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Cell-to-cell DNA Transfer among Thermus Species
栖热菌属细胞间DNA的转移   

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Abstract

The ability to transfer DNA via direct cell-to-cell contact-dependent process similar to conjugation has been described in Thermus thermophilus (Tth). Here, we detail the mating experiment protocol involving the lateral transfer of thermostable antibiotic resistance markers (i.e., kanamycin: KmR; hygromycin: HygR) between Thermus cells, enabling the selection and quantification of the transfer frequencies. Briefly, liquid cultures of both mates are mixed and laid onto a nitrocellulose filter on a TB plate. After incubation at 60 °C, filters are resuspended upon selective plating. The contribution of DNA uptake by transformation is abolished by the addition of DNase I to the mix. This protocol can be used for the transfer of large DNA fragments (> 10 kb) to Thermus species.

Background

Conjugation, as the chief mechanism for horizontal gene transfer for most bacteria, is a highly specialized process by which DNA is transferred between two cells which are in direct contact (Lederberg and Tatum, 1946). Classical conjugation involves the unidirectional transfer of a DNA molecule, generally plasmid-encoded, from a donor to a recipient cell, which remains passive. However, alternative models have been described for a wide variety of bacteria. In the laboratory, conjugation experiments are fruitful for marker-exchange mutagenesis, among others. Over other methods of genetic transfer, conjugation is advantageous in terms of minimal disruption of the bacterial envelope and the feasibility to transfer large DNA fragments, including large chromosomal regions (> 10 kb).

A conjugation-like process among T. thermophilus cells was reported a decade ago, where chromosomal markers were transferred following a high frequency of recombinants Hfr-like process, as evidenced by interrupted mating assays employing liquid mixes of different strains (Ramírez-Arcos et al., 1998). Recently, the model proposed for Thermus thermophilus describes the transfer as a two-step bidirectional process where a functional competence apparatus is required in the recipient cell but not in the donor, bestowing this phenomenon the name of ‘transjugation’ (Blesa et al., 2015). Compared to liquid mating assays aforementioned, transfer frequencies obtained with the protocol described here are higher and more robust. Higher reproducibility of the assays is reached with this protocol compared to mating tests in liquid. Besides, validation of the conjugative transfer is ensured by the addition of DNase.

Materials and Reagents

  1. Sterile plastic Petri dish plates (standard size; 100 x 15 mm)
  2. Sterile 1.5 ml microcentrifuge tubes (STARSTEDT, catalog number: 72.690.001 )
  3. Sterile tips for micropippettes
  4. Nitrocellulose filters, 0.22 μm (EMD Millipore, catalog number: GSWP02500 )
  5. Donor and recipient Thermus strains, containing selectable markers (i.e., Thermus sp geneA::kat and Thermus sp geneB::hyg)*
  6. Tryptone (Conda, catalog number: 1612 )
  7. Yeast extract (Conda, catalog number: 1702 )
  8. Sodium chloride (NaCl) (EMD Millipore, catalog number: 1.06404.1000 )
  9. Agar (grade A) (Conda, catalog number: 1800 )
  10. Thermus water (carbonate-rich mineral water)
  11. Appropriate thermostable selectable markers (i.e., kanamycin: KmR; Hygromycin: HygR)**
  12. Kanamycin sulphate (Sigma-Aldrich, catalog number: K1377-5G )
  13. Hygromycin B (Sigma-Aldrich, catalog number: BH7772-1G )
  14. DNase I, grade II from bovine pancreas (1 mg/ml) and its buffer solution (Roche Diagnostics, catalog number: 10104159001 )
  15. TB liquid medium (see Recipes)
  16. TB agar plates (see Recipes)

Notes:

  1. *Several Thermus thermophilus strains such as HB27, NAR1 and HB8, have been successfully employed in mating assays (Ramírez-Arcos et al., 1998; César et al., 2011; Blesa et al., 2015). All parental Thermus strains can be acquired at the DSMZ ollection.
  2. **Vectors containing thermostable selectable markers are available at RIKEN repository

