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Rhodobacter capsulatus Gene Transfer Agent (RcGTA) Activity Bioassays
荚膜红细菌基因转移因子(RCGTA)活性的生物测定   

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Abstract

RcGTA, a small phage-like particle produced by Rhodobacter capsulatus, was initially identified in culture filtrates as a DNase-resistant form of DNA transfer between R. capsulatus cells. This gene transfer assay has been used to identify RcGTA-producing strains and to help determine the roles of genes thought to be responsible for RcGTA production.

Keywords: Transduction(转导), Gene exchange(基因交换), Bacteriophage(噬菌体), Gene disruption(基因中断), Gene knockout(基因敲除)

Materials and Reagents

  1. GTA donor strain (see Notes 1)
  2. Recipient strain (see Notes 1)
  3. RCV broth (Beatty and Gest, 1981)
  4. YPS broth (Wall et al., 1975)
  5. YPS agar (2 plates per recipient-donor combination, one plate per donor, and one plate per recipient)
  6. 0.22 μm filtered GTA buffer (Solioz and Marrs, 1977)
  7. 0.45 μm low protein binding (PVDF) syringe filters (e.g. EMD Millipore, catalog number: SLHV033RB )
  8. Plate spreader (Including 95% ethanol + flame)
  9. 1 ml syringes
  10. Test tubes for aerobic culturing (e.g. Fisher brand brand, catalog number: 14-961-30 ) w/caps
  11. Culture tubes for anaerobic/photosynthetic culturing (e.g. Fisher brand, catalog number: 14-959-37A w/screw caps)
  12. Polypropylene sterile culture tubes (e.g. Simport, catalog number: T405-2A )
  13. Optional (if transferring photosynthesis marker): Chambers/packs for anaerobic plate growth
  14. RCV Medium (see Recipes)
  15. YPS Medium (see Recipes)
  16. GTA buffer (see Recipes)

Equipment

  1. Shaking incubator
  2. Microcentrifuge
  3. Incandescent light-box or light incubator
  4. 1 ml pipettes
  5. 1.5 ml Microcentrifuge tubes

Procedure

  1. Preparation:
    1. 3 days prior to the assay, inoculate GTA donor strains in RCV broth and grow aerobically overnight at 35 °C (200-250 rpm).
    2. 2 days prior, measure optical densities (OD) of overnight cultures of GTA donor strains and normalize them by dilution with RCV broth. It is simplest to dilute all ODs to match the lowest, as the actual OD is irrelevant so long as all cultures are at the same final density. Use a 1% v/v inoculum of normalized donor to inoculate YPS broth without antibiotics to grow anaerobically and photosynthetically over two days at 35 °C without shaking, mixing occasionally by inverting the culture tubes. These culture tubes should be filled to the brim with YPS broth and sealed tightly, to create anaerobic conditions, and placed equidistant from the light source. Incandescent bulbs are better than fluorescent bulbs, but the heat generated from the bulbs must be dissipated (e.g. by having the culture tubes in a water tank) in order to maintain culturing temperature.
    3. 1 day prior, inoculate recipient strain in RCV broth and grow aerobically overnight at 35 °C (200-250 RPM).

  2. Assay:
    1. Pass donor strain cultures through a 0.45 μm filter, collecting the filtrates in polypropylene tubes.
    2. Centrifuge 1 ml of recipient cultures, decant the supernatant and re-suspend in an equal volume of GTA buffer.
    3. Mix the following in a polypropylene tube:
      -Filtrate Controls (for each Donor filtrate): 0.5 ml GTA buffer, 0.1 ml filtrate.
      -Recipient Controls (for each Recipient Strain): 0.5 ml GTA buffer, 0.1 ml recipient cells.
      -Experimental samples: 0.4 ml GTA buffer, 0.1 ml filtrate, 0.1 ml recipient cells.
    4. Incubate tubes at 35 °C for 1 h with no shaking.
    5. Add 0.9 ml RCV broth to each tube.
    6. Incubate tubes at 35 °C for 3 h with shaking at ~200 rpm.
    7. Transfer the 1.5 ml mixtures from the polypropylene tubes to individual 1.5 ml microcentrifuge tubes.
    8. Plate 150 μl of each filtrate + recipient mix on plates to represent 10% of the total. This is not necessary for the recipient and filtrate controls.
    9. Centrifuge all tubes, decant supernatant, and resuspend pellets in the small (~100 μl) volume that remains. Spread these resuspensions on plates to represent 90% and 100% for experimental and control assays, respectively. These plates should be selective for the transfer of the marker, or grown in conditions that select for a transferred marker.
    10. Grow for 2-3 days at 30-35 °C.
    11. Count colonies. Determine ratios of colonies on experimental plates over the number of colonies found on a positive control (e.g. a wild type strain).

