Evolution of Escherichia coli to Macrophage Cell Line

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PLOS Pathogens
Dec 2013


The genomes of species of Escherichia coli (E. coli) show an extraordinary amount of diversity, which include commensal strains and strains belonging to different pathovars. Many strains of E. coli, which can cause mild or severe pathologies in humans, have a commensal ancestor. Understanding the evolutionary changes that can lead to a transition from commensal to pathogen is an important task, which requires integration of different methodologies. One method is experimental evolution of bacteria, in controlled environments, that mimic some of the selective pressures, likely to be important during the transition to pathogenesis. The success of such a transition will depend, at least partially, on ability of E. coli to adapt to the presence of cells of the immune system. Here, we describe a protocol for performing experimental evolution of a commensal strain of E. coli, a derivative of the well studied K12, under the constant selective pressure imposed by cells of the innate immune system, specifically RAW 264.7 murine macrophage cell line.

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

Materials and Reagents

  1. RAW 264.7 murine macrophage cell line (MΦ) (Sigma-Aldrich, catalog number: 91062702 )
  2. E. coli strains, marked with constitutive expression of yellow (YFP) and cyan (CFP) fluorescent proteins (e. g. E. coli- MC4100, galK::CFP/YFP, AmpR StrepR)
  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) (Life Technologies, Gibco®, catalog number: 10500-064 )
  8. GlutaMAX (100X) (Life Technologies, Gibco®, catalog number: 35050-038 )
  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 (5´-TCCATGACGTTCCTGACGTT-3´) (Sigma-Aldrich)
  15. RPMI-complete media (see Recipes)
  16. RPMI-Strep media (see Recipes)


  1. CO2 incubators
  2. Microscope
  3. Luria-Bertani (LB) agar plates (Panreac Life-sciences, catalog number: 596661.1210 )
  4. Centrifuge (Sigma-Aldrich, model: 4K15 with a rotor 11150 )
  5. Neubauer cell counting chamber (VWR International, catalog number: 631-1114 )
  6. Air flow chamber
  7. 12-well microtiter plates (Costar, catalog number: 3513 )
  8. Cell culture flasks (25 cm2 and 75 cm2 growth area) (Corning, catalog numbers: 430639 and 430641 )
  9. 15 ml Falcon tubes
  10. Cell scrapers (16 cm and 25 cm) (SARSTEDT AG, catalog numbers: 83.1832 and 83.1830 )
  11. 2 μm size beads (Sphero AccuCount Blank Particles) (Spherotech, catalog number: ACBP-20-10 )
  12. LSR Fortessa Flow cytometer