Equipment

  1. 50 ml Erlenmeyer flask
  2. Rotary shaker incubator reaching 70 °C (150 rpm) (Thermo Scientific, Thermo ScientificTM, catalog number: SHKE 420HP )
  3. Glass plating beads or a spreader
  4. Autoclave device (CertoClav, model: EL 18L )
  5. Tweezers (EMD Millipore, catalog number: XX6200006P )
  6. Vortex Minishaker MS2 (IKA, model: MS2 )
  7. Spectrophotometer (Hitachi, model: U-2000 )
  8. Pipette P2, 0.2-2 μl (Gilson, Pipetman classicTM, catalog number: F144801 )
  9. Pipette P20, 2-20 μl (Gilson, Pipetman classicTM, catalog number: F123600 )
  10. Pipette P100, 20-100 μl (Gilson, Pipetman classicTM, catalog number: F123615 )
  11. Pipette P200, 50-200 μl (Gilson, Pipetman classicTM, catalog number: F123601 )
  12. 70 °C heater (Gemini, model: Memmert UE400 )
  13. Bench-top microcentrifuge with rotor for 1.5 ml microcentrifuge tubes (Eppendorf, model: Mini Spin® plus )
  14. Humid chambers
    Notes:
    1. Tupperware devices are preferentially used. Humidified plastic bags are also valid.
    2. Addition of a paper towel, soaked on Thermus water and laid on the bottom of the chamber would enhance humidity maintenance. Ventilation should be avoided in order to conserve humidity. An example is provided in the photographs beneath (Figure 1).


      Figure 1. Humid chambers. Descriptive photographs of the humid chambers used to incubate Thermus plates. Place a piece of paper soaked with Thermus water and a bottle cap filled with the same water on the bottom of the chambers to maintain high humidity.

Procedure

A schematic summary is also provided underneath (Figure 2).

  1. Culture preparation
    1. Use 50 μl of each saturated T. thermophilus liquid growth culture to inoculate 10 ml TB medium containing the appropriate antibiotics (KmR [30 μg/ml] or HygR [100 μg/ml]) or inoculate the culture from the glycerol stock or from a frozen pellet.
    2. Incubate overnight the culture at 65 °C in a shaker incubator at 150 rpm.
      Note: If cultures have not grown optimally overnight, it is highly recommended to delay the assay.
  2. Mating mix
    1. Take 100 μl of each saturated overnight grown cells (OD600 ~1.5-2), place them on sterile microcentrifuge tubes and centrifuged them separately for 4 min at 1,500 x g at room temperature (~24 °C).
      Note: In general, 1:1 cells ratio (both saturated cultures; OD600 ~1.5-2) is employed unless specific tests are accomplished or one of the mates has grown in lesser amount.
    2. Discard the supernatant and resuspend each pellet in 6 μl of warm TB medium (pre-heated at 60 °C) and supplement each of them with 0.6 units of DNase I (~0.8 μl; Roche Diagnostics) and its buffer (~1.5 μl of buffer 10x).
      Note: Room temperature TB medium can also be employed for resuspension.
    3. Mix both mates and gently vortex for 5 sec.
      Note: The purpose for vortexing is just to enhance the mixing. It should not be vigorous nor prolonged to avoid harnessing the cells.
    4. Lay the cell mix on a sterile nitrocellulose filter which had been placed on a non-selective pre-warmed TB agar plate.
      Note: A pre-warmed plate facilitates the attachment of the cells to the filter.
  3. Mating transfer
    1. Place the TB agar plate with the cell-containing nitrocellulose filter on a heater at 60 °C for 4 h.
      Note: The plate does not need to be incubated inside a humid chamber.
  4. Mating interruption
    1. Take the agar plate out from the heater and pick the filter containing the mating strains with tweezers.
    2. Soak the filter in 1 ml of sterile TB medium in a microcentrifuge tube at room temperature.
      Note: Pre-heated medium (~60 °C) is recommended as it fastens the detachment of the cells from the filters.
    3. Vortex vigorously each tube to detach the cells from the filter.
      Note: It is preferable to perform several vortex times (e.g., 4 times, max 30-40 sec) than one prolonged one (larger than 1.5 min) to minimize the disturbance upon the cells.
  5. Plating
    1. Prepare serial dilutions of the cell mix in TB medium in sterile microcentrifuge tubes.
    2. Plate 100 μl onto selective TB agar plates using glass beads or a spreader, one with both antibiotics (KmR and HygR) and also plate 100 μl of the mix on its appropriate dilution and selective marker (KmR or HygR).
      Note: Appropriate dilutions should be plated, for instance, 10-6 for each parental strain. Despite differences among mating efficiencies and thus, transconjugants arousal, in general, transconjugants should be plated 10-1 and 10-3 dilutions, which are common in standard matings.
    3. Incubate the TB agar plates at 60 °C for 48 h inside humid chambers.
      Note: Humidity preservation inside the humid chambers is essential for proper growth of colonies.
  6. Transfer frequencies’ calculation
    1. Count the CFUs on each selective plates.
    2. Express the frequency of transfer as the CFUs grown in both markers (KmR and HygR) divided by the CFUs of the recipient mate (for instance, HygR).


