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Bacterial Conjugation in Rhodobacter capsulatus
荚膜红细菌中的细菌接合   

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Abstract

Bacterial conjugation of plasmids is the common method of introducing foreign DNA into Rhodobacter capsulatus because transformational systems have not been shown as efficient methods of introducing DNA to R. capsulatus. For R. capsulatus bacterial conjugation using an Escherichia coli donor can be used to introduce replicating vectors, and non-replicating vectors for targeted chromosomal modifications.

Materials and Reagents

  1. R. capsulatus recipient strain
  2. Escherichia coli donor strain (containing plasmid to be conjugated) capable of conjugation (e.g. S17-1) or E. coli donor strain containing plasmid to be conjugated and a helper strain containing the tra genes [e.g. HB101 (pRK2013)]. For a review on conjugation and tra genes see Willetts et al. (1984)
  3. Plasmid to be conjugated into R. capsulatus (e.g. pXCA601; Tetracycline resistance)
  4. Appropriate antibiotic (resistance specified by plasmids and bacterial strains)
  5. Thiamine hydrochloride
  6. H3BO3
  7. MnSO4·H2O
  8. Na2MoO4·2H2O
  9. ZnSO4·7H2O
  10. Cu(NO3)·3H2O
  11. D, L-malic acid
  12. Na2EDTA
  13. MgSO4·7H2O
  14. CaCl2·2H2O
  15. FeSO4·7H2O
  16. 10 mM potassium phosphate buffer
  17. 0.3% Difco yeast extract
  18. 0.3% Difco bactopeptone
  19. Bacto-tryptone
  20. Yeast extract
  21. Trace element solution (see Recipes)
  22. RCV broth (see Recipes)
  23. RCV agar (see Recipes)
  24. LB broth (see Recipes)
  25. LB agar (see Recipes)
  26. YPS agar (see Recipes)

Equipment

  1. 30 °C and 37 °C shakers
  2. 30 °C and 37 °C incubator
  3. Test tubes
  4. Petri plates
  5. Sterile 1.7 ml microcentrifuge tubes
  6. Inoculation loop
  7. Pipetmen (10 μl to 1 ml range) and appropriate tips
  8. Graduated pipette (5 ml range) and aspiration bulb
  9. Bench-top microcentrifuge with rotor for 1.7 ml microcentrifuge tubes

Procedure

  1. Streak recipient R. capsulatus strain on RCV agar plate (with appropriate antibiotics) and incubate at 30 °C for 2-3 days.
  2. Streak donor E. coli strain (and helper E. coli) on LB agar plate with appropriate antibiotics and incubate at 37 °C overnight.
  3. Inoculate 4 ml of RCV broth (with appropriate antibiotics) with a single colony of the recipient R. capsulatus strain and incubate at 30 °C in a 200-250 rpm shaker for 2 days.
  4. One day later inoculate 4 ml of LB broth (with appropriate antibiotics) with donor E. coli strain, and 4 ml of LB broth with helper E. coli strain if applicable (see Materials and Reagents for examples), and incubate at 37 °C in a 200-250 rpm shaker overnight.
  5. In separate sterile microcentrifuge tubes transfer 100 μl of donor E. coli strain, 100 μl of helper E. coli strain (if applicable), and 200 μl of recipient R. capsulatus strain. Each strain should be in mid- to late- log phase.
  6. Spin microcentrifuge tubes containing cultures at 3,500 x g for 1 min. in bench-top centrifuge.
  7. Decant all supernatant from microcentrifuge tubes.
  8. Resuspend cell pellets in 500 μl RCV broth per microcentrifuge tube to wash away residual antibiotics and LB broth.
  9. Spin microcentrifuge tubes containing resuspended cultures at 4,000 x g for 1 min in bench-top centrifuge.
  10. Decant all supernatant from microcentrifuge tubes.
  11. Resuspend donor E. coli strain cell pellet in 50 μl RCV broth.
  12. Transfer all of the resuspended donor E. coli strain to helper E. coli strain cell pellet and resuspend (if applicable).
  13. Transfer all of the resuspended donor E. coli strain (and helper E. coli strain) to the recipient R. capsulatus strain and resuspend.
  14. Aliquot 10 μl drops of donor-helper-recipient mix onto a dry RCV agar plate (no antibiotics) and allow for the drops to dry.
  15. Incubate plate upside down at 30 °C for 1-2 days. R. capsulatus but not E. coli will grow on the RCV agar plate and the cells are ready when the conjugation spots have a red ring around it. The middle of the spot will likely be pale pink.
  16. Streak the conjugation spots onto RCV agar plates containing appropriate antibiotic to select for the cell containing the plasmid (see Materials and Reagents for plasmid example). Do this by scraping the red ring around the conjugation spot up with an inoculation loop.
  17. Incubate streaked plate at 30 °C for 3-4 days or until you see colonies.
  18. Optional (this will also be done in step 20): Test the R. capsulatus colonies for plasmid using your choice method, such as colony PCR.
  19. Restreak colony on YPS agar plate containing appropriate antibiotics and incubate at 30 °C for 2-3 days to ensure that it is “clean” of E. coli cells. Although E. coli does not grow on RCV, it can survive. E. coli will grow on YPS agar plates. This YPS agar plate will isolate R. capsulatus cells containing the conjugated plasmid from the E. coli survivors as individual colonies. You can visually identify single R. capsulatus colonies on this YPS agar plate. It will be pink/maroon in colour compared to the cream colored E. coli colonies.
  20. Test the “non-contaminated” R. capsulatus colonies for the conjugated plasmid by colony PCR.