  1. Grow MΦ in two 75 cm2 culture flasks (20 ml total volume) in RPMI- complete media until 80% (flask A) and 25% (flask B) confluency (Note 1).
    Condition: The cell cultures are maintained in an incubator with 5% CO2 and 37 °C, including incubation steps.
    Day 1
  2. Remove old media, add fresh media (same volume) and detach MΦ with a 25 cm cell scraper from the flask A.
  3. Carefully pipette culture up and down until pellet is dissolved.
  4. Leave 5 ml of the cell suspension in the flask A and add 15 ml of RPMI-complete media.
  5. Centrifuge remaining 15 ml of MΦ cell suspension in two 15 ml Falcon tubes at 1,200 rpm for 5 min (Note 2).
  6. Remove supernatant and re-suspend pellet in the same volume of RPMI-Strep media (7.5 ml for each falcon tube) (Note 3).
  7. Count MΦ in the Neubauer cell counter with Trypan blue viability dye (use 10 μl of cell suspension with 10 μl of dye) and use approximately 0.7-1.3 x 106 cells/ml for activation. 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.
  8. Transfer this amount of cells into a 25 cm2 flask (8 ml total volume) and activate them with CpG-ODN at a final concentration of 2 µg/µl for 24 h (Note 4).
  9. Grow E. coli - CFP (or YFP) bacterial population from the frozen stock in RPMI-Strep media in the 12-well plates (3 ml of the total volume for each well) for 24 h in the same conditions as the cell cultures (CO2 incubator at 37 °C and 5% CO2) (Note 5).
    Day 2
  10. Repeat steps 2-8 with MΦ from the flask B.
  11. Repeat steps 2-3 with activated MΦ from flask A (use a 16 cm scraper).
  12. Centrifuge MΦ cell suspension in one 15 ml Falcon tube at 1,000 rpm for 5 min and resuspend using RPMI-Strep at the same volume.
  13. After counting the cells, seed activated MΦ at 0.8-1.6 x 106 cells/ml in 12-well microtiter plates (3 ml of total volume for each well).
  14. Allow cells to attach in the 37 °C, 5% CO2 incubator for 2 h.
  15. Count numbers of grown bacteria by using 2 μm size beads in the Flow cytometer (Note 6).
  16. Wash activated and attached MΦ with RPMI-Strep media (remove old and add new media, same volume).
  17. Infect activated MΦ at the multiplicity of infection (MOI) of 1 to 1 (106 bacteria/ml to 106 MΦ/ml).
    Day 3
  18. Repeat steps 2-8 with MΦ from the flask A.
  19. Repeat steps 2-3 with activated MΦ from flask B (use a 16 cm scraper).
  20. Centrifuge MΦ cell suspension in one 15 ml falcon tube at 1,000 rpm for 5 min and resuspend using RPMI-Strep at the same volume.
  21. After counting the cells, seed activated MΦ at 0.8-1.6 x 106 cells/ml in 12-well microtiter plates (3 ml of total volume for each well) and allow cells to attach in the 37 °C 5% CO2 incubator for 2 h.
  22. After 24 h of infection (12-well infection plate from the Day 2), detach MΦ with a 16 cm cell scraper and centrifuge culture at 4,000 rpm for 10 min. This procedure lyses MΦ and releases intracellular bacteria (Note 7).
  23. Discard the supernatant and resuspend each pellet in 3 ml of PBS.
  24. Dilute bacteria in PBS, count their numbers in flow cytometer.
  25. Use this bacteria to infect activated MΦ again (from flask B) at the multiplicity of infection (MOI) of 1 to 1 (106 bacteria/ml to 106 MΦs/ml).
  26. Freeze a sample of the bacterial culture every day and keep your stocks (“fossil records”) at -80 °C.
  27. Repeat steps from Day 2 and Day 3 to continue with evolution experiment (Note 8).


  1. To be able to repeat the same protocol every day, maintain MΦ always in the two 75 cm2 culture flasks. Use MΦ from the flask A for every odd and the flask B for every even day of evolution experiment. To estimate confluency compare the amount of space covered by cells with unoccupied spaces (check: http://www.abcam.com/ps/pdf/protocols/cell_culture.pdf).
  2. If 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 enable the growth of STR resistant E. coli in the media and control for possible contaminations by other bacteria. If you use different antibiotic, test if antibiotic is not toxic for MΦ cell line.
  4. This dilution is necessary because MΦ 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. How to prepare a bacterial stock for evolution experiment:
    1. Streak a bacterial culture on LB plate and grow for 24 h at 37 °C.
    2. Isolate and inoculate a single bacterial colony into RPMI-Strep media.
    3. After 24 h of growth, freeze culture in glycerol solution [500 µl of bacterial culture with 500 µl of glycerol (30% stock solution)].
    4. Use this stock to start evolution experiment.
  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 MΦ, 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.
  7. To perform phenotypic characterization of the evolved E. coli check our other protocol “Fitness Measurements of Evolved Esherichia coli” (Miskinyte and Gordo, 2014) and original paper (Miskinyte et al., 2013).


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


This protocol was adapted or modified from Lenski et al. (1991). 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.


  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). Fitness measurements of evolved Esherichia coli. Bio-protocol 4(17): e1228.
  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.