      Figure 2. Schematic representation of the Thermus transjugation procedure. Thermus derivatives PH1, a hygromycin resistant strain (in green, H-termed) harbouring the hyg cassette at the megaplasmid (P-termed) is mated to CK1, a kanamycin resistant strain (in blue, K-termed) harbouring the kat cassette in the chromosome (C-termed). Saturated cultures of each of these strains are mixed in equal proportion (100 μl each) and incubated together in the presence of DNase I and then diluted for plating in different selective plates.

      This transjugation protocol was optimized for Thermus thermophilus HB27, which shows the highest rates of cell-to-cell transfer. Less efficient strains should be incubated for longer periods or larger culture volumes employed in order to achieve reproducible data and maximize the transjugants yields.
      Qualitative assay of transjugation can be also assayed by laying 107 Thermus cells (for instance, 10 μl of saturated culture of CK1) on top of a pre-dried double selective agar plate (KmR and HygR) and topped with 107 cells of the counterpart strain (for instance, 10 μl of saturated culture of PH1). One unit of DNase I is added to the spot so as to prevent transformation (Figure 3). The plate is incubated in humid chambers, like aforementioned.


      Figure 3. Schematic representation of the qualitative assay of Thermus transjugation procedure. A double selective Thermus agar plate (KmR and HygR) is amended with a 10 μl drop of saturated cultures of Thermus derivatives PH1, a hygromycin resistant strain (in green, H-termed) harbouring the hyg cassette at the megaplasmid (P-termed) which is then topped with an equal volume of a saturated culture of CK1, a kanamycin resistant strain (in blue, K-termed) harbouring the kat cassette in the chromosome (C-termed). Both cultures are incubated together in the presence of DNase I and incubated at 60 °C in humid chambers.

Data analysis

  1. Transfer frequencies are calculated as aforementioned, dividing the number of CFU transconjugants grown in double selective plates (KmR and HygR) by the CFUs grown in single selective plate (for instance, HygR). Data can be analysed through a set of statistical procedures, as described in Blesa et al. (2015). In general, to examine differences among frequencies of transfers, Student’s t-test and Mann-Whitney U-tests may be used. Transfer efficiencies among strains tagged alike can be compared using these tests too. Besides, Wilcoxon-tests may be used when comparing sets of transfer frequencies of different strains to assess whether the transfer frequency mean differs. When the purpose is to compare the ease for transfer a marker placed in different regions of the genome, one-way ANOVA assays will fit as differences among and within various loci transfer frequencies can be addressed using these tests. Simple linear regression test can be implemented for modelling the relationship between loci and frequency of transfer. Post-hoc Tukey and Bonferroni tests should be applied when convenient.
  2. At least three independent experiments should be performed for reliable and reproducible data.

