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[14C] Linoleic Acid Uptake and Fractionation Assay in Vibrio cholerae
霍乱弧菌中[14C] 亚油酸的摄取和分级分离试验   

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Abstract

The gram-negative curved bacillus Vibrio cholerae (V. cholerae) causes the severe diarrheal illness cholera. The work presented here is to assess whether unsaturated fatty acids (UFAs), such as linoleic acid, have the potential to directly affect proteins involved in DNA binding because they are able to enter the cell. In this protocol, we show how to measure linoleic acid entering V. cholerae when added exogenously and determine whether it is able to enter the cytoplasm. This protocol will quantify how much linoleic acid is able to enter the cell and then identify the amount of linoleic acid that stays in the membrane or ultimately enters the cytoplasm.

Keywords: Vibrio cholerae(霍乱弧菌), Cholera(霍乱), Linoleic acid(亚油酸), Unsaturated fatty acids(不饱和脂肪酸)

Materials and Reagents

  1. Scintillation vials (Thermo Fisher Scientific, catalog number: 03-337-20 )
  2. Autoclaved 1.7 ml microcentrifuge tubes (BioExpress, catalog number: C-3262-1 )
  3. Test Tubes (Thermo Fisher Scientific, catalog number: 14-955E )
  4. 15 ml conical screw cap tubes (BioExpress, catalog number: C-3394-2 )
  5. Vibrio cholerae classical biotype strain O395
  6. 14C linoleic acid (PerkinElmer, catalog number: NEC501050UC )
  7. Scintillation cocktail (Thermo Fisher Scientific, catalog number: SX18-4 )
  8. Tryptone (Thermo Fisher Scientific, catalog number: B211705 )
  9. Yeast Extract (Thermo Fisher Scientific, catalog number: B288620 )
  10. Sodium chloride (NaCl) (Thermo Fisher Scientific, catalog number: BP358-212 )
  11. Potassium chloride (KCl) (Thermo Fisher Scientific, catalog number: BP366-500 )
  12. Sodium phosphate (Na2HPO4) (Thermo Fisher Scientific, catalog number: BP332-500 )
  13. Potassium phosphate (KH2PO4) (Thermo Fisher Scientific, catalog number: BP362-500 )
  14. Tris-Base (Thermo Fisher Scientific, catalog number: BP152-500 )
  15. 95% ethanol (Decon Labs, catalog number: 2805HC )
  16. Dry ice
  17. LB-Lennox (pH 6.5) (see Recipes)
  18. 10x PBS (see Recipes)
  19. 20 mM Tris-Base (pH 8.5) (see Recipes)
  20. 500 mM NaCl (see Recipes)

Equipment

  1. Shaker-capable of shaking at 200 rpm at 37 °C (VWR International, New Brunswick Scientific, model: Excella E25 )
  2. Water bath shaker-capable of shaking at 30 °C at 200 rpm (VWR International, New Brunswick Scientific, model: Classic C76 )
  3. LS6000IC liquid scintillation counting system (Beckman Coulter)
  4. Timer
  5. Autoclave
  6. Biomate 3S Spectrophotometer-capable of reading at OD600 (Thermo Fisher Scientific)
  7. Semimicro Cuvettes (Thermo Fisher Scientific, catalog number: 14-955-127 )
  8. Table top Centrifuge-capable of spinning at 15,000 rpm at 4 °C (Eppendorf, catalog number: 5424 )
  9. Micropipettes (1,000 μl, 200 μl, 20 μl)
  10. 250 ml Erlenmeyer flask