大肠杆菌物种的基因组(大肠杆菌)显示出非凡的多样性,其包括共生菌株和属于不同病原体的菌株。许多菌株。大肠杆菌,其可引起人类的轻度或严重病理,具有共生祖先。了解可导致从共生转变为病原体的进化变化是一项重要任务,需要整合不同的方法。一种方法是在受控环境中的细菌的实验进化,其模拟一些选择性压力,在向发病过渡期间可能是重要的。这种转变的成功将至少部分地取决于E的能力。大肠杆菌以适应免疫系统细胞的存在。在这里,我们描述了用于进行共生应变的实验进化的方案。大肠杆菌,在充分研究的K12的衍生物,在先天免疫系统的细胞施加的恒定选择压力下,特别是RAW 264.7鼠巨噬细胞细胞系。

关键字:实验性的进化, 健身, 宿主和微生物


  1. RAW 264.7鼠巨噬细胞细胞系(MΦ)(Sigma-Aldrich,目录号:91062702)
  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 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. 1M 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)(Life Technologies,Gibco ,目录号:10500-064)
  8. GlutaMAX(100X)(Life Technologies,Gibco ,目录号:35050-038)
  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(5'-TCCATGACGTTCCTGACGTT-3')(Sigma-Aldrich)
  15. RPMI完整媒体(参见配方)
  16. RPMI-Strep培养基(参见配方)


  1. CO <2>孵化器
  2. 显微镜
  3. Luria-Bertani(LB)琼脂平板(Panreac Life-sciences,目录号:596661.1210)
  4. 离心机(Sigma-Aldrich,型号:4K15,转子11150)
  5. Neubauer细胞计数室(VWR International,目录号:631-1114)
  6. 气流室
  7. 12孔微量滴定板(Costar,目录号:3513)
  8. 细胞培养瓶(25cm 2和75cm 2生长区域)(Corning,目录号:430639和430641)
  9. 15 ml Falcon管
  10. 细胞刮刀(16cm和25cm)(SARSTEDT AG,目录号:83.1832和83.1830)
  11. 2μm粒珠(Sphero AccuCount Blank Particles)(Spherotech,目录号:ACBP-20-10)
  12. LSR Fortessa流式细胞仪


  1. 在两个75cm 2培养瓶(总体积20ml)中在RPMI完全培养基中生长Mφ,直到80%(烧瓶A)和25%(烧瓶B)融合(注1)。
    条件:将细胞培养物保持在具有5%CO 2和37℃的培养箱中,包括孵育步骤。
  2. 取出旧培养基,加入新鲜培养基(体积相同),用25cm细胞刮刀从瓶A中取出MΦ
  3. 小心地将培养物上下移动,直到颗粒溶解
  4. 将5ml细胞悬浮液留在烧瓶A中,加入15ml RPMI完全培养基
  5. 在两个15ml Falcon管中以1,200rpm离心5mlMΦ细胞悬浮液5分钟(注2)。
  6. 取出上清液并在相同体积的RPMI-Strep培养基(每个falcon管7.5 ml)中重悬细胞(注3)。
  7. 用台盼蓝活力染料(使用10μl具有10μl染料的细胞悬浮液)在Neubauer细胞计数器中计数MΦ,并使用约0.7-1.3×10 6个细胞/ml激活。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吸收染料。
  8. 将该量的细胞转移到25cm 2烧瓶(总体积8ml)中,并用终浓度为2μg/μl的CpG-ODN活化24小时(注4)。
  9. 成长。在与细胞培养物相同的条件下,从12孔板中的RPMI-Strep培养基中的冷冻储液(每个孔的总体积3ml)中大肠杆菌 - CFP(或YFP)细菌群体24小时(CO 2培养箱,37℃和5%CO 2)(注5)。
  10. 使用来自烧瓶B的MΦ重复步骤2-8
  11. 使用来自烧瓶A的活化MΦ重复步骤2-3(使用16cm刮刀)
  12. 将MΦ细胞悬浮液在一个15ml Falcon管中以1,000rpm离心5分钟,并使用相同体积的RPMI-Strep重悬。
  13. 在计数细胞后,在12孔微量滴定板(每孔3ml总体积)中以0.8-1.6×10 6个细胞/ml接种活化的MΦ。
  14. 允许细胞在37℃,5%CO 2培养箱中附着2小时。
  15. 通过在流式细胞仪中使用2μm大小的珠子计数生长的细菌数(注6)。
  16. 用RPMI-Strep培养基洗涤活化和附着的MΦ(除去旧的并添加新培养基,体积相同)
  17. 以1至1(10 6个细菌/ml至10 6个MΦ/ml)的感染复数(MOI)感染活化的MΦ。
  18. 从瓶A中用MΦ重复步骤2-8
  19. 使用来自烧瓶B的活化MΦ重复步骤2-3(使用16cm刮刀)
  20. 将MΦ细胞悬浮液在一个15ml falcon管中以1,000rpm离心5分钟,并使用相同体积的RPMI-Strep重悬。
  21. 在计数细胞后,在12孔微量滴定板中以0.8-1.6×10 6个细胞/ml种子活化MΦ(每个孔3ml总体积),并允许细胞以37°附着 C 5%CO 2 2培养箱中培养2小时
  22. 感染24小时后(第2天的12孔感染板),用16cm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7)
  23. 弃去上清液,并将每个沉淀重悬于3ml PBS中
  24. 在PBS中稀释细菌,在流式细胞仪中计数它们的数量。
  25. 使用该细菌以1至1(10 6个细菌/ml至10 6个细胞/ml)的感染复数(MOI)再次感染活化的MΦ(来自烧瓶B) ml)。
  26. 每天冻结细菌培养物的样品,并保持你的股票("化石记录")在-80°C。
  27. 重复从第2天和第3天的步骤,继续进化实验(注8)。


