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The gene transfer agent (GTA) is a bacteriophage-like particle that transfers genomic DNA from a donor to a recipient bacterium. The Rhodobacter capsulatus GTA (RcGTA) was the first to be studied and this protocol has been optimized for RcGTA transduction, although it could be modified for other bacteria containing a GTA. The RcGTA transduction assay can be used to determine transduction efficiencies, to create gene knock-outs, or to create new strains by transferring alleles from one strain to another.

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Rhodobacter capsulatus Gene Transfer Agent Transduction Assay
荚膜红细菌基因转移因子转导试验

微生物学 > 微生物遗传学 > DNA > 染色体
作者: Molly M. Leung
Molly M. LeungAffiliation: Department of Microbiology and Immunology, University of British Columbia, Vancouver, Canada
Bio-protocol author page: a240
 and John Thomas Beatty
John Thomas BeattyAffiliation: Department of Microbiology and Immunology, University of British Columbia, Vancouver, Canada
For correspondence: jbeatty@interchange.ubc.ca
Bio-protocol author page: a241
Vol 3, Iss 4, 2/20/2013, 4095 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.334

[Abstract] The gene transfer agent (GTA) is a bacteriophage-like particle that transfers genomic DNA from a donor to a recipient bacterium. The Rhodobacter capsulatus GTA (RcGTA) was the first to be studied and this protocol has been optimized for RcGTA transduction, although it could be modified for other bacteria containing a GTA. The RcGTA transduction assay can be used to determine transduction efficiencies, to create gene knock-outs, or to create new strains by transferring alleles from one strain to another.

[Abstract] 该实验方案的中文版正在准备中...

Materials and Reagents

  1. RcGTA donor strain (e.g. R. capsulatus Y262 or RifR strain)
  2. Recipient strain (e.g. R. capsulatus DW5 [photosynthesis mutant] or RifS strain to test the transfer of the RifR allele)
  3. Potassium phosphate buffer
  4. L-malic acid (Sigma-Aldrich, catalog number: 240176 )
  5. Thiamine hydrochloride (Sigma-Aldrich, catalog number: T1270 )
  6. G-buffer (filter sterilize) (see Recipes)
  7. RCV broth/agar1 (see Recipes)
  8. YPS broth/agar3 (autoclaved) (see Recipes)

Equipment

  1. Variable temperature shaker for test tubes (30-35 °C)
  2. Anaerobic jars for photosynthetic growth (if using photosynthesis mutant for recipient)
  3. Light box for photosynthetic growth
  4. 30 °C incubator
  5. Test tubes
  6. 17 ml glass screwcap tubes
  7. 10 ml plastic culture tubes
  8. Glass beads
  9. 0.45 μm pore-size filter
  10. 10 ml syringe