Notes

  1. Transjugants should be re-streaked twice on selective plates and further grown in liquid under each particular selective condition so as to stabilize the integration of the markers.
  2. DNAse I is compulsory to defeat transformation transfer.
  3. Matings can also be performed on liquid TB in presence of DNase I. Standard experiments involving 100 μl of each mating pair (unless otherwise required) are centrifuged independently for 4 min at 1,500 x g and resuspended in 50 μl of TB medium mixed in 1.5-ml microcentrifuge tubes amended with 5 units of DNase I, centrifuged for 4 min at 1,500 x g and resuspended in 100 μl of TB medium, supplementing the mix with 5 units of DNase I and incubated for 5 h at 60 °C under shaking (180 rpm). After incubation, appropriate dilutions are plated on selective plates, as described above.
  4. For other markers or properties, appropriate selection should be applied. For example, in transjugation of the large denitrification island, the ordinary protocol may be adapted to anoxic conditions for selection. Further adaptations for the transfer of the denitrification island can be reviewed in Álvarez et al. (2014). Briefly, overnight cultures grown at 70 °C are harvested and washed together in liquid TB medium and supplemented with 5 units of DNase I. Then, 60 μl of the mix is laid onto 0.22 μm nitrocellulose filter and incubated at 60 °C for 24 h. Cells are then detached, resuspended in 1 ml of TB medium and added to 10 ml TB medium in anaerobiosis tubes, amended with potassium nitrate (40 mM) and the appropriate antibiotics for selection. Tubes are incubated for 48 h at 70 °C without agitation. Two re-inoculation steps under the same conditions are required prior to plating. Individual colonies should be streaked and tested for denitrifying respiration and presence of denitrifying genes should be confirmed by polymerase chain reaction (PCR).
  5. Erlenmeyer flasks do not require any special sealing. Standard lids are enough to prevent evaporation and preserve sterile conditions. 

Recipes

  1. TB liquid medium
    0.8 % tryptone (w/v)
    0.4 % yeast extract (w/v)
    50 mM NaCl, pH 7.5
    Thermus water (carbonated-rich mineral water)*
    pH may be adjusted by the addition of NaOH
  2. TB agar plates
    0.8% tryptone (w/v)
    0.4% yeast extract (w/v)
    50 mM NaCl, pH 7.5
    1.8% agar (grade A) (w/v)
    Thermus water (carbonated-rich mineral water)*

*Note: The analytical composition of the spring mineral water employed, per mg/ml, is listed beneath (Table 1). Other mineral waters easily purchasable such as Evian water or a laboratory solution resembling this recipe, can be employed.

Table 1. Mineral composition of the spring water from Jaraba Spring in Zaragoza (Spain)

Acknowledgments

This work has been funded by Grants BIO2013-44963-R, and FP7-PEOPLE-2012-IAPP No. 324439. A. Blesa held an FPI contract from the Spanish Ministry of Economy and Competitivity.

References

  1. Alvarez, L., Bricio, C., Blesa, A., Hidalgo, A. and Berenguer, J. (2014). Transferable denitrification capability of Thermus thermophilus. Appl Environ Microbiol 80(1): 19-28.
  2. Blesa, A., Cesar, C. E., Averhoff, B. and Berenguer, J. (2015). Noncanonical cell-to-cell DNA transfer in Thermus spp. is insensitive to argonaute-mediated interference. J Bacteriol 197(1): 138-146.
  3. César, C. E., Alvarez, L., Bricio, C., van Heerden, E., Littauer, D. and Berenguer, J. (2011). Unconventional lateral gene transfer in extreme thermophilic bacteria. Int Microbiol 14(4): 187-199.
  4. Lederberg, J. and Tatum, E. L. (1946). Gene recombination in Escherichia coli. Nature 158(4016): 558.
  5. Ramirez-Arcos, S., Fernandez-Herrero, L. A., Marín, I. and Berenguer, J. (1998). Anaerobic growth, a property horizontally transferred by an Hfr-like mechanism among extreme thermophiles. 180(12): 3137-3143.