Recipes

  1. Trace element solution (in 250 ml dH2O)
    0.7 g H3BO3
    398 mg MnSO4·H2O
    188 mg Na2MoO4·2H2O
    60 mg ZnSO4·7H2O
    10 mg Cu(NO3)·3H2O
  2. RCV broth/agar (Beatty et al., 1981) (in 1 L; autoclaved)
    4 g D, L-malic acid
    1 g (NH4)2SO4
    10 mM potassium phosphate buffer
    200 mg MgSO4·7H2O
    75 mg CaCl2·2H2O
    12 mg FeSO4·7H2O
    20 mg Na2EDTA
    1 ml trace element solution
    1 mg thiamine hydrochloride
    Adjust pH to 6.8 with NaOH before autoclaving
    (for agar add 1.5% Agar)
  3. YPS broth/agar (Wall et al., 1975) (autoclaved)
    0.3% Difco yeast extract
    0.3% Difco bactopeptone
    2 mM CaCl2
    2 mM MgSO4
    (for agar add 1.5% agar)
  4. LB Broth (Sambrook et al., 1989) (in 1 L; autoclaved)
    10 g Bacto-tryptone
    5 g Yeast extract
    10 g NaCl
    Adjust pH to 7.5 with NaOH before to autoclaving
    (for agar add 1.5% Agar)

Acknowledgments

The development of this protocol was funded by a grant to J.T.B. from the Canadian Institutes of Health Research.

References

  1. Beatty J. T. and Gest H. (1981). Generation of succinyl-coenzyme A in photosynthetic bacteria. Arch Microbiol 129(5): 335-340.
  2. Leung M.M., Brimacombe C.A., Spiegelman G.B., and Beatty, J.T. (2012). The GtaR protein negatively regulates transcription of the gtaRI operon and modulates gene transfer agent (RcGTA) expression in Rhodobacter capsulatus. Mol Microbiol 83(4):759-74.
  3. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). Molecular cloning: a laboratory manual (2nd edn). Plainview: New York: Cold Spring Harbor Laboratory Press.
  4. Wall J.D., Weaver P.F., et al. (1975). Gene transfer agents, bacteriophages, and bacteriocins of Rhodopseudomonas capsulata. Arch Microbiol 105(3): 217-224.
  5. Willetts,N., and Wilkins, B. (1984). Processing of plasmid DNA during bacterial conjugation. Microbiol Rev 48: 24-41.