Procedure

     Day 1

  1. Streak Rhodobacter capsulatus Y262 containing plasmid containing gene to be transferred (or other gene donor), and DW5 (or other desired recipient strain) on RCV agar plates with appropriate antibiotics.
  2. Incubate plates aerobically at 30 °C for 2 days (or until colonies form)
    Day 3
  3. In a 17 ml screwcap tube (should contain sterile glass beads to help with mixing the cells during growth) inoculate 17 ml of YPS broth (NO antibiotic) with Y262 (containing plasmid) from a single colony.
  4. Grow photosynthetically at 30 °C until culture is well into the stationary phase (cell density should be around 450-500 Klett units).
    Day 4
  5. In test tubes inoculate 7 ml RCV broth with R. capsulatus DW5 or recipient strain(s) from a single colony.
  6. Incubate at 30 °C, shaking at 250 RPM for 2 days (cell density should be around 300-350 Klett units).
    Day 6
  7. Spin down 7 ml of DW5 and recipient cultures at ~20,000 x g (5,000 rpm in a Beckman JA-20 rotor) for 5 min.
  8. Decant and resuspend cell pellets in 3.5 ml G-buffer.
  9. Spin down 17 ml of Y262 (plasmid) donor strain at ~20,000 x g for 5 min to pellet most of the cells.
  10. Filter the remaining culture supernatant with a 0.45 μm pore-size filter into a sterile plastic (polystyrene) tube (not glass because it is suspected that RcGTA particles stick to glass).
  11. Prepare the transduction mix in a 10 ml plastic culture tube:
    0.1 ml filtrate (donor)
    0.2 ml recipient
    0.4 ml G-buffer
    0.7 ml total volume
    (Negative controls: 1) lacking the recipient and 2) lacking the RcGTA donor; positive control: DW5 with photosynthetically capable donor RcGTA).
  12. Incubate the transduction mix for 1-1.5 h at 30-35 °C with slow shaking (150 rpm).
  13. Add 0.9 ml RCV broth to each tube and incubate for 3-4 h at 30-35 °C with slow shaking (~150 rpm).
  14. Transfer transduced culture to a 1.7 ml microcentrifuge tube and pellet cells at maximum rpm for 30 sec.
  15. Decant the majority of the supernatant leaving about 50 μl.
  16. Resuspend cells in remaining supernatant and spread cells on an agar plate appropriate for selection (for DW5 positive control may use YPS medium).
  17. Incubate plates in conditions appropriate for selection for 3-4 days (for positive control, select for anaerobic photosynthetic growth).
  18. Test colonies for desired insertion.

Recipes

  1. G-buffer (filter sterilize)
    10 mM Tris-HCl (pH 7.8)
    1 mM MgCl2
    1 mM CaCl2
    1 mM NaCl
    0.5 mg/ml BSA
  2. RCV broth/agar1 (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
    pH adjusted to 6.8 with NaOH before autoclaving
    (for agar add 1.5 % agar)
    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
  3. YPS broth/agar3 (autoclaved)
    0.3% Difco yeast extract
    0.3% Difco Bactopeptone
    2 mM CaCl2
    2 mM MgSO4
    (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. This protocol was adapted from the assay developed by Solioz et al. (1975).

References

  1. Beatty, J. T. and H. Gest (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):759-774.
  3. Solioz, M., Yen, H. C. and Marris, B. (1975). Release and uptake of gene transfer agent by Rhodopseudomonas capsulata. J Bacteriol 123(2): 651-657.
  4. Wall, J. D., Weaver, P. F. and Gest, H. (1975). Gene transfer agents, bacteriophages, and bacteriocins of Rhodopseudomonas capsulata. Arch Microbiol 105(3): 217-224.

材料和试剂

  1. RcGTA供体菌株(例如,荚膜囊菌Y262或Rif R 菌株)
  2. 为了测试Rif R 转移的受体菌株(例如荚膜囊泡DW5 [光合作用突变体]或Rif sup>等位基因)
  3. 磷酸钾缓冲液
  4. L-苹果酸(Sigma-Aldrich,目录号:240176)
  5. 盐酸硫胺素(Sigma-Aldrich,目录号:T1270)
  6. G缓冲(过滤灭菌)(参见配方)
  7. RCV肉汤/琼脂 1 (参见配方)
  8. YPS肉汤/琼脂3(高压灭菌)(参见Recipes)

设备

  1. 用于试管(30-35℃)的可变温度摇床
  2. 厌氧罐用于光合生长(如果使用光合作用突变体用于受体)
  3. 光合增长的灯箱
  4. 30& C孵育器
  5. 试管
  6. 17毫升玻璃螺丝帽管
  7. 10ml塑料培养管
  8. 玻璃珠
  9. 0.45μm孔径过滤器
  10. 10毫升注射器