简介

通过与共轭相似的直接细胞间接触依赖性过程转移DNA的能力已在Thermus thermophilus(Tth)中描述。在这里,我们详细描述了包括热稳定抗生素抗性标记(即卡那霉素:Km),潮霉素:Hyg R 的横向转移的交配实验方案>)在 细胞之间,使得能够选择和量化转移频率。简言之,将两种配合物的液体培养物混合并铺在TB板上的硝酸纤维素滤膜上。在60℃温育后,在选择性平板培养基上重悬浮过滤器。通过向混合物中加入DNA酶I消除通过转化的DNA摄取的贡献。该方案可用于将大DNA片段(> 10kb)转移到Thermus 物种。

[背景] 是大多数细菌水平基因转移的主要机制,是一种高度专业化的过程,通过其DNA在直接接触的两个细胞之间转移(Lederberg和Tatum,1946)。经典缀合涉及DNA分子(通常是质粒编码的)从供体到受体细胞的单向转移,其仍然是被动的。然而,已经描述了用于多种细菌的替代模型。在实验室中,缀合实验对于标志物交换诱变是有效的。在其它遗传转移方法中,缀合在细菌包膜的最小破坏和转移大DNA片段(包括大的染色体区域(> 10kb))的可行性方面是有利的。  在T之间的共轭样过程。嗜热细菌在十年前被报道,其中染色体标记在重组HFR - 样过程的高频率后被转移,如通过使用不同菌株的液体混合物的中断交配测定所证明的(Ramírez- Arcos等人,1998)。最近,为嗜热栖热菌(Thermus thermophilus)提出的模型将转移描述为两步双向过程,其中在受体细胞中需要功能性能力装置,而不是在供体中,赋予这种现象的名称为" '(Blesa 。,2015)。与上述液体交配测定相比,用本文所述方案获得的转移频率更高和更强。与液体中的交配测试相比,使用该方案达到了测定的更高的再现性。此外,通过添加DNase来确保接合转移的验证。

材料和试剂

  1. 无菌塑料培养皿(标准尺寸; 100×15mm)
  2. 无菌1.5ml微量离心管(STARSTEDT,目录号:72.690.001)
  3. 微博的无菌提示
  4. 硝化纤维素滤膜,0.22μm(EMD Millipore,目录号:GSWP02500)
  5. 供体和接受者Thermus 菌株,其含有可选择标记(即。, geneA :: kat > Thermus sp geneB :: hyg )*
  6. 胰蛋白胨(Conda,目录号:1612)
  7. 酵母提取物(Conda,目录号:1702)
  8. 氯化钠(NaCl)(EMD Millipore,目录号:1.06404.1000)
  9. 琼脂(A级)(Conda,目录号:1800)
  10. 水(富含碳酸盐的矿泉水)
  11. 合适的热稳定性可选择标记(即卡那霉素:Km sup);潮霉素:Hyg R )**
  12. 硫酸卡那霉素(Sigma-Aldrich,目录号:K1377-5G)
  13. 潮霉素B(Sigma-Aldrich,目录号:BH7772-1G)
  14. 来自牛胰腺(1mg/ml)的DNase I,II级及其缓冲溶液(Roche Diagnostics,目录号:10104159001)
  15. TB液体介质(参见配方)
  16. TB琼脂平板(见配方)

注意:

  1. 几种嗜热栖热菌菌株例如HB27,NAR1和HB8已经成功地用于交配测定(Ramírez-Arcos等人,1998;César等人,2011; Blesa等人,2015)。所有亲本Thermus菌株均可在以下地址获取: DSMZ < a>  ollection。
  2. **包含热稳定可选择标记的载体可在RIKEN 知识库中获得

