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Fitness Measurements of Evolved Esherichia coli
进化后的大肠杆菌的适应性测定   

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Abstract

Bacteria can adapt very rapidly to novel selective pressures. In the transition from commensalism to pathogenicity bacteria have to face and adapt to the host immune system. Specifically, the antagonistic interaction imposed by one of the first line of defense of innate immunity cells, macrophages, on commensal bacteria, such as Escherichia coli (E. coli), can lead to its rapid adaptation. Such adaptation is characterized by the emergence of clones with mutations that allow them to better escape macrophage phagocytosis. Here, we describe how to quantify the amount of fitness increase of bacterial clones that evolved under the constant selective pressure of macrophages, from a murine cell line RAW 264.7. The most widely used assay for measuring fitness changes along an evolutionary laboratory experiment is a competitive fitness assay. This assay consists of determining how fast an evolved strain outcompetes the ancestral in a competition where each starts at equal frequency. The strains compete in the same environment of the evolution experiment and if the evolved strain has acquired strong beneficial mutations it will become significantly overrepresented in repeated competitive fitness assays.

Keywords: Experimental evolution(实验性的进化), Host-microbe(宿主和微生物), Fitness(健身)

Materials and Reagents

  1. RAW 264.7 murine macrophage cell line (MΦs)
  2. E. coli strains, marked with constitutive expression of yellow (YFP) and cyan (CFP) fluorescent proteins (e.g. E. coli- MC4100, galK::CFP/YFP, AmpRStrepR)
  3. RPMI media1640 (Life Technologies, Gibco®, catalog number: 11875-093 )
  4. 2-mercaptoethanol solution (Life Technologies, InvitrogenTM, catalog number: 31350-010 )
  5. 1 M HEPES buffer (Life Technologies, InvitrogenTM, catalog number: 15630-056 )
  6. 100 mM sodium pyruvate (Life Technologies, InvitrogenTM, catalog number: 11360-039 )
  7. Heat-inactivated fetal bovine serum (FBS)
  8. 200 mM L-glutamine (Life Technologies, Gibco®, catalog number: 25030-081 )
  9. 10,000 U/ml penicillin/streptomycin (Life Technologies, Gibco®, catalog number: 15140-122 )
  10. Streptomycin sulfate salt (Sigma-Aldrich, catalog number: S9137 )
  11. 50 mg/ml Gentamycin solution (Sigma-Aldrich, catalog number: G1397 )
  12. 1x Phosphate-buffered saline (PBS)
  13. Trypan blue solution (Sigma-Aldrich, catalog number: T8154 )
  14. CpG-ODN -1826 (Sigma-Aldrich) (5´-TCCATGACGTTCCTGACGTT-3´)
  15. RPMI-complete media (see Recipes)
  16. RPMI-strep media (see Recipes)

Equipment

  1. 37 °C, 5% CO2 cell culture incubator
  2. Microscope
  3. Centrifuge (Sigma-Aldrich, model: 4K15 with a rotor 11150)
  4. Neubauer cell counting chamber
  5. Air flow chamber
  6. 12-well microtiter plates
  7. Cell culture flasks (25 cm2 and 75 cm2 growth area)
  8. 15 ml Falcons tubes
  9. Cell scrapers
  10. 2 μm size beads (Sphero AccuCount Blank Particles)
  11. Flow cytometer