Notes

  1. One common bioassay employs monitoring the transfer of the puhA gene to puhA deletion mutant DW5 (Wong et al., 1996), with selection for transfer of the puhA gene being the ability to grow photosynthetically. This bioassay shows no spontaneous mutation background. Another bioassay is the transfer of rifampicin resistance, which is a property of some strains of R. capsulatus, to rifampicin-sensitive strains such as the natural isolate strain B10 (Weaver et al., 1975). This bioassay has a low but detectable rate of spontaneous mutation background that must be accounted for in the data analysis.
  2. It is essential to compare transfer of the same marker, as marker sizes can affect bioassay results (Hynes et al., 2012), presumably affecting both packaging and recombination rates.
  3. Bioassay absolute numbers can vary greatly depending on growth state of donor and recipient cells, batch of media (e.g. the batch of Yeast Extract was used for YPS), so it is essential to compare ratios within one bioassay, and perform multiple independent replicates. When performing bioassays for or into strains/mutants with impaired growth or viability, it can help to normalize transfer rate to the number of viable cells (either donors or recipients, whichever is impaired) by performing viable cell counts alongside the bioassay.

Recipe

  1. RCV Medium
    7.5 mM (NH4)2SO4
    30 mM DL-malate, pH 6.8 with NaOH
    54 μM EDTA
    0.8 mM MgSO4
    0.51 mM CaCl2
    43 μM FeSO4
    3 μM Thiamine-HCl
    9.5 μM MnSO4
    45 μM H3BO3
    0.2 μM Cu(NO3)2
    0.83 μM ZnSO4
    3 μM NaMoO4
    4.5 mM KH2PO4
    5.1 mM K2HPO4
    pH to 6.8
  2. YPS Medium
    3.0 g/L Yeast Extract
    3.0 g/L Peptone
    2 mM MgSO4
    2 mM CaCl2
    pH to 7.0 with NaOH or HCl, as needed
    Add 1.5% Agar for solid media
  3. GTA Buffer
    10mM Tris-HCl (pH 7.8) with NaOH
    1.0 mM MgCl2
    1.0 mM CaCl2
    1.0 mM NaCl
    500 μg/ml BSA (Fraction V)
    Filter sterilize with 0.22 μm filter

Acknowledgments

APH was supported by fellowships from Memorial University School of Graduate Studies and the Natural Sciences and Engineering Research Council (NSERC) of Canada. This research in ASL’s lab was supported by grants from NSERC, the Canada Foundation for Innovation, and the Newfoundland and Labrador Research & Development Corporation. This protocol is based on work originally reported in: Hynes et al. (2012).

References

  1. Beatty, J. T. and H. Gest (1981). Generation of succinyl-coenzyme A in photosynthetic bacteria. Arch Microbiol 129(5): 335-340.
  2. Hynes, A. P., R. G. Mercer, D. E. Watton, C. B. Buckley and A. S. Lang (2012). DNA packaging bias and differential expression of gene transfer agent genes within a population during production and release of the Rhodobacter capsulatus gene transfer agent, RcGTA. Mol Microbiol 85(2): 314-325. 
  3. Solioz, M. and B. Marrs (1977). The gene transfer agent of Rhodopseudomonas capsulata. Purification and characterization of its nucleic acid. Arch Biochem Biophys 181(1): 300-307.
  4. Wall, J. D., P. F. Weaver and H. Gest (1975). Genetic transfer of nitrogenase-hydrogenase activity in Rhodopseudomonas capsulata. Nature 258(5536): 630-631.
  5. Weaver, P. F., J. D. Wall and H. Gest (1975). Characterization of Rhodopseudomonas capsulata. Arch Microbiol 105(3): 207-216. 
  6. Wong, D. K., W. J. Collins, A. Harmer, T. G. Lilburn and J. T. Beatty (1996). Directed mutagenesis of the Rhodobacter capsulatus puhA gene and orf 214: pleiotropic effects on photosynthetic reaction center and light-harvesting 1 complexes. J Bacteriol 178(8): 2334-2342. 

