 min. at 80C. Dilute samples 1:100 (for triplicate samples, add 5L to 495L PBS).2. Add 100L diluted sample (plus 5, 0.5, 0.05 g Psl standards and PBS blank) to appropriate wells and allow to attach overnight at 4C. Samples and blank in triplicate, standards in at least duplicate (Nunc Elisa plate: Nunc 449824, Fisher: 12-565-137)3. Wash 3x with 200L per well PBS + 0.05% Tween-20.4. Block with 200L per well PBS + 10% NCS overnight (or overday) at 4C.5. Wash 3x with 200L per well PBS + 0.05% Tween-20.6. Add 100L primary anti-Psl antibody at 1:25000 in PBS (Make 1:10000 with 0.5L in 5 mL, then dilute 2.5x) 2h at 4C.7. Wash 5x with 200L per well PBS + 0.05% Tween-20.8. Add 100L secondary donkey anti-rabbit HRP antibody at 1:10000 in PBS 1h at RT.9. Wash 7x with 200L per well PBS + 0.05% Tween-20.10. Add 100L TMB substrate (tetramethylbenzidine) for 30 min. at RT.11. Stop with 50L 2N sulfuric acid and read at 450nm within 30 min.Biparental Spot Mating（启动子加lacZ）-Plate out E.coli SM10 strain containing algZ-lacZ transcriptional fusion (Woz# 1611; pDJW647; Tc15) and chosen Pseudomonas strain(s)-Select isolated colony, and make overnight cultures or grow to late log phase (overday)-For the spot mating (5-6 spots per mating):-Drop 7l of Pseudomonas strain on LANS plate; allow spot to dry-Drop 7l of SM10 strain over the spot and allow to dry-Incubate matings at 37C overnight-Streak spot matings for isolation on LANS + Tet100 + Irg25 (selects for insert but kills E.coli)-Incubate overnight at 37C-Streak out E.coli SM10 strain containing pFLP2 plasmid (Woz#1303; Amp)-Incubate overnight at 30CPCR screen can be performed on isolated clones using attB2 and CTX1 as primers, giving a 950bp product-Choosing isolated colonies, make overnight cultures or grow to late log phase (overday)-For the spot mating (5-6 spots per mating):-Drop 7l of Pseudomonas strain on LANS plate; allow spot to dry-Drop 7l of SM10 strain over the spot and allow to dry-Incubate matings at 37C overnight-Streak spot matings for isolation on LANS + Carbenicillin250 + Irg25-Incubate overnight at 37C-Pass isolated colonies on LANS-Incubate overnight at 37C-To get rid of the pFLP2 plasmid, streak isolated colonies on LANS + 5% sucrose-Incubate overnight at 30C-Patch clones on LANS, LANS + Carbenicillin 250, and LANS + Tet100-Positive screening control at this step is Timna Strain #66-Incubate overnight at 37C-Look for colonies that are Carbenicillin sensitive (loss of pFLP2) and Tet sensitive (loss of mini-CTX-lacZ)-Screen Crbs/Tets clones by PCR from colony-Use attB4 and attB5 as primers (yields 450bp product)-Timnas Strain #66 serves as a positive screening controlPellicle assay1. 过夜培养PAO1。2. 转接PAO1至jenson培养基，培养3h至OD600 0.5.3. 向24孔板中加入300ul培养液，30吸附1h4. 弃去24孔板中的培养基，加入1ml新鲜jenson培养基，适当温度培养适当时间（为防止营养缺失对膜产生影响，可以放至室温条件过夜培养，及时观察）。5. 膜长好后小心弃去培养液，只留下膜。将膜小心挑到1.5ml离心管中，加适量水测其OD600值。Sytox Green染色观察菌落（朱斌）1. B. subtilis strains were picked from overnight growth on LB plates and cultured in LB at 37 C. 2. Saturated culture was grown in MSgg5 mM potassium phosphate (pH 7.0), 100 mM 3-(N-morpholino)propane-sulfonic acid (pH 7.0), 2 mM MgCl2, 700 M CaCl2,50 M MnCl2, 100 M FeCl3,1 MZnCl2,2 M thiamine, 0.5% glycerol, 0.5% glutamate for 1 h and wasthen spotted on an MSgg plate (MSgg medium supplemented with 1.5%agar, 3 mm thickness, dried overnight) and incubated at 30 C. 3. When appropriate, Sytox deadcell stain (Invitrogen) was supplemented into MSgg medium, with a nalconcentration of 0.5 M and 1 M for Sytox Green and Blue (Invitrogen),respectively. 4. For cross-section images of