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Measurement of Intracellular cAMP Levels Using the Cyclic Nucleotide XP Enzymatic Immunoassay Kit in Bacteria
使用环核苷酸XP酶免疫分析试剂盒测量细菌的胞内cAMP含量   

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Abstract

Cyclic AMP (cAMP) is a ubiquitous secondary signaling molecule, commonly associated with many bacterial processes, including the regulation of virulence factors, such as biofilms, pellicles and motility (Wolfgang, 2003). The quantity of available cAMP is controlled by the interplay between the synthesis of adenosine triphosphate (ATP) to cAMP by adenylyl cyclases, and the degradation of cAMP by phosphodiesterase (McDonough et al., 2012). Adequate quantification of cAMP levels within a bacterial cell is an important step in identifying the impact that secondary signaling molecules play on the regulatory pathway within the cell. The principle of this method is to measure total cAMP levels within a bacterial cell, using crude bacterial whole cell lysate. The Cyclic AMP XPTM Assay kit used in this protocol was originally designed to be used for determining the level of cAMP in eukaryotic cells, however, the antibodies used in coating the wells will react with cAMP from any species and thus can be used for determining levels in bacterial cells. The measurement of cAMP in prokaryotic cells described here is a simple and cost effective method of producing quantifiable results.

Materials and Reagents

  1. 1 ml cuvettes (SARSTEDT AG & Co., catalog number: 67.742 )
  2. 15 ml tubes (Falcon Tube) (DKSH, catalog number: LP021015 )
  3. 50 ml tubes (Falcon Tubes) (DKSH, catalog number: LP21050 )
  4. 96-well microtitre tray (tissue culture plate) (DKSH, catalog number: LP031096 )
  5. Aluminum foil
  6. Bacterial cell culture
  7. Mueller Hinton (MH) broth (prepared as per manufacturer’s instructions) (Oxoid Australia, catalog number: CM0405 )
  8. MH agar media (25 ml/Petri dish) (prepared as per manufacturer’s instructions) (Oxoid Australia, catalog number: CM0337 )
  9. Cyclic AMP XPTM Assay kit (Cell Signaling Technology, catalog number: 4339 )
  10. Protein assay DCTM reagents A & B (Bio-Rad Laboratories, catalog number: 500-121 )
  11. Protein assay bovine serum albumin (BSA) standards (0, 0.125, 0.25, 0.5, 1, 2.5, 5 mg/ml of protein)
  12. Milli Q water
  13. Sodium Chloride (NaCl) (Chem Supply, catalog number: SA046-5 kg )
  14. Potassium Chloride (KCl) (Thermo Fisher Scientific, Ajax Finechem, catalog number: A383-500 g )
  15. Sodium Phosphate (Na2HPO4) (Merck Millipore Corporation, catalog numer: 7558-79-4 )
  16. Potassium Phosphat (KH2PO4) (Merck Millipore Corporation, catalog number: 7778-77-0 )
  17. Phosphate buffered saline (PBS) (see Recipes)

Equipment

  1. VIS spectrophotometer able to read OD600 nm [e.g. DU®640 (Beckman Coulter)]
  2. ELISA plate reader able to read absorption at 450 nm [e.g. Multiskan EX original (Adelab Scientific)]
  3. French pressure cell able to operate at 30,000 psi or cell disruptor [e.g. One Shot Head from the TS series bench top disruptor (Constant Systems)]
  4. Ice bucket
  5. Sterile inoculating loop
  6. Shaking incubator (37 °C, at 200 rpm)
  7. Centrifuge (able to hold 50 ml tubes, spin at 8,000 x g and hold a pre-cooled temperature of 4 °C) [e.g. Benchtop Centrifuge Hermle Z383K (DKSH)]
  8. Horizontal orbital shaker (at ~30 rpm) [e.g. OM5 (Ratek, Adelab Scientific)]

Software

  1. Software for use with the ELISA plate reader that can export raw OD600 nm values (e.g. Microsoft Excel)
  2. GraphPad prism (GraphPad Software Inc)