简介

质粒的细菌缀合是将外来DNA引入红细胞脓杆菌的常见方法,因为转化系统未显示为将DNA引入R的有效方法。 capsulatus 。 对于 R。 使用大肠杆菌供体的胶质细胞共轭可用于引入复制载体和用于靶向染色体修饰的非复制载体。

材料和试剂

  1. R。 胶囊受体菌株
  2. 能够偶联的大肠杆菌供体菌株(含有待偶联的质粒)(例如S17-1)或E。 含有待结合的质粒的大肠杆菌供体菌株和含有基因[例如HB101(pRK2013)]的辅助菌株。 关于结合和tra基因的综述参见Willetts等人(1984)
  3. 待结合到质粒中的质粒。 capsulatus (例如 pXCA601;四环素抗性)
  4. 适当的抗生素(质粒和细菌菌株指定的抗性)
  5. 盐酸硫胺素
  6. H 3 BO 3
  7. MnSO 4 H·H 2 O·m/2
  8. Na 2 MoO 4 4·2H 2 O··············································································································
  9. ZnSO 4·7H 2 O·m/2
  10. Cu(NO 3)·3H 2 O·m/2
  11. D,L-苹果酸
  12. Na 2 EDTA
  13. MgSO 4·7H 2 O·h/v
  14. CaCl 2 2·2H 2 O·m/2
  15. FeSO 4 7H·7H 2 O·m/2
  16. 10mM磷酸钾缓冲液
  17. 0.3%Difco酵母提取物
  18. 0.3%Difco细菌蛋白胨
  19. 细菌胰蛋白酶
  20. 酵母提取物
  21. 微量元素溶液(参见配方)
  22. RCV肉汤(请参阅食谱)
  23. RCV琼脂(见配方)
  24. LB肉汤(见配方)
  25. LB琼脂(见配方)
  26. YPS琼脂(见Recipes)

设备

  1. 30℃和37℃摇床
  2. 30℃和37℃孵育器
  3. 试管
  4. 培养皿
  5. 无菌1.7 ml微量离心管
  6. 接收回路
  7. 移液管(10μl至1 ml范围)和适当的提示
  8. 刻度移液管(5 ml量程)和抽吸灯泡
  9. 台式微量离心机,带有转子,用于1.7ml微量离心管

程序

  1. 条纹收件人。 在RCV琼脂平板(含有合适的抗生素)上涂布荚膜囊泡菌株并在30℃下孵育2-3天。
  2. 在LB琼脂平板上用适当的抗生素条纹供体大肠杆菌菌株(和辅助大肠杆菌),并在37℃温育过夜。
  3. 用受体R的单个菌落接种4ml RCV肉汤(具有合适的抗生素)。 囊泡菌株,并在30℃下在200-250rpm振荡器中孵育2天。
  4. 一天后,用供体E接种4ml LB肉汤(具有合适的抗生素)。 大肠杆菌菌株和4ml具有辅助E的LB肉汤。 大肠杆菌菌株(例如参见材料和试剂),并在37℃下在200-250rpm振荡器中孵育过夜。
  5. 在单独的无菌微量离心管中转移100微升供体。 大肠杆菌菌株,100μl辅助大肠杆菌菌株(如果适用)和200μl受体荚膜囊泡菌株。 每个菌株应处于对数中期至晚期。
  6. 旋转微量离心管,其含有在3500xg下的培养物1分钟。 在台式离心机中
  7. 从微量离心管中倒出所有上清液
  8. 重悬细胞沉淀在500微升RCV肉汤每个离心管洗去残留的抗生素和LB肉汤。
  9. 在台式离心机中以4,000×g离心1分钟旋转含有重悬浮培养物的微量离心管。
  10. 从微量离心管中倒出所有上清液
  11. 重悬供体。大肠杆菌株细胞沉淀在50μlRCV肉汤中
  12. 将所有重悬的供体大肠杆菌菌株转移至辅助大肠杆菌菌株细胞沉淀并重悬(如果适用)。
  13. 将所有重悬的供体大肠杆菌菌株(和辅助大肠杆菌菌株)转移至接受体R。 capsulatus 紧张和重新悬浮
  14. 将10微升供体 - 辅助受体混合物滴在干燥的RCV琼脂板(无抗生素)上,并使滴剂干燥。
  15. 将板在30℃下颠倒孵育1-2天。 R。 capsulatus 而不是大肠杆菌将在RCV琼脂平板上生长,当结合点周围有红色环时,细胞就准备好了。地点的中间可能是淡粉红色。
  16. 将结合点连接到含有合适抗生素的RCV琼脂平板上,以选择含有质粒的细胞(参见材料和试剂的质粒实施例)。通过用接种环刮擦缀合点周围的红色环来实现这一点
  17. 将条纹板在30°C孵育3-4天,或直到你看到殖民地
  18. 可选(这也将在步骤20中进行):使用您的选择方法(例如菌落PCR)测试质粒的 R.capsulatus 菌落。
  19. 在含有适当抗生素的YPS琼脂平板上再分裂菌落,并在30℃下孵育2-3天,以确保它是"清洁的"E。大肠杆菌细胞。虽然大肠杆菌不在RCV上生长,它可以存活。大肠杆菌将在YPS琼脂平板上生长。该YPS琼脂板将分离R。含有来自大肠杆菌存活菌的共轭质粒的胶质细胞作为单个菌落。您可以直观地识别单个R。在该YPS琼脂平板上的荚膜囊泡菌落。与奶油色的 E相比,它将是粉红色/栗色。大肠杆菌菌落
  20. 测试"无污染"的R。通过菌落PCR检测结合的质粒的囊泡菌落