程序

     第1天

  1. 条纹的含有待转移的基因(或其他基因供体)的质粒Y262和DW5(或其它所需的受体菌株)在含有适当抗生素的RCV琼脂平板上。
  2. 在30℃有氧条件下培养平板2天(或直到菌落形成)
    第3天
  3. 在17ml螺旋盖管(应含有无菌玻璃珠以帮助在生长期间混合细胞)接种来自单个菌落的17ml YPS肉汤(NO抗生素)与Y262(含有质粒)。
  4. 在30℃生长光合作用,直到培养物进入稳定期(细胞密度应在约450-500Klett单位)。
    第4天
  5. 在试管中用R接种7ml RCV肉汤。 荚膜囊泡 DW5或来自单个菌落的受体菌株
  6. 在30℃孵育,以250RPM摇动2天(细胞密度应为约300-350Klett单位)。
    第6天
  7. 在〜20,000×g(5000rpm,Beckman JA-20转头)中旋转7ml DW5和接受培养物5分钟。
  8. 倾析和重悬细胞沉淀在3.5毫升G缓冲液
  9. 在〜20,000×g下旋转17ml Y262(质粒)供体菌株5分钟,以沉淀大部分细胞。
  10. 用0.45μm孔径的过滤器将剩余的培养物上清液过滤到无菌塑料(聚苯乙烯)管(不是玻璃,因为怀疑RcGTA颗粒粘在玻璃上)。
  11. 在10ml塑料培养管中制备转导混合物:
    0.1ml滤液(供体) 0.2 ml收件人
    0.4ml G-缓冲液
    总量为0.7 ml
    (阴性对照:1)缺乏受体和2)缺乏RcGTA供体; 阳性对照:具有光合作用能力的供体RcGTA的DW5)
  12. 在30-35℃,缓慢摇动(150rpm)下孵育转导混合物1-1.5小时。
  13. 向每个管中加入0.9ml RCV肉汤,并在30-35℃下缓慢摇动(〜150rpm)孵育3-4小时。
  14. 将转导培养物转移到1.7ml微量离心管中,并以最大rpm速度沉淀细胞30秒
  15. 倾析大部分上清液留下约50μl
  16. 重悬细胞在剩余的上清液和扩散细胞在适于选择的琼脂板上(对于DW5阳性对照可以使用YPS培养基)。
  17. 在适合选择3-4天的条件下孵育平板(对于阳性对照,选择厌氧光合生长)。
  18. 测试所需插入的菌落。

食谱

  1. G缓冲(过滤灭菌)
    10mM Tris-HCl(pH7.8) 1mM MgCl 2
    1mM CaCl 2
    1mM NaCl 0.5 mg/ml BSA
  2. RCV肉汤/琼脂1(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> 7H 2 O 75mg CaCl 2 2 2H 2 12mg FeSO 4 sub 7H 2 O 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将pH调节至6.8 (用于琼脂加1.5%琼脂) 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 O
    188mg Na 2 MoO 4 2H 2 60mg ZnSO 4 7H 2 O 10毫克Cu(NO 3)n。3 H 2 O 2。
  3. YPS肉汤/琼脂3(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)

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。 该方案改编自Solioz等人(1975)开发的测定法。

参考文献

  1. Beatty,J.T.和H.Gest(1981)。 在光合细菌中产生琥珀酰辅酶A。 Arch Microbiol   129(5):335-340。
  2. Leung MM,Brimacombe CA,Spiegelman GB和Beatty JT(2012)。  GtaR蛋白 负调节αRI的转录 操纵子并调节荚膜红细菌中的基因转移剂(RcGTA)表达。 Mol Microbiol   83(4):759-774。
  3. Solioz,M.,Yen,H.C.and Marris,B。(1975)。 通过红细胞藻(Rhodopseudomonas capsulata)释放和摄取基因转移剂。 123(2):651-657。
  4. Wall,J.D.,Weaver,P.F.and Gest,H。(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
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How to cite this protocol: Leung, M. M. and Beatty, J. T. (2013). Rhodobacter capsulatus Gene Transfer Agent Transduction Assay. Bio-protocol 3(4): e334. DOI: 10.21769/BioProtoc.334; Full Text



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