Procedure

  1. Grow an overnight culture (~16 h) of the required strain on an agar plate, including any antibiotic selection if required. Prepare MH broth media for culture and sterile PBS.
  2. Next Day: Under sterile conditions, using a sterile inoculating loop remove a single colony from the agar plate and inoculate 5 ml of MH broth in a 50 ml tube including any antibiotic selection if required. Grow culture overnight (~16 h) at 37 °C at 200 rpm.
  3. Next Day: Make a 1:100 dilution of the overnight culture into 15 ml of MH broth in a 50 ml tube. Grow culture at 37 °C shaking at 200 rpm until the culture reaches an OD600 = 0.7 (approximately 3-4 h), measure using a spectrophotometer.
  4. While the bacterial culture is growing, pre-cool the centrifuge to 4 °C, prepare ice bucket, pre-cool PBS on ice.
  5. Remove bacterial cells from incubator and put on ice, centrifuge sample for 20 min, 4 °C at 8,000 x g, decant supernatant and keep cell pellet on ice; sample can be frozen at -20 °C for up to 6 months.
  6. Resuspend the pellet in 3 ml of cooled PBS buffer and keep sample on ice.
  7. Set up cell disruptor with a one shot head (pre-cooled to 4 °C); set to 30,000 psi, and process sample, collect lysed cells into a new 15 ml tube on ice.
  8. Centrifuge sample at 8,000 x g for 20 min at 4 °C, remove the supernatant and store at -80 °C until required, up to 6 months.
  9. Remove 10 μl of sample to quantify total protein in sample using the Protein assay DCTM kit following manufacturer’s instructions.
  10. Using a microtitre tray, add 20 μl of Reagent A across 7 wells of row A; this is to be used for the BSA standard samples. Add 20 μl of Reagent A across 3 wells of row B; this is to be used for your sample.
  11. To the 20 μl of Reagent A in row A, add 5 μl of each BSA protein standard (0, 0.125, 0.25, 0.5, 1, 2.5, 5 mg/ml) to the 7 individual wells. To the 20 μl of Reagent A in row B, add 5 μl of your sample to each of the three wells.
  12. Subsequently add 200 μl of Reagent B into all wells used and leave samples to react for 3 min at room temperature.
  13. Measure the absorbance of the wells using a Multiskan at 600 nm; create a standard curve using the BSA protein standards in an Excel spread sheet. Then using the equation of the line calculate the amount of protein in your sample. See Note 3 for experimental details, as well as Table 1 and Figure 1 as a representative example.
  14. Using the calculated protein concentration of your samples from step 13 adjust the final protein concentration to 1 mg/ml in PBS for cAMP analysis.
  15. Put all Cyclic AMP XPTM assay kit solutions and the cAMP XPTM Rabbit mAb coated microwell strips at room temperature.
  16. Dilute the 20x wash buffer in Milli-Q to 1x buffer.
  17. Prepare a 1x cell lysis buffer from the 10x stock solution provided in Milli-Q water, then add 1 mM of phenylmethylsulfonyl fluoride provided.
  18. Make fresh cAMP standards in 1x cell lysis buffer: In 450 μl of 1x cell lysis buffer add 50 μl of cAMP standard (2.4 M) to make 240 nM of cAMP.
  19. Using the 240 nM cAMP solution make a 1:3 serial dilution in the cell lysis buffer producing the following concentrations: 80 nM, 26.7 nM, 8.9 nM, 3.0 nM, 1 nM, 0.3 nM and 0 nM.
  20. For the unknown samples add 50 μl of the crude protein extract (at 1 mg/ml) in triplicate into the microwell strips. Subsequently, add 50 μl of provided horseradish peroxidase-linked cAMP solution to all standards and samples. Cover with aluminum foil and incubate at room temperature for 3 h on a horizontal orbital plate shaker (~30 rpm).
  21. Tip out the solution in the microwell strips and wash 4 times with 200 μl/well of 1x wash buffer; discard all liquid each time but do not allow wells to dry.
  22. Add 100 μl of 3, 3', 5, 5'-tetramethylbenzidinesubstrate solution provided in the kit and incubate the plate at room temperature for 30 min. Continue to monitor the wells as they will have a transparent color at the start of the reaction which will change to a yellow. Monitor wells for color change and when an intense yellow color is seen the reaction should be stopped; this may be necessary to do prior to the 30 min incubation time-frame described in the kit.
  23. Add 100 μl of stop solution and then immediately measure the absorbance at 450 nm see representative data 3 for example (Table 2 and Figure 2).
  24. Using the inverse for the standards derived from a standard curve, and adjusted for the amount of total protein added from the protein assay; calculate the amount of cAMP in nM/1 mg of crude protein extract.

Representative data

  1. A representative experiment demonstrating quantification of cAMP in the bacterium Acinetobacter baumannii can be found in Giles et al. (2015) Investigation of genes essential for pellicle formation in Acinetobacter baumannii.
  2. Below is an example of the excel BSA standard curve.