Procedure

  1. [14C] linoleic acid uptake
    1. Use one colony of V. cholerae classical biotype strain O395 to start an overnight culture in a test tube containing 5 ml standard LB at 37 °C and 200 rpm shaking.
    2. After overnight growth, subculture the bacteria 250 μl in 10 ml LB pH 6.5 (1:40 ratio) in an Erlenmeyer flask, and grow for 2 h in a water bath shaker at 30 °C.
    3. At 2 h, record the OD600 of the culture. Expect an OD600 of between 0.2 and 0.3.
    4. Transfer the culture to a 15 ml falcon tube and add 0.1 μCi (1 μl) of 14C-radiolabeled linoleic acid for each milliliter of the subculture (final concentration of about 3.2 mM linoleic acid). This tube can stay on the bench top for the duration of the experiment or, if desired, put at 37 °C without agitation between aliquots.
    5. Upon addition of the radiolabeled linoleic acid, extract 1 ml of the culture and immediately centrifuge in a 1.5 ml microcentrifuge tube at 15,000 rpm for 3 min at room temperature. This is t = 0. In order to compare the counts per minute (CPM) of 14C in the supernatant and the cell pellet, transfer the supernatant in a 1.5 ml tube and wash the cell pellet 3 times with 1 ml of 1x PBS and centrifuge each for 3 min.
    6. Resuspend the cell pellet in 100 μl of 1x PBS and add to 5 ml of scintillation cocktail per scintillation vial. In order to compare the amount of 14C-radiolabeled linoleic acid not taken up by V. cholerae, add 100 μl of the supernatant fraction to another vial with 5 ml scintillation cocktail.
    7. The same procedure can be repeated for other aliquots of the subculture at times 5, 15, and 30 min or whatever time points are desired. All scintillation vials can be stored at room temperature and read together after the last time point.
    8. After uptake, counts per minute are measured for each time point using a scintillation counting system.
    9. Data are then analyzed using the cpm determined from the cell pellet fraction and an equation of best fit determined. To determine how much 14C-radiolabeled linoleic acid is not taken up as a check of accuracy, use cpm determined from the supernatant fraction and multiply by 10 as only 100 μl of the 1 ml was used. The amount of 14C-radiolabeled linoleic acid in the cell pellet is graphed below for each time point:


      Figure 1. 14C-radiolabeled linoleic acid uptake by V. cholerae. The equation of best fit is y = 24622 + 156786*(1 - e-0.036x) with an R2 value of 0.989.

  2. Fractionation of V. cholerae to determine localization of [14C] linoleic acid
    1. After overnight growth at 37 °C as described in step A1, subculture V. cholerae O395 classical biotype 250 μl in 10 ml LB (pH 6.5) (1:40 ratio) and grow in a water bath shaker at 30 °C for 2 h.
    2. At 2 h, add 0.1 μCi of 14C-radiolabeled linoleic acid to 1 ml of the subculture and incubate at room temperature for 1 h without agitation. This time is chosen to ensure the bacteria are still in log-phase growth.
    3. Harvest the bacteria by centrifugation (15,000 rpm) at room temperature for 3 min and wash the pellet three times with 1 ml 1x PBS at 15,000 rpm for 3 min each time.
    4. Resuspend the bacteria in a 500 μl solution of 20 mM Tris-Base (pH 8.5) and 500 mM NaCl. Freeze the suspension in an ethanol (at least 95%) and dry ice bath for 2 minutes ensuring the tube is fully submerged, and then put at 37 °C until thawed. Repeat this freeze-thaw process for a total of three freeze-thaw cycles to ensure complete fractionation.
    5. Fractionate the bacteria by centrifugation for 10 min at 15,000 x g at 4 °C to separate the membrane and cytoplasm. The cytoplasm is taken as the supernatant and the pellet (cell envelope fraction) is washed 3 times in 500 μl of 1x PBS by centrifuging for 3 min at 15,000 x g. Expect a very small pellet at the bottom of the tube. The amount of 14C-linoleic acid in each fraction is determined by adding the entire fraction to 5 ml scintillation cocktail, followed by measurement of CPM in a scintillation counter.
    6. Data are then analyzed as percent 14C-linoleic acid in each fraction. A representative graph is shown below:


      Figure 2. Percentages of 14C-radiolabeled linoleic acid in cytoplasm and envelope fractions

Recipes

  1. LB-Lennox (500 ml) (pH 6.5)
    5 g tryptone
    2.5 g NaCl
    2.5 g yeast extract
    Add about 400 ml of water to dissolve and mix the components
    pH to 6.5 with HCl and bring up to 500 ml with water
    Autoclave before use
  2. 10x PBS (1 L)
    80 g NaCl
    2 g KCl
    14.4 g Na2HPO4
    2.4 g KH2PO4
    To use, dilute 1:10 (such as 10 ml brought up to 100 ml with autoclaved water)
  3. 20 mM Tris-base (pH 8.5) (100 ml)
    0.24 g Tris-base
    pH to 8.5 with HCl and bring up to 100 ml with water
    Autoclave before use
  4. 500 mM NaCl (100 ml)
    2.92 g NaCl

Acknowledgments

This work was supported by P. H. S. grants K22AI071011 and R56AI093622 and Bill and Melinda Gates Foundation grant OPP1068124 (to J. H. W.).
This work is modified from previous work done in our laboratory (Thomson and Withey, 2014).