Procedure

  1. Grow MΦs (in the 37 °C, 5% CO2 incubator) in 75 cm2 culture flask in RPMI- complete media until 80% confluency (Note 1).
  2. Change old media (remove old media by aspiration and add fresh RPMI-complete media) and detach Mϕs with a cell scraper.
  3. Carefully pipette culture up and down until pellet is dissolved.
  4. Centrifuge in 15 ml Falcon tubes at 1,200 rpm for 5 min (Note 2).
  5. Remove supernatant and re-suspend pellet in the same volume of RPMI-Strep media (Note 3).
  6. Count MΦs in the Neubauer cell counter with Trypan blue dye (use 10 μl of cell suspension with 10 μl of dye). Allow the dye to stain for approximately 5 to 15 min. If cells are exposed to extended period of time to this dye, viable cells, as well as nonviable cells, may begin to uptake dye.
  7. Approximately 0.7-1.3 x 106 cells/ml activate with CpG-ODN at a final concentration of 2 µg/µl for 24 h in 10 cm2 flask (8 ml total volume) (Note 4).
  8. Grow separately evolved E. coli - CFP (or YFP) and ancestral E. coli - YFP (or CFP) bacterial populations from the frozen stock in RPMI-Strep media (3 ml of the total volume for each well) in the 12-well plates for 24 h (for the evolved E. coli - CFP (or YFP) please see our other protocol “Evolution of Escherichia coli to Macrophage Cell Line” (Miskinyte and Gordo, 2014).
  9. After 24 h repeat steps 2-6, but for the step 4 use 1,000 rpm instead of 1,200.
  10. Seed 0.8-1.6 x 106 cells/ml in 12-well microtiter plates (3 ml total volume for each well).
  11. Allow cells to attach in the 37 °C, 5% CO2 incubator for 2 h.
  12. Mix E. coli - CFP (evolved) with E. coli - YFP (ancestral) at a ratio of 1 to 1.
  13. Count numbers of CFP (Nie) and YFP (Nia) bacteria before competition in the mixture by using 2 μm size beads in the flow cytometer (Note 5).
  14. Wash MΦs with RPMI-Strep media (remove old media by aspiration and add fresh RPMI-Strep media).
  15. Infect MΦs at the multiplicity of infection (MOI) of 1 to 1 (mixture of 106 bacteria/ml to 106 MΦs/ml).
  16. After 24 h of infection, detach MΦs with a cell scraper and centrifuge culture at 4,000 rpm for 10 min. This procedure lyses MΦs and releases intracellular bacteria (Note 6).
  17. Discard the supernatant and resuspend the pellet in 3 ml of PBS.
  18. Dilute bacteria in PBS and count CFP (Nfe) and YFP (Nfa) bacterial numbers after competition in Flow cytometer.
  19. Use Formula 1 to calculate relative fitness increase of the evolved bacteria over the ancestral.


    Formula 1. Selection coeficient, a measure of relative fitness increase. Scoef is the selective advantage of the evolved strain e over the ancestral strain a, Nfe and Nfa are the numbers of evolved (e) and ancestral (a) bacteria after competition and Nia and Nie are the initial numbers, before the competition.

Notes

  1. You should compare the amount of space covered by cells with unoccupied spaces to estimate approximately percent confluency (check: http://www.abcam.com/ps/pdf/protocols/cell culture.pdf).
  2. If the MΦ culture has many dead (aggregated or detached cells) centrifuge at 800 rpm. This way healthy cells will pellet at the bottom of the tube and dead cells will remain mainly in the supernatant.
  3. Streptomycin is used to reduce the possibility of contamination. The strain of E. coli is STR resistant. If you use a different antibiotic, test for its toxicity on the MΦs cell line.
  4. This dilution is necessary because MΦs will grow in 24 h. We use CpG-ODN-1826, because ODN 1826 is a B-class CpG ODN specific for mouse TLR9 that leads to strong immunostimulatory effects after 24 h of activation. If you intend to use other cell line (e.g. ODN 2395 is specific for class C human TLR9) choose an appropriate ODN.
  5. You can use the following dilution to count in the flow cytometer: 10 µl of bacterial culture (diluted 1: 100), 10 µl of beads and 180 ml PBS.
  6. You can also detach MΦs, by bending a blue 1,000 µl pipette tip and using it as a scraper. Remember that for each replicate well you need to use a new sterile scraper.