    Table 1. BSA protein standards and their relative absorbance at OD600

    aAbsorbance was determined using a Protein Assay DCTM kit and read using a Multiskan EX. Data was subsequently plotted as seen in Figure 1.


    Figure 1. BSA protein standard curve. The absorbance at OD600 (nm) was determined for a range of BSA protein standards from 0-5 mg/ml. Protein concentration was determined using the Protein Assay DCTM kit. The line of best fit was plotted using y=mx+c (Excel 2003, MicrosoftTM).

  3. A representative of the cAMP standards diluted in 1x cell lysis buffer is on page 1 of the Cyclic AMP XPTM assay kit manufacturer’s instruction; an example (Table 2 and Figure 2) is also provided below.

    Table 2. cAMP standards and their relative absorbance at OD450 (nm)

    aThe amount of cAMP (nM) was determined using the Cyclic AMP XPTM Assay kit and absorbance read using a Multiskan EX. Data was subsequently plotted as seen in Figure 2.


    Figure 2. cAMP concentration standard curve. The absorbance at OD450 (nm) was determined for a range of cAMP standards from 0.3-80 nM. cAMP concentration was determined using the Cyclic AMP XPTM Assay kit. The line of best fit was plotted using y=mln(x)+c (Excel 2003, MicrosoftTM). Using this standard curve the quantity of cAMP can be determined for cell lysates.

Notes

  1. This protocol is optimized for measuring intracellular cAMP levels in Acinetobacter baumannii. For other bacteria, use the appropriate growth medium, antibiotics, and growth conditions.
  2. The method for reading OD600: Under sterile conditions pipette 1 ml of culture into a 1 ml cuvette and measure cellular density at an OD600 length in the spectrophotometer; repeat this every 1 h or until the culture reaches the required OD600 = 0.7.
  3. Protein assay using the Multiskan at OD600: after waiting 3 min at room temperature measure the samples in the microtitre tray, then output the data to an excel spread sheet for data manipulation and save. Spread sheet 1 should contain your Multiskan raw data. Then in spread sheet 2 column A row 1 type in BSA standard, column B type in OD600; In column A rows 2-8 type in the BSA standards numbers, in column B type in the Multiskan reads for the BSA standards (see Table 1). Then highlight all the data and make a scatter plot with no markers, add a trend line, add the equation of the line as well as a title and axis labels (see Figure 1). Your samples are in triplicate, average your samples, then insert your average number into the equation of the line as the Y intersect and find X: example Y = 0.066 X + 0.0732: if Y is 0.2, then 0.2 - 0.0732 = 0.1268/0.066 =1.92 mg/ml of protein in your sample.
  4. When waiting for the color change to occur from clear to yellow before adding the stop solution, it is imperative that the microwell plates are continually monitored throughout this reaction. After approximately 15 min, depending on the concentration of cAMP within a sample, the development of an intense yellow color will occur. Once the entire well is yellow add the stop solution.
  5. Cyclic AMP is stable for long periods of time when frozen (approximately 6 months at -20 °C), it can be stored for 2 weeks at 4 °C.
  6. The use of the Constant System one shot head results in reliable disruption of Acinetobacter baumannii cells. The efficiency of cell disruption using other mechanical of chemical methods or the use of differing species will need to be determined empirically.

Recipes

  1. Phosphate buffer saline (PBS)
    For 500 ml
    4.0 grams NaCl
    0.1 grams KCl
    0.7 grams Na2HPO4
    0.1 grams KH2PO4
    Add dH2O up to 500 ml
    pH sample with HCl pH 7.4 final

Acknowledgments

This protocol was modified and adapted from the manufacturer’s instructions specifically for eukaryotic cells using the Cyclic AMP XPTM Assay kit. This project was financially supported by the National Health and Medical Research Council Australia Project Grant 535053 and a Flinders Medical Research Foundation Grant. SKG is the recipient of a Flinders University Research Scholarship (FURS).

References

  1. Giles, S. K., Stroeher, U. H., Eijkelkamp, B. A. and Brown, M. H. (2015). Identification of genes essential for pellicle formation in Acinetobacter baumannii. BMC Microbiol 15: 116.
  2. McDonough, K. A. and Rodriguez, A. (2012). The myriad roles of cyclic AMP in microbial pathogens: from signal to sword. Nat Rev Microbiol 10(1): 27-38.
  3. Wolfgang, M. C., Lee, V. T., Gilmore, M. E. and Lory, S. (2003). Coordinate regulation of bacterial virulence genes by a novel adenylate cyclase-dependent signaling pathway. Dev Cell 4(2): 253-263.