References

  1. Plecha, S. C. and Withey, J. H. (2015). Mechanism for inhibition of Vibrio cholerae ToxT activity by the unsaturated fatty acid components of bile. J Bacteriol 197(10): 1716-1725.
  2. Thomson, J. J. and Withey, J. H. (2014). Bicarbonate increases binding affinity of Vibrio cholerae ToxT to virulence gene promoters. J Bacteriol 196(22): 3872-3880.

简介

革兰氏阴性弯曲杆菌霍乱弧菌(<霍乱弧菌)导致严重的腹泻病霍乱。 这里介绍的工作是评估是否不饱和脂肪酸(UFAs),如亚油酸,有可能直接影响参与DNA结合的蛋白质,因为他们能够进入细胞。 在这个协议,我们显示如何测量亚麻酸进入。 霍乱菌,并且确定它是否能够进入细胞质。 该方案将量化亚油酸能够进入细胞多少,然后鉴定保留在膜中或最终进入细胞质的亚油酸的量。

关键字:霍乱弧菌, 霍乱, 亚油酸, 不饱和脂肪酸

材料和试剂

  1. 闪烁瓶(Thermo Fisher Scientific,目录号:03-337-20)
  2. 高压灭菌1.7ml微量离心管(BioExpress,目录号:C-3262-1)
  3. 试管(Thermo Fisher Scientific,目录号:14-955E)
  4. 15ml锥形螺旋盖管(BioExpress,目录号:C-3394-2)
  5. 霍乱弧菌经典生物型菌株O395
  6. 14亚麻油酸(PerkinElmer,目录号:NEC501050UC)
  7. 闪烁混合物(Thermo Fisher Scientific,目录号:SX18-4)
  8. 胰蛋白胨(Thermo Fisher Scientific,目录号:B211705)
  9. 酵母提取物(Thermo Fisher Scientific,目录号:B288620)
  10. 氯化钠(NaCl)(Thermo Fisher Scientific,目录号:BP358-212)
  11. 氯化钾(KCl)(Thermo Fisher Scientific,目录号:BP366-500)
  12. 磷酸钠(Na 2 HPO 4)(Thermo Fisher Scientific,目录号:BP332-500)
  13. 磷酸钾(KH 2 PO 4)(Thermo Fisher Scientific,目录号:BP362-500)
  14. Tris-Base(Thermo Fisher Scientific,目录号:BP152-500)
  15. 95%乙醇(Decon Labs,目录号:2805HC)
  16. 干冰
  17. LB-Lennox(pH 6.5)(参见配方)
  18. 10x PBS(请参阅配方)
  19. 20 mM Tris-Base(pH 8.5)(参见配方)
  20. 500 mM NaCl(见配方)

设备

  1. 能够在37℃下以200rpm振动的振荡器(VWR International,New Brunswick Scientific,型号:Excella E25)
  2. 能够在30℃以200rpm振动的水浴摇动器(VWR International,New Brunswick Scientific,型号:Classic C76)
  3. LS6000IC液体闪烁计数系统(Beckman Coulter)
  4. 计时器
  5. 高压灭菌器
  6. Biomate 3S分光光度计 - 能够在OD 600(Thermo Fisher Scientific)读数
  7. 半微型比色杯(Thermo Fisher Scientific,目录号:14-955-127)
  8. 台式离心机 - 能够在4℃以15,000rpm旋转(Eppendorf,目录号:5424)
  9. 微量移液器(1000μl,200μl,20μl)
  10. 250ml锥形瓶

程序

  1. [14 C]亚油酸吸收量
    1. 使用一个殖民地的 V。霍乱菌经典生物型菌株O395启动 在含有5ml标准LB的试管中于37℃过夜培养 和200rpm摇动
    2. 过夜生长后,继代培养 细菌在10ml LB pH6.5(1:40比例)中的250μl在锥形瓶中, 并在30℃的水浴摇动器中生长2小时。
    3. 在2小时,记录培养物的OD <600>。期望OD <600> 在0.2和0.3之间。
    4. 转移培养到15毫升falcon管,加入0.1μCi(1微升) 的每一毫升传代培养物中的14 C-放射性标记的亚油酸 (终浓度约3.2mM亚油酸)。这管可以留下 在实验期间的台面上,或者如果需要,放置 在37℃,在等分试样之间无搅拌
    5. 加入后 ?放射性标记的亚油酸,提取1ml培养物并立即 ?在1.5ml微量离心管中以15,000rpm离心3分钟 室内温度。这是t = 0。为了比较每个计数 在上清液和细胞沉淀中的14分钟(CPM),转移 ?上清液在1.5ml管中,并用1ml洗涤细胞沉淀3次 ?的1x PBS,并各离心3分钟
    6. 重悬细胞 在100μl的1×PBS中沉淀,并加入5ml闪烁混合物 ?闪烁瓶。为了比较放射性标记的14 C的量 亚油酸不被V吸收。霍乱菌,加入100μl上清液 ?部分与另一个小瓶用5ml闪烁混合物
    7. 的 可以对其他等份的亚文化重复相同的程序 时间5,15和30分钟或任何期望的时间点。所有 闪烁瓶可以在室温下储存并一起阅读 最后一个时间点后。
    8. 摄取后,使用闪烁计数系统测量每个时间点的每分钟计数
    9. 然后使用从细胞沉淀物确定的cpm分析数据 ?分数和最佳拟合方程。为了确定多少14 C-放射性标记的亚油酸没有被用作精确度检查, 使用从上清液部分确定的cpm并乘以10 仅使用100μl的1ml。放射性标记的14 C的量 在每个时间点,细胞沉淀中的亚油酸被绘制在下面:


      图1. 14 C-放射性标记的亚油酸的摄取。霍乱。 最佳拟合方程为y = 24622 + 156786 *(1-e <-0.036x ),其中R 2的值为0.989。

  2. 分馏。霍乱以确定[14 C]亚油酸的定位。
    1. 在如步骤A1中所述在37℃下过夜生长后,继代培养。 霍乱菌O395经典生物型250μl在10ml LB(pH 6.5)(1:40比例) ?并在30℃的水浴摇床中生长2小时。
    2. 在2小时,添加 将0.1μCi的14 C放射性标记的亚油酸加入1ml的传代培养物中, 在室温下孵育1小时,无需搅拌。这一次是 选择以确保细菌仍处于对数期生长
    3. 通过在室温下离心(15,000rpm)收获细菌 处理3分钟,并用1ml 1x PBS以15,000rpm洗涤沉淀三次 ?每次3分钟。
    4. 将细菌重悬在500μl溶液中 ?的20mM Tris-Base(pH8.5)和500mM NaCl。冻结悬浮液 乙醇(至少95%)和干冰浴2分钟,确保 管完全浸没,然后置于37℃直至解冻。重复此操作 ?冻融过程共进行三次冻融循环,以确保 完全分馏。
    5. 分馏细菌 在4℃下以15,000×g离心10分钟以分离膜 ?和细胞质。将细胞质作为上清液和沉淀 ?(细胞包膜级分)在500μl的1×PBS中洗涤3次 在15,000×g离心3分钟。期待一个非常小的丸 底部。每个级分中的14 C-亚油酸的量 通过将全部级分加入到5ml闪烁混合物中来测定, ?然后在闪烁计数器中测量CPM
    6. 然后将数据分析为每个级分中的14 C-亚油酸的百分比。代表图如下所示:


      图2.细胞质和包膜部分中14 C-放射性标记的亚油酸的百分比

食谱

  1. LB-Lennox(500ml)(pH6.5) 5克胰蛋白酶
    2.5克NaCl 2.5g酵母提取物
    加入约400ml水溶解并混合组分
    用HCl将pH调节至6.5,用水调节至500ml 使用前高压灭菌
  2. 10x PBS(1L)
    80克NaCl
    2克KCl
    14.4g Na 2 HPO 4
    2.4g KH 2 PO 4 4/
    使用时,稀释1:10(例如10ml用高压灭菌水加至100ml)
  3. 20mM Tris-碱(pH8.5)(100ml) 0.24克Tris-碱
    用HCl调节pH至8.5,用水调节至100ml 使用前高压灭菌
  4. 500mM NaCl(100ml) 2.92g NaCl

致谢

该工作由P.H.S.支持,授予K22AI071011和R56AI093622以及Bill和Melinda Gates基金会授予OPP1068124(授予J.H.W。)。
这项工作是从我们实验室以前的工作(Thomson和Withey,2014)修改的。

参考文献

  1. Plecha,S.C。和Withey,J.H。(2015)。 通过不饱和脂肪酸组分抑制霍乱弧菌的机制 ToxT活性of bile。 J Bacteriol 197(10):1716-1725。
  2. Thomson,J. J.和Withey,J. H.(2014)。 碳酸氢盐增加霍乱弧菌毒素ToxT与毒力基因启动子的结合亲和力。 J Bacteriol 196(22):3872-3880。
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引用:Plecha, S. C. and Withey, J. H. (2015). [14C] Linoleic Acid Uptake and Fractionation Assay in Vibrio cholerae. Bio-protocol 5(24): e1682. DOI: 10.21769/BioProtoc.1682.
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