Recipes

  1. RPMI-complete media
    500 ml RPMI 1640
    50 ml FBS
    5 ml sodium pyruvate
    5 ml HEPES
    5 ml L-glutamine
    0.5 ml 2-mercaptoethanol solution
    0.5 ml gentamycin solution
    5 ml penicillin/streptomycin solution
    Stored at 4 °C
  2. RPMI-strep media
    500 ml RPMI 1640
    50 ml FBS
    5 ml sodium pyruvate
    5 ml HEPES
    5 ml L-glutamine
    0.5 ml 2-mercaptoethanol solution
    1 ml of 50 mg/ml streptomycin solution
    Stored at 4 °C

Acknowledgments

This protocol was adapted or modified from Richard E. Lenski, Michael R. Rose, Suzanne C. Simpson and Scott C. Tadler 1991, Long-Term Experimental Evolution in Escherichia coli. I. Adaptation and Divergence During 2,000 Generations. The American Naturalist, Vol. 138, No. 6, pp. 1315-1341. The research received funding from the European Research Council under the European Community's Seventh Framework Programme (FP7/2007–2013)/ERC grant agreement no 260421 – ECOADAPT. IG acknowledges the salary support of LAO/ITQB & FCT.

References

  1. Lenski, R. E., Rose, M. R., Simpson, S. C. and Tadler, S. C. (1991). Long-term experimental evolution in Escherichia coil. I. Adaptation and divergence during 2,000 generations. American Naturalist 138:1315-1341.
  2. Miskinyte, M. and Gordo, I. (2014). Evolution of Escherichia coli to macrophage cell line. Bio-protocol 4(17): e1227.
  3. Miskinyte, M., Sousa, A., Ramiro, R. S., de Sousa, J. A., Kotlinowski, J., Caramalho, I., Magalhaes, S., Soares, M. P. and Gordo, I. (2013). The genetic basis of Escherichia coli pathoadaptation to macrophages. PLoS Pathog 9(12): e1003802.

简介

细菌可以非常迅速地适应新的选择压力。在从共生到致病性的转变中,细菌必须面对并适应宿主免疫系统。具体地,由先天免疫细胞(巨噬细胞)的第一防御线对共生细菌如大肠杆菌(大肠杆菌)(大肠杆菌)施加的拮抗相互作用可以导致其快速适应。这种适应的特征在于具有允许它们更好地逃脱巨噬细胞吞噬的突变的克隆的出现。在这里,我们描述如何量化在巨噬细胞的恒定选择压力下,从鼠细胞系RAW 264.7进化的细菌克隆的适应增加的量。用于测量沿进化实验室实验的适应度变化的最广泛使用的测定法是竞争性健身测定法。该测定法包括确定进化的菌株在竞争中胜过祖先的速度,其中每个以相同的频率开始。菌株在进化实验的相同环境中竞争,并且如果进化菌株已经获得强的有益突变,其将在重复的竞争性健康测定中变得显着过表达。

关键字:实验性的进化, 宿主和微生物, 健身

材料和试剂

  1. RAW 264.7鼠巨噬细胞细胞系(MΦs)
  2. E。 标记有黄色(YFP)和青色(CFP)荧光蛋白(例如大肠杆菌MC4100,galK :: CFP/YFP,Amp R)的组成型表达的大肠杆菌 Strep R
  3. RPMI media1640(Life Technologies,Gibco ,目录号:11875-093)
  4. 2-巯基乙醇溶液(Life Technologies,Invitrogen TM ,目录号:31350-010)
  5. 1 M HEPES缓冲液(Life Technologies,Invitrogen TM ,目录号:15630-056)
  6. 100mM丙酮酸钠(Life Technologies,Invitrogen TM ,目录号:11360-039)
  7. 热灭活的胎牛血清(FBS)
  8. 200mM L-谷氨酰胺(Life Technologies,Gibco ,目录号:25030-081)
  9. 10,000 U/ml青霉素/链霉素(Life Technologies,Gibco ,目录号:15140-122)
  10. 链霉素硫酸盐(Sigma-Aldrich,目录号:S9137)
  11. 50mg/ml庆大霉素溶液(Sigma-Aldrich,目录号:G1397)
  12. 1×磷酸盐缓冲盐水(PBS)
  13. 台盼蓝溶液(Sigma-Aldrich,目录号:T8154)
  14. CpG-ODN-1826(Sigma-Aldrich)(5'-TCCATGACGTTCCTGACGTT-3')
  15. RPMI完整媒体(参见配方)
  16. RPMI-strep培养基(参见配方)