简介

环AMP(cAMP)是普遍存在的次级信号分子,通常与许多细菌过程相关,包括调节毒力因子,如生物膜,薄膜和运动性(Wolfgang,2003)。可用的cAMP的量由腺苷酸环化酶对cAMP的腺苷三磷酸(ATP)合成和磷酸二酯酶对cAMP的降解之间的相互作用控制(McDonough等人,2012)。细菌细胞内cAMP水平的充分量化是鉴定次级信号传导分子对细胞内调节途径起作用的重要步骤。该方法的原理是使用粗细菌全细胞裂解物测量细菌细胞内的总cAMP水平。本方案中使用的环状AMP XP sup试剂盒最初设计用于测定真核细胞中cAMP的水平,然而,用于涂覆孔的抗体将与来自任何物种的cAMP反应并因此可用于测定细菌细胞中的水平。这里描述的原核细胞中cAMP的测量是产生可定量结果的简单且成本有效的方法。

材料和试剂

  1. 1ml比色杯(SARSTEDT AG& Co.,目录号:67.742)
  2. 15ml管(Falcon Tube)(DKSH,目录号:LP021015)
  3. 50ml管(Falcon管)(DKSH,目录号:LP21050)
  4. 96孔微量滴定盘(组织培养板)(DKSH,目录号:LP031096)
  5. 铝箔
  6. 细菌细胞培养
  7. Mueller Hinton(MH)肉汤(按照制造商的说明制备)(Oxoid Australia,目录号:CM0405)
  8. MH琼脂培养基(25ml /培养皿)(根据制造商的说明书制备)(Oxoid Australia,目录号:CM0337)
  9. 循环AMP XP TM 测定试剂盒(Cell Signaling Technology,目录号:4339)
  10. 蛋白质测定DC< sup>试剂A& B(Bio-Rad Laboratories,目录号:500-121)
  11. 蛋白质测定牛血清白蛋白(BSA)标准品(0,0.125,0.25,0.5,1,2.5,5mg/ml蛋白质)
  12. Milli Q水
  13. 氯化钠(NaCl)(Chem Supply,目录号:SA046-5kg)
  14. 氯化钾(KCl)(Thermo Fisher Scientific,Ajax Finechem,目录号:A383-500g)
  15. 磷酸钠(Na 2 HPO 4)(Merck Millipore Corporation,目录编号:7558-79-4)
  16. 磷酸钾(KH 2 PO 4)(Merck Millipore Corporation,目录号:7778-77-0)
  17. 磷酸盐缓冲盐水(PBS)(见Recipes)

设备

  1. VIS分光光度计,能够读取OD 600nm [例如640(Beckman Coulter)]。
  2. ELISA平板阅读器能够在450nm读取吸光度[例如Multiskan EX原件(Adelab Scientific)]
  3. 法国压力传感器能够在30,000 psi或者细胞破碎器下操作[例如来自TS系列台式破碎机(恒定系统)的单射头]
  4. 冰桶
  5. 无菌接种环
  6. 摇匀培养箱(37℃,200rpm)
  7. 离心机(能够容纳50ml管,以8,000×g旋转并保持4℃的预冷却温度)[例如 Benchtop Centrifuge Hermle Z383K(DKSH)] < br />
  8. 水平轨道摇床(?30rpm)[例如OM5(Ratek,Adelab Scientific)]

软件

  1. 用于ELISA读板仪的软件,可以导出原始OD600 nm值(例如Microsoft Excel)
  2. Graphpad prism(Graphpad Software Inc)