食谱

  1. 微量元素溶液(在250ml dH 2 O中) 0.7 g H sub 3 BO sub 3
    398mg MnSO 4 H·H 2 O·m / 188mg Na 2 MoO 4 4·2H 2 O·m 2 / 60mg ZnSO 4·7H 2 O·h/v 10mg Cu(NO 3)·3H 2 O·m/2
  2. RCV肉汤/琼脂(Beatty等人,1981)(在1L中;高压灭菌)
    4克D,L-苹果酸 1 g(NH 4)2 SO 2 4
    10mM磷酸钾缓冲液 200mg MgSO 4·7H 2 O·h/v 75mg CaCl 2·2H 2 O·h/v 12mg FeSO 4·7H 2 O x/v 20mg Na 2 EDTA 1 ml微量元素溶液
    1mg盐酸硫胺素 在高压灭菌之前用NaOH调节pH至6.8 (用于琼脂加1.5%琼脂)
  3. YPS肉汤/琼脂(Wall et al。,1975)(高压灭菌) 0.3%Difco酵母提取物
    0.3%Difco细菌蛋白胨 2mM CaCl 2 2 / 2mM MgSO 4 (用于琼脂加1.5%琼脂)
  4. LB Broth(Sambrook等人,1989)(在1L中;高压灭菌)
    10克细菌用胰蛋白胨 5克酵母提取物
    10克NaCl
    在高压灭菌之前用NaOH调节pH至7.5 (用于琼脂加1.5%琼脂)

致谢

该方案的开发由授予J.T.B.的资助。 来自加拿大健康研究所。

参考文献

  1. Beatty J.T.和Gest H.(1981)。 在光合细菌中产生琥珀酰 - 辅酶A. rch Microbiol 129(5):335-340。
  2. Leung M.M.,Brimacombe C.A.,Spiegelman G.B.,and Beatty,J.T。 (2012)。 GtaR蛋白质负调控 gtaRI 操纵子的转录并调节基因转移 (RcGTA)在 Rhodobacter capsulatus中的表达。 83 83(4):759-74。
  3. Sambrook,J.,Fritsch,E.F。,和Maniatis,T。(1989)。 分子克隆:实验室手册(2 nd edn)。 Plainview: New York:Cold Spring Harbor Laboratory Press
  4. Wall J.D.,Weaver P.F.,et al。 (1975)。 红景天藻(Rhodopseudomonas capsulata)的基因转移剂,噬菌体和细菌素。 Arch Microbiol 105(3):217-224
  5. Willetts,N.和Wilkins,B。(1984)。 在细菌共轭过程中处理质粒DNA。 Microbiol Rev 48:24-41。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Leung, M. M. and Beatty, J. T. (2013). Bacterial Conjugation in Rhodobacter capsulatus. Bio-protocol 3(13): e804. DOI: 10.21769/BioProtoc.804.
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