设备

  1. 37℃,5%CO 2细胞培养箱中培养
  2. 显微镜
  3. 离心机(Sigma-Aldrich,型号:4K15,转子11150)
  4. Neubauer细胞计数室
  5. 气流室
  6. 12孔微量滴定板
  7. 细胞培养瓶(25cm 2和75cm 2生长区域)
  8. 15毫升Falcons管
  9. 细胞刮刀
  10. 2μm尺寸珠(Sphero AccuCount Blank Particles)
  11. 流式细胞仪

程序

  1. 在75cm 2培养瓶中在RPMI-完全培养基中生长MΦ(在37℃,5%CO 2培养箱中)直到80%汇合(注1)。
  2. 更换旧介质(通过吸气除去旧介质,添加新的RPMI完全介质),并用细胞刮刀分离Mφ。
  3. 小心地将培养物上下移动,直到颗粒溶解
  4. 在15ml Falcon管中以1,200rpm离心5分钟(注2)
  5. 移除上清液,并在相同体积的RPMI-Strep培养基(注3)中重悬浮沉淀。
  6. 使用台盼蓝染料(使用10μl含有10μl染料的细胞悬浮液)计数Neubauer细胞计数器中的MΦ。 使染料染色约5至15分钟。 如果细胞长时间暴露于这种染料,活细胞以及无活力的细胞可能开始摄取染料。
  7. 大约0.7-1.3×10 6个细胞/ml用在10cm 2烧瓶中的2μg/μl的终浓度的CpG-ODN活化24小时(总共8ml体积)(注4)。
  8. 分别成长。大肠杆菌 - CFP(或YFP)和祖先。在12孔板中在RPMI-Strep培养基(3ml每个孔的总体积)中冷冻库存的大肠杆菌 - YFP(或CFP)细菌群体24小时(对于进化的E 。大肠杆菌 - CFP(或YFP)请参见我们的其他方案"大肠杆菌的进化到巨噬细胞细胞系"(Miskinyte和Gordo,2014)
  9. 24小时后重复步骤2-6,但对于步骤4,使用1,000rpm而不是1,200。
  10. 在12孔微量滴定板(每孔3ml总体积)中种子0.8-1.6×10 6个细胞/ml。
  11. 允许细胞在37℃,5%CO 2培养箱中附着2小时。
  12. 混合E。大肠杆菌 - CFP(进化)。大肠杆菌 -YFP(祖先),比例为1比1
  13. 在混合物竞争之前计数CFP( Ni )和YFP( Ni a
  14. 用RPMI-Strep培养基洗涤MΦ(通过抽吸除去旧培养基并加入新鲜的RPMI-Strep培养基)。
  15. 在感染复数(MOI)为1比1(10 6个细菌/ml至10 6个MΦs/ml的混合物)时感染MΦ。
  16. 感染24小时后,用细胞刮刀分离MΦ,并以4,000rpm离心培养10分钟。 该程序溶解MΦ并释放细胞内细菌(注6)
  17. 弃去上清液并将沉淀重悬在3ml PBS中
  18. 在PBS中稀释细菌并在竞争后计数CFP( Nf )和YFP( Nf a 流式细胞仪
  19. 在混合物竞争之前计数CFP( Ni )和YFP( Ni a
  20. 用RPMI-Strep培养基洗涤MΦ(通过抽吸除去旧培养基并加入新鲜的RPMI-Strep培养基)。
  21. 在感染复数(MOI)为1比1(10 6个细菌/ml至10 6个MΦs/ml的混合物)时感染MΦ。
  22. 感染24小时后,用细胞刮刀分离MΦ,并以4,000rpm离心培养10分钟。 该程序溶解MΦ并释放细胞内细菌(注6)
  23. 弃去上清液并将沉淀重悬在3ml PBS中
  24. 在PBS中稀释细菌并在竞争后计数CFP( Nf )和YFP( Nf a 流式细胞仪
  25. ...
  26. Streptomycin is used to reduce the possibility of contamination. The strain of E. coli is STR resistant. If you use a different antibiotic, test for its toxicity on the MΦs cell line.
  27. This dilution is necessary because MΦs will grow in 24 h. We use CpG-ODN-1826, because ODN 1826 is a B-class CpG ODN specific for mouse TLR9 that leads to strong immunostimulatory effects after 24 h of activation. If you intend to use other cell line (e.g. ODN 2395 is specific for class C human TLR9) choose an appropriate ODN.
  28. You can use the following dilution to count in the flow cytometer: 10 µl of bacterial culture (diluted 1: 100), 10 µl of beads and 180 ml PBS.
  29. You can also detach MΦs, by bending a blue 1,000 µl pipette tip and using it as a scraper. Remember that for each replicate well you need to use a new sterile scraper.