程序

  1. 在琼脂平板上生长所需菌株的过夜培养物(约16小时),包括任何抗生素选择(如果需要)。准备用于培养的MH肉汤培养基和无菌PBS
  2. 第二天:在无菌条件下,使用无菌接种环从琼脂平板除去单个菌落,并接种在50ml管中的5ml MH肉汤,包括任何抗生素选择(如果需要)。在37℃下以200rpm生长培养物过夜(?16h)
  3. 次日:将过夜培养物的1:100稀释液置于50ml管中的15ml MH肉汤中。在37℃下以200rpm振荡培养培养物直至培养物达到OD 600 = 0.7(约3-4小时),使用分光光度计测量。
  4. 当细菌培养物生长时,将离心机预冷至4℃,制备冰桶,在冰上预冷PBS。
  5. 从培养箱中取出细菌细胞并置于冰上,以8,000×g离心样品20分钟,4℃,倾析上清液并将细胞沉淀保存在冰上;样品可以在-20℃下冷冻最多6个月
  6. 将沉淀重悬于3ml冷却的PBS缓冲液中,并将样品保存在冰上
  7. 用一次性头设置细胞破碎器(预冷却至4℃);设置为30,000psi,并且处理样品,在冰上将溶解的细胞收集到新的15ml管中。
  8. 在4℃下以8,000×g离心样品20分钟,除去上清液并在-80℃下储存,直到需要时,最多6个月。
  9. 取出10μl样品,使用蛋白质测定DC TM 试剂盒按照生产商的说明书定量样品中的总蛋白。
  10. 使用微量滴定盘,在A行的7个孔中加入20μl试剂A;这是用于BSA标准样品。在B行的3个孔中加入20μl的试剂A;这将用于您的样品。
  11. 向20μlA行中的试剂A中,向7个单独的孔中加入5μl的每种BSA蛋白标准品(0,0.125,0.25,0.5,1,2.5,5mg/ml)。向20μlB行中的试剂A中,向每个孔中加入5μl样品
  12. 随后向所有使用的孔中加入200μl试剂B,使样品在室温下反应3分钟
  13. 使用Multiskan在600nm测量孔的吸光度;使用BSA蛋白质标准品在Excel电子表格中创建标准曲线。然后使用线的方程计算样品中的蛋白质的量。参见注释3的实验细节,以及作为代表性实例的表1和图1
  14. 使用计算的来自步骤13的样品的蛋白质浓度,将PBS中的最终蛋白质浓度调节至1mg/ml以用于cAMP分析。
  15. 将所有Cyclic AMP XPTM测定试剂盒溶液和cAMP XPTM兔mAb涂布的微孔条在室温下。
  16. 将Milli-Q中的20x洗涤缓冲液稀释为1x缓冲液
  17. 从Milli-Q水中提供的10x储备液中制备1x细胞裂解缓冲液,然后加入1mM苯甲基磺酰氟。
  18. 在1x细胞裂解缓冲液中制备新鲜的cAMP标准品:在450μl的1x细胞裂解缓冲液中加入50μlcAMP标准品(2.4 M)以制备240 nM cAMP。
  19. 使用240nM cAMP溶液在细胞裂解缓冲液中进行1:3连续稀释,产生以下浓度:80nM,26.7nM,8.9nM,3.0nM,1nM,0.3nM和0nM。
  20. 对于未知样品,将50μl粗蛋白提取物(1mg/ml)一式三份加入微孔板中。随后,向所有标准品和样品中加入50μl提供的辣根过氧化物酶连接的cAMP溶液。用铝箔覆盖并在室温下在水平轨道板振荡器(?30rpm)上孵育3小时。
  21. 提取微孔条中的溶液,并用200μl/孔的1x洗涤缓冲液洗涤4次;每次丢弃所有液体,但不允许井干
  22. 加入100μl提供的试剂盒中的3,3',5,5'-四甲基联苯胺底物溶液,并在室温下温育平板30分钟。继续监测孔,因为它们将在反应开始时具有透明的颜色,这将变成黄色。监测孔的颜色变化,当看到强烈的黄色时,应停止反应;这可能需要在试剂盒中描述的30分钟孵育时间框架之前进行
  23. 加入100μl终止液,然后立即测量450 nm处的吸光度,参见代表性数据3(表2和图2)。
  24. 使用来自标准曲线的标准的倒数,并对从蛋白质测定中添加的总蛋白的量进行调整;计算cAMP的量,以nM/1mg粗蛋白提取物计

代表数据

  1. 表明细菌鲍氏不动杆菌中cAMP定量的代表性实验可以在Giles等人(2015)对鲍氏不动杆菌中的薄膜形成所必需的基因的研究
  2. 以下是excel BSA标准曲线的实例。

    表1. BSA蛋白标准品及其在OD 600的相对吸光度

    使用蛋白测定试剂盒测定吸光度并使用Multiskan EX读数。随后绘制数据,如图1所示。


    图1. BSA蛋白标准曲线。 对于范围为0-5mg/ml的BSA蛋白标准品,测定OD 600nm(nm)处的吸光度。使用蛋白质测定DCTM试剂盒测定蛋白质浓度。使用y = mx + c(Excel 2003,Microsoft TM)绘制最佳拟合线。