  1. 为了能够每天重复相同的方案,保持MΦ总是在两个75cm 2培养瓶中。对于每个奇数使用来自烧瓶A的MΦ,对于进化实验的每偶数天使用烧瓶B.要估计汇合,请比较具有未占用空间的单元格覆盖的空间量(检查: http: //www.abcam.com/ps/pdf/protocols/cell_culture.pdf )。
  2. 如果MΦ培养物具有许多死亡(聚集或分离)细胞以800rpm离心,这样健康的细胞将在管底部沉淀,死细胞将主要保留在上清液中。
  3. 链霉素用于使STR抗性E生长。大肠杆菌在媒体和控制其他细菌可能的污染。如果使用不同的抗生素,测试抗生素对MΦ细胞系是否有毒
  4. 这种稀释是必要的,因为MΦ将在24小时内生长。我们使用CpG-ODN-1826,因为ODN 1826是小鼠TLR9特异性的B类CpG ODN,其在激活24小时后导致强的免疫刺激作用。如果您打算使用其他细胞系(例如 ODN 2395特异于C类人TLR9),请选择合适的ODN。如何准备用于进化实验的细菌原液:
    1. 将细菌培养物在LB平板上划线并在37℃下生长24小时
    2. 将单个细菌菌落分离并接种到RPMI-Strep培养基中
    3. 生长24小时后,在甘油溶液[500μl细菌培养物与500μl甘油(30%储备液)]中冷冻培养。
    4. 使用此股票开始进化实验。
  5. 您可以使用以下稀释度在流式细胞仪中计数:10μl细菌培养物(稀释1:100),10μl珠和180 ml PBS。
  6. 你也可以通过弯曲一个蓝色的1000微升移液器尖,并使用它作为刮刀分离MΦ。 记住,对于每个重复,你需要使用一个新的无菌刮刀
  7. 进行进化的E的表型表征。 大肠杆菌检查我们的其他方案"大肠杆菌的健康测量 a>"(Miskinyte和Gordo,2014)和原始论文(Miskinyte等人,2013年)。


  1. RPMI完整媒体
    500ml RPMI 1640
    50ml FBS
    5ml丙酮酸钠 5ml肝脏
    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肝脏
    5ml L-谷氨酰胺 0.5ml 2-巯基乙醇溶液 1ml 50mg/ml链霉素溶液


该协议由Lenski等人(1991)修改或修改。 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资助 协议no 260421 - ECOADAPT。 IG确认LAO/ITQB& FCT。


  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。 <美国自然主义 138:1315-1341
  2. Miskinyte,M。和Gordo,I.(2014)。 进化大肠杆菌的体能测量 生物协议 4(17):e1228。
  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). Evolution of Escherichia coli to Macrophage Cell Line. Bio-protocol 4(17): e1227. DOI: 10.21769/BioProtoc.1227.
  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.