简介

最初在培养滤液中鉴定了由红细菌产生的小噬菌体样颗粒RcGTA作为DNA之间DNA转移的DNA酶抗性形式。 胶囊细胞。 该基因转移测定已经用于鉴定产生RcGTA的菌株并帮助确定被认为负责RcGTA产生的基因的作用。

关键字:转导, 基因交换, 噬菌体, 基因中断, 基因敲除

材料和试剂

  1. GTA供体毒株(见注1)
  2. 受体菌株(见注1)
  3. RCV肉汤(Beatty和Gest,1981)
  4. YPS肉汤(Wall等人,1975)
  5. YPS琼脂(每个受体 - 供体组合2个平板,每个供体一个平板,每个受体一个平板)
  6. 0.22μm过滤的GTA缓冲液(Solioz和Marrs,1977)
  7. 0.45μm低蛋白结合(PVDF)注射器过滤器(例如EMD Millipore,目录号:SLHV033RB)
  8. 平板摊铺机(包括95%乙醇+火焰)
  9. 1 ml注射器
  10. 用于有氧培养的试管(例如 Fisher品牌品牌,目录号:14-961-30)
  11. 用于厌氧/光合培养的培养管(例如Fisher品牌,目录号:14-959-37A /螺旋盖)
  12. 聚丙烯无菌培养管(例如Simport,目录号:T405-2A)
  13. 可选(如果转移光合作用标记物):用于厌氧板生长的箱/包
  14. RCV Medium(见配方)
  15. YPS中等(见配方)
  16. GTA缓冲区(参见配方)

设备

  1. 摇动培养箱
  2. 微量离心机
  3. 白炽灯箱或光孵化器
  4. 1ml移液管
  5. 1.5 ml微量离心管

程序

  1. 制备:
    1. 在测定前3天,在RCV肉汤中接种GTA供体菌株,并在35℃(200-250rpm)有氧生长过夜。
    2. 2天前,测量GTA供体菌株的过夜培养物的光密度(OD),并通过用RCV肉汤稀释将其标准化。稀释所有OD以匹配最低浓度是最简单的,因为只要所有培养物处于相同的最终密度,实际OD是不相关的。使用1%v/v的标准化供体的接种物接种没有抗生素的YPS肉汤,在35℃下在无振荡的情况下在两天内无氧生长和光合作用生长,偶尔通过颠倒培养管混合。这些培养管应该用YPS肉汤填充到边缘并密封,以产生无氧条件,并与光源等距离放置。白炽灯泡比荧光灯泡更好,但是从灯泡产生的热必须消散(例如通过在水箱中具有培养管)以维持培养温度。
    3. 1天前,在RCV肉汤中接种受体菌株,并在35℃(200-250RPM)下有氧生长过夜。

  2. 测定:
    1. 使供体菌株培养物通过0.45μm过滤器,在聚丙烯管中收集滤液。
    2. 离心1ml接受者培养物,倾析上清液并重悬于等体积的GTA缓冲液中。
    3. 将以下物质在聚丙烯管中混合:
      - 滤液对照(对于每种供体滤液):0.5ml GTA缓冲液,0.1ml滤液 - 受体对照(对于每种受体菌株):0.5ml GTA缓冲液,0.1ml受体细胞 实验样品:0.4ml GTA缓冲液,0.1ml滤液,0.1ml受体细胞
    4. 孵育管在35°C 1小时,没有摇晃。
    5. 向每个管中加入0.9ml RCV肉汤。
    6. 孵育管在35℃下3小时,在〜200 rpm摇动。
    7. 将1.5毫升混合物从聚丙烯管转移到单独的1.5毫升微量离心管中
    8. 板在板上150μl每种滤液+受体混合物以代表总量的10%。 对于受体和滤液控制,这不是必需的
    9. 离心所有试管,倾析上清液,并重悬在小(〜100微升)体积剩余的颗粒。 将这些重悬浮在板上分别代表90%和100%的实验和对照测定。 这些平板应当对标记的转移具有选择性,或在选择转移的标记的条件下生长。
    10. 在30-35℃生长2-3天。
    11. 计数菌落。确定实验平板上的集落相对于阳性对照(例如野生型菌株)上发现的集落数的比率。