  3. 在1x细胞裂解缓冲液中稀释的cAMP标准品的代表在Cyclic AMP XP TM supec TM测定试剂盒制造商的说明书的第1页;下面还提供了一个示例(表2和图2)。

    表2.cAMP标准品及其在OD 450nm(nm)的相对吸光度

    a 使用Cyclic AMP XP TM测定试剂盒测定cAMP的量(nM),并使用Multiskan EX读取吸光度。随后绘制数据,如图2所示。


    图2.cAMP浓度标准曲线。对于范围从0.3-80nM的cAMP标准品测定OD 450nm(nm)处的吸光度。 cAMP浓度使用Cyclic AMP XPTM Assay试剂盒测定。使用y = mln(x)+ c(Excel 2003,Microsoft TM )绘制最佳拟合线。使用该标准曲线,可以测定细胞裂解物的cAMP的量

笔记

  1. 该方案被优化用于测量鲍曼不动杆菌中的细胞内cAMP水平。对于其他细菌,使用适当的生长培养基,抗生素和生长条件
  2. 读取OD 600的方法:在无菌条件下,将1ml培养物吸入1ml比色杯中,并在分光光度计中测量OD 600长度的细胞密度;每1小时重复一次或直到培养物达到所需的OD <600> <0.7>
  3. 在OD 600下使用Multiskan的蛋白质测定:在室温下等待3分钟后,测量微量滴定板中的样品,然后将数据输出到excel电子表格以进行数据操作并保存。扩展表1应包含您的Multiskan原始数据。然后在电泳片2柱A中,1号BSA型标准品,B柱型号OD 600;在A列中,行2-8键入BSA标准品编号,在B列中显示BSA标准品的Multiskan读数(见表1)。然后突出显示所有数据,制作没有标记的散点图,添加趋势线,添加线的方程以及标题和轴标签(参见图1)。你的样本是一式三份,平均你的样本,然后插入你的平均数到线的方程作为Y相交,找到X:例如Y = 0.066 X + 0.0732:如果Y是0.2,则0.2 - 0.0732 = 0.1268/0.066 = 1.92 mg/ml蛋白质在您的样品中
  4. 当在加入终止溶液之前等待颜色从透明变为黄色时,必须在整个反应过程中连续监测微孔板。在约15分钟后,取决于样品中cAMP的浓度,将发生强烈的黄色。一旦整个井是黄色的,添加停止溶液。
  5. 环状AMP当冷冻时(在-20℃下约6个月)可长期稳定,其可在4℃下储存2周。
  6. 使用恒定系统单击头导致鲍曼不动杆菌细胞的可靠破坏。使用其他机械化学方法或使用不同物种的细胞破碎的效率需要根据经验确定。

食谱

  1. 磷酸盐缓冲液(PBS)
    对于500毫升
    4.0克NaCl
    0.1克KCl
    0.7克Na 2 HPO 4
    0.1克KH 2 PO 4 sub/
    O高达500毫升
    pH为7.4的pH样品最终

致谢

该方案根据制造商的说明书专门针对真核细胞使用Cyclic AMP XP TM supay TM测定试剂盒进行修改和改编。该项目得到国家卫生和医学研究委员会澳大利亚项目拨款535053和弗林德斯医学研究基金会赠款的资助。 SKG是弗林德斯大学研究奖学金(FURS)的接受者。

参考文献

  1. Giles,S.K.,Stroeher,U.H.,Eijkelkamp,B.A。和Brown,M.H。(2015)。 鉴定鲍氏不动杆菌中的薄膜形成所必需的基因。 BMC Microbiol 15:116.
  2. McDonough,K.A。和Rodriguez,A。(2012)。 环AMP在微生物病原体中的无数作用:从信号到剑。 Nat Rev Microbiol 10(1):27-38。
  3. Wolfgang,M.C.,Lee,V.T.,Gilmore,M.E.and Lory,S。(2003)。 通过新型腺苷酸环化酶依赖性信号通路协调细菌毒力基因的调节。 em> Dev Cell 4(2):253-263。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Giles, S. K., Stroeher, U. H. and Brown, M. H. (2016). Measurement of Intracellular cAMP Levels Using the Cyclic Nucleotide XP Enzymatic Immunoassay Kit in Bacteria. Bio-protocol 6(8): e1792. DOI: 10.21769/BioProtoc.1792.
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