设备

  1. 50ml锥形瓶
  2. 旋转摇床培养箱达到70℃(150rpm)(Thermo Scientific,Thermo Scientific ,目录号:SHKE 420HP)
  3. 玻璃电镀珠或吊具
  4. 高压釜设备(CertoClav,型号:EL 18L)
  5. 镊子(EMD Millipore,目录号:XX6200006P)
  6. Vortex Minishaker MS2(IKA,型号:MS2)
  7. 分光光度计(日立,型号:U-2000)
  8. 移液管P2,0.2-2μl(Gilson,Pipetman classic TM ,目录号:F144801)
  9. 移液管P20,2-20μl(Gilson,Pipetman classic TM ,目录号:F123600)
  10. 移液管P100,20-100μl(Gilson,Pipetman classic TM ,目录号:F123615)
  11. PipetteP200,50-200μl(Gilson,Pipetman classic TM ,目录号:F123601)
  12. 70℃加热器(Gemni,型号:Memmert UE400)
  13. 带有用于1.5ml微量离心管(Eppendorf,型号:Mini Spin plus)的转子的台式微量离心机
  14. 湿气室
    注意:
    1. 优先使用Tupperware设备。加湿的塑料袋也有效。
    2. 添加浸在Thermus水上并置于室底部的纸巾将增强湿度维持。应避免通风,以保存湿度。下面的照片中提供了一个示例(图1)。


      图1.潮湿室。用于孵育Thermus板的潮湿室的描述性照片。将一块浸有Thermus 水的纸和一个装满相同水的瓶盖放在小室底部,以保持高湿度。

程序

下面还提供了示意图(图2)。

  1. 文化准备
    1. 使用50微升每个饱和的。嗜热链球菌液体生长培养物接种含有适当抗生素(Km缓冲液[30μg/ml]或Hyg [100μg/ml])的10ml TB培养基, )或从甘油原液或冷冻颗粒中接种培养物
    2. 在65℃下在振荡培养箱中以150rpm培养过夜培养物 注意:如果培养物在一夜之间没有最佳生长,强烈建议延迟测定。
  2. 配对混合
    1. 取100μl的每个饱和的过夜生长的细胞(OD <600>〜1.5-2),将它们置于无菌微量离心管上,并以1500×g离心它们4分钟室温(〜24℃) 注意:一般来说,使用1:1细胞比率(饱和培养物; OD <600>〜1.5-2),除非完成特定测试或者其中一个配偶生长较少。
    2. 弃去上清液并将每个沉淀物重悬于6μl温热的TB培养基(在60℃下预热)中,并用0.6单位的DNase I(〜0.8μl; Roche Diagnostics)和其缓冲液(〜1.5μl缓冲区10x)。
      注意:室温TB培养基也可用于重悬。
    3. 混合两个伙伴,轻轻涡旋5秒 注意:涡旋的目的只是增强混合。它不应该是有力的或长期的,以避免使用细胞。
    4. 将细胞混合物放置在放置在非选择性预热的TB琼脂平板上的无菌硝酸纤维素滤膜上。
      注意:预热板有助于细胞附着到过滤器。
  3. 配对转移
    1. 将含有细胞的硝酸纤维素滤膜的TB琼脂平板放在60℃的加热器上4小时 注意:板不需要在潮湿的室内孵育。
  4. 配合中断
    1. 从加热器取出琼脂板,用镊子取出含有交配菌株的过滤器
    2. 在室温下将过滤器浸泡在微量离心管中的1ml无菌TB培养基中 注意:建议使用预热介质(约60°C),因为它可以固定细胞与过滤器的分离。
    3. 大力涡旋每个管以从过滤器分离细胞 注意:优选地,比一个延长的(大于1.5分钟)进行几个涡流时间(例如,4倍,最大30-40秒),以最小化对细胞的干扰。
  5. 电镀
    1. 在无菌微量离心管中,在TB培养基中制备连续稀释的细胞混合物
    2. 使用玻璃珠或扩散器将100μl板置于选择性TB琼脂平板上,所述玻璃珠或扩散器具有两种抗生素(Km引物和Hyg ),并且还将100μl混合物适当稀释和选择标记(Km R 或Hyg R )。
      注意:应对每个亲本菌株铺板适当的稀释液,例如10 -6 。尽管交配效率之间存在差异,并且因此,转座结合物的唤醒,一般来说,转接合子应该接种在标准交配中常见的10 -1和10 -3稀释度。 em>
    3. 在60℃下在潮湿室内孵育TB琼脂平板48小时 注意:潮湿室内的湿度保持对于菌落的正常生长至关重要。
  6. 传输频率计算
    1. 计算每个选择性板上的CFU
    2. 表达转移的频率作为在两种标记(Km R 和Hyg R )中生长的CFU除以受者配偶的CFU(例如,Hyg R )。