Recipes

  1. RPMI完整媒体
    500ml RPMI 1640
    50ml FBS
    5ml丙酮酸钠 5ml HEPES
    5ml L-谷氨酰胺 0.5ml 2-巯基乙醇溶液 0.5 ml庆大霉素溶液 5ml青霉素/链霉素溶液 储存在4°C
  2. RPMI-strep培养基
    500ml RPMI 1640
    50ml FBS
    5ml丙酮酸钠 5ml HEPES
    5ml L-谷氨酰胺 0.5ml 2-巯基乙醇溶液 1ml 50mg/ml链霉素溶液
    储存在4°C

致谢

该方案由Richard E. Lenski,Michael R.Rose,Suzanne C.Simpson和Scott C.Tadler 1991,"大肠杆菌长期实验进化"改编或修改。一,二代的适应和分歧。美国自然主义, 138,No.6,pp。1315-1341。研究得到欧洲研究理事会根据欧洲共同体第七框架计划(FP7/2007-2013)/ERC赠款协议第260421号 - ECOADAPT的资助。 IG确认LAO/ITQB& FCT。

参考文献

  1. Lenski,R.E.,Rose,M.R.,Simpson,S.C.and Tadler,S.C。(1991)。在大肠杆菌中的长期实验进化。I.在2,000代中的适应和发散。
  2. Miskinyte,M。和Gordo,I.(2014)。 大肠杆菌对巨噬细胞细胞系的演变 生物协议 4 17):e1227。
  3. Miskinyte,M.,Sousa,A.,Ramiro,R.S.,de Sousa,J.A.,Kotlinowski,J.,Caramalho,I.,Magalhaes,S.,Soares,M.P.和Gordo, 大肠杆菌的遗传基础对巨噬细胞的适应性。 PLoS Pathog 9(12):e1003802。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Miskinyte, M. and Gordo, I. (2014). Fitness Measurements of Evolved Esherichia coli. Bio-protocol 4(17): e1228. DOI: 10.21769/BioProtoc.1228.
  2. Miskinyte, M., Sousa, A., Ramiro, R. S., de Sousa, J. A., Kotlinowski, J., Caramalho, I., Magalhaes, S., Soares, M. P. and Gordo, I. (2013). The genetic basis of Escherichia coli pathoadaptation to macrophages. PLoS Pathog 9(12): e1003802.
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