欢迎您, 登录 | 注册

首页 | English

X
加载中

Choline is a methylated nitrogen compound that is widespread in nature. It is a precursor of several metabolites that perform numerous biological functions and it is predominantly used for the synthesis of essential lipid components of the cell membranes. Since there is no evidence that prokariotes can synthesize choline de novo and because choline uptake from exogenous sources is energetically more favorable than de novo synthesis, bacteria have evolved different uptake mechanisms for choline transport across the bacterial membrane. This protocol describes an easy and high sensitive method to assess choline uptake in bacteria using as tracer [3H]-choline chloride. The protocol was originally intended for Brucella abortus but could be applied for any bacteria with the corresponding modifications depending on the bacteria growth requirements (composition of the culture medium, temperature for growth, etc.). It can be useful to determine the choline uptake ability of several bacterial species under different growth conditions.

Thanks for your further question/comment. It has been sent to the author(s) of this protocol. You will receive a notification once your question/comment is addressed again by the author(s).
Meanwhile, it would be great if you could help us to spread the word about Bio-protocol.

X

Choline Uptake Assay in Bacterial Cells
细菌细胞中的胆碱摄取分析

微生物学 > 微生物新陈代谢 > 营养运输
作者: Lucas Bukata
Lucas BukataAffiliation: Instituto de Investigaciones Biotecnológicas IIB-UNSAM CONICET, Universidad Nacional de San Martín, San Martín, Buenos Aires, Argentina
Bio-protocol author page: a837
Claudia K. Herrmann
Claudia K. HerrmannAffiliation: Instituto de Investigaciones Biotecnológicas IIB-UNSAM CONICET, Universidad Nacional de San Martín, San Martín, Buenos Aires, Argentina
Bio-protocol author page: a838
 and Diego J. Comerci
Diego J. ComerciAffiliation: Instituto de Investigaciones Biotecnológicas IIB-UNSAM CONICET, Universidad Nacional de San Martín, San Martín, Buenos Aires, Argentina
For correspondence: dcomerci@iibintech.com.ar
Bio-protocol author page: a519
Vol 3, Iss 18, 9/20/2013, 2338 views, 0 Q&A, How to cite
DOI: http://dx.doi.org/10.21769/BioProtoc.902

[Abstract] Choline is a methylated nitrogen compound that is widespread in nature. It is a precursor of several metabolites that perform numerous biological functions and it is predominantly used for the synthesis of essential lipid components of the cell membranes. Since there is no evidence that prokariotes can synthesize choline de novo and because choline uptake from exogenous sources is energetically more favorable than de novo synthesis, bacteria have evolved different uptake mechanisms for choline transport across the bacterial membrane. This protocol describes an easy and high sensitive method to assess choline uptake in bacteria using as tracer [3H]-choline chloride. The protocol was originally intended for Brucella abortus but could be applied for any bacteria with the corresponding modifications depending on the bacteria growth requirements (composition of the culture medium, temperature for growth, etc.). It can be useful to determine the choline uptake ability of several bacterial species under different growth conditions.

Keywords: Choline(胆碱), Uptake assay(吸收法), Choline transporter(胆碱转运), Brucella(布鲁氏菌), Brucellosis(布氏杆菌病)

Materials and Reagents

  1. Brucella abortus or the bacteria species you want to test
  2. Minimal medium (MM) such as M9 or equivalent
    In the case of Brucella abortus we use Gerhardt-Wilson (GW) minimum medium (Gerhardt, 1958)
  3. Choline Chloride ([Methyl-3H]-, 250 μCi (9.25 MBq)) (PerkingElmer, catalog number: NET109250UC)
  4. [3H]-choline/cold choline chloride (1:100)
  5. Liquid Scintillation (liquid Optiphase HISAFE 3) (PerkingElmer, catalog number: 1200-437)

Equipment

  1. Microplate reader or Spectrophotometer
  2. Liquid scintillation spectrometer (Gemini BV, model: WinSpectralTM 1414)

Procedure

  1. For radioactive choline uptake analyses, cultures of bacteria grown in an appropriate minimal medium (MM) at mid log phase were harvested, washed three times with ice-chilled MM and adjusted to an optical density at 600 nm (OD600) of 0.5 with fresh MM.
    Note: Bacterial culture is harvested at OD600= 1-1.2 that correspond to a mid log phase for Brucella under this growth conditions.
  2. Reactions were initiated by addition of [3H]-choline chloride (80.6 Ci/mmol) to a final concentration of 3.3 μM, incubated at 37 °C and aliquots (around 300 μl) were taken at different time points (0 to 30 min).
  3. Samples were immediately chilled on ice, washed five times with the same volume of ice-chilled MM, centrifuged at 10,000 x g, 15 min at 4 °C and cell pellets were resuspended in 500 μl of scintillation liquid.
  4. The radioactivity in the cell pellet was determined with a liquid scintillation spectrometer.
  5. To assess uptake kinetics at different choline concentrations, bacteria were incubated 7 min at 37 °C in MM with a mix of [3H]-choline/cold choline (1:100) ranging from 6.25 x 10-2 μM to 64 μM (total choline concentration) and incorporated radioactivity in the cell pellet was determined as describe above.

Acknowledgments

This protocol was adapted from Herrmann et al. (2013).

References

  1. Gerhardt, P. (1958). The nutrition of brucellae. Bacteriol Rev 22(2):81-98.
  2. Herrmann, C. K., Bukata, L., Melli, L., Marchesini, M. I., Caramelo, J. J. and Comerci, D. J. (2013). Identification and characterization of a high-affinity choline uptake system of Brucella abortus. J Bacteriol 195(3): 493-501.


How to cite this protocol: Bukata, L., Herrmann, C. K. and Comerci, D. J. (2013). Choline Uptake Assay in Bacterial Cells . Bio-protocol 3(18): e902. DOI: 10.21769/BioProtoc.902; Full Text



可重复性反馈:

  • 添加图片
  • 添加视频

我们的目标是让重复别人的实验变得更轻松,如果您已经使用过本实验方案,欢迎您做出评价。我们鼓励上传实验图片或视频与小伙伴们(同行)分享您的实验心得和经验。(评论前请登录)

问题&解答:

  • 添加图片
  • 添加视频

(提问前,请先登陆)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。


登陆 | 注册
分享
Twitter Twitter
LinkedIn LinkedIn
Google+ Google+
Facebook Facebook