笔记

  1. 一种常见的生物测定法使用监测puhA基因转移到puhA缺失突变体DW5(Wong等人,1996)的转移,其中选择转移的puhA 基因是光合生长的能力。该生物测定显示没有自发突变背景。另一种生物测定是利福平抗性的转移,其是一些R菌株的特性。 (例如天然分离菌株B10(Weaver等人,1975))的利福平敏感性菌株。该生物测定具有低但可检测的自发突变背景的速率,其必须在数据分析中考虑
  2. 比较相同标记的转移是必要的,因为标记大小可影响生物测定结果(Hynes等人,2012),可能影响包装和重组率。
  3. 生物测定绝对数可以根据供体和受体细胞,培养基批次(例如,将一批酵母提取物用于YPS)的生长状态极大地变化,因此必须比较 一个生物测定,并执行多个独立的重复。 当对具有受损的生长或存活力的菌株/突变体进行生物测定时,通过在生物测定旁边进行活细胞计数,其可以帮助将转移速率标准化为活细胞数目(供体或受体,无论哪种受损)。 >

食谱

  1. RCV中
    7.5mM(NH 4)2 SO 4 4/
    30mM DL-苹果酸盐,pH 6.8,用NaOH洗涤 54μMEDTA
    0.8mM MgSO 4 0.51mM CaCl 2·h/v 43μMFeSO 4
    3μM硫胺素-HCl
    9.5μMMnSO 4
    45μMH sub 3 BO 3
    0.2μMCu(NO 3)2 sub。 0.83μMZnSO 4
    3μMNaMoO 4
    4.5mM KH 2 PO 4>/
    5.1mM K 2 HPO 4
    pH至6.8
  2. YPS中等
    3.0 g/L酵母提取物
    3.0 g/L蛋白胨 2mM MgSO 4 2mM CaCl 2 2 / 根据需要用NaOH或HCl将pH调节至7.0 为固体培养基添加1.5%琼脂
  3. GTA缓冲区
    10mM Tris-HCl(pH7.8)和NaOH 1.0mM MgCl 2·h/v 1.0mM CaCl 2/v/v 1.0 mM NaCl
    500μg/ml BSA(组分V)
    用0.22μm过滤器过滤灭菌

致谢

APH得到了来自纪念大学研究生学院和加拿大自然科学和工程研究理事会(NSERC)的奖学金的支持。 这项在ASL实验室的研究得到了NSERC,加拿大创新基金会和纽芬兰和拉布拉多研究与发展基金会的资助。 发展公司。 该协议基于最初报道的工作:Hynes等人(2012)。

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. Hynes,A.P.,R.G.Mercer,D.E.Watton,C.B.Buckley和A.S.Lang(2012)。 DNA包装偏差和基因转移剂基因在生产和释放期间的群体中的差异表达em> Rhodobacter capsulatus gene transfer agent,RcGTA。 Mol Microbiol 85(2):314-325。 
  3. Solioz,M。和B.Marrs(1977)。 红景天藻(Rhodopseudomonas capsulata)的基因转移剂。其核酸的纯化和表征。 Arch Biochem Biophys 181(1):300-307。
  4. Wall,J.D.,P.F.Weaver和H.Gest(1975)。 在Rhodopseudomonas capsulata中的氮化酶 - 氢化酶活性的遗传转移 。 自然 258(5536):630-631。
  5. Weaver,P.F.,J.D.Wall和H.Gest(1975)。 Rhodopseudomonas capsulata的表征 Arch Microbiol 105(3):207-216。 
  6. Wong,D.K.,W.J.Collins,A.Harmer,T.G.Lilburn和J.T.Batty(1996)。 耻链球菌的定向诱变 puhA基因和orf 214:多效性 对光合反应中心和光收获1复合物的影响。细菌 178(8):2334-2342。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Hynes, A. P. and Lang, A. S. (2013). Rhodobacter capsulatus Gene Transfer Agent (RcGTA) Activity Bioassays. Bio-protocol 3(2): e317. DOI: 10.21769/BioProtoc.317.
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