      图2. Thermus 转导程序的示意图 衍生物PH1,潮霉素抗性菌株(P命名)与染色体(C)中携带kat 盒的卡那霉素抗性菌株(蓝色,K-命名)CK1相配合的大肠杆菌定量)。将这些菌株的饱和培养物以等比例(每种100μl)混合,并在DNase I存在下一起温育,然后稀释以在不同选择性平板中接种。
      这种转染方案针对嗜热栖热菌(Thermus thermophilus)HB27进行优化,其显示细胞至细胞转移的最高速率。效率较低的菌株应该孵育更长的时间或更大的培养体积,以获得可重复的数据并最大化转化体的产量。 也可以通过在预干燥的双选择性琼脂上放置10 7个栖热菌细胞(例如,10μl的CK1的饱和培养物)来测定转移的定性测定平板(Km引物和Hyg引物),并且用对应物菌株的10个支持细胞(例如,10μl的PH1的饱和培养物)。将一单位DNA酶I加入到斑点中以防止转化(图3)。如上所述,将板在潮湿的室中孵育

      图3.热栖热菌交配程序的定性测定的示意图。双选择性Thermus琼脂平板(Km R 和Hyg R )用10μl的Thermus衍生物PH1的饱和培养物修改,所述PH1是一种潮霉素抗性菌株(绿色,H型),其含有

数据分析

  1. 如上所述计算转移频率,将在双选择性平板中生长的CFU转导结合物的数目(Km R 和Hyg R )除以在单选择板中生长的CFU ,Hyg )。可以通过一组统计程序分析数据,如Blesa等人中所述。 (2015)。一般来说,为了检查转移频率之间的差异,可以使用学生的测试和曼 - 惠特尼的测试。使用这些测试也可以比较标记的菌株之间的转移效率。此外,当比较不同菌株的转移频率组以评估转移频率平均值是否不同时,可以使用Wilcoxon 测试。当目的是比较放置在基因组的不同区域中的标记的转移的容易性时,单因素ANOVA测定将适合作为使用这些测试可以解决各种基因座转移频率之间和之内的差异。可以实现简单的线性回归测试来建立轨迹和转移频率之间的关系。 Post-hoc 在方便时,应该应用Tukey和Bonferroni测试。
  2. 应至少进行三次独立实验,以获得可靠和可重现的数据

笔记

  1. 应当将突变体在选择性平板上重新划线两次,并在每种特定选择条件下在液体中进一步生长,以稳定标记的整合。
  2. DNAse我是强制性的,以击败转化转移
  3. 也可以在DNA酶I的存在下对液体TB进行接合。将包括100μl每个接合对(除非另有要求)的标准实验在1,500xg下独立离心4分钟,并重悬于50μl TB培养基在用5单位DNA酶I修饰的1.5ml微量离心管中混合,在1,500×g离心4分钟,并重悬于100μlTB培养基中,用5单位DNA酶I补充混合物,在60℃下在振荡(180rpm)下孵育5小时。孵育后,如上所述将适当的稀释液涂布在选择性平板上
  4. 对于其他标记或属性,应应用适当的选择。例如,在大型脱氮岛的转化中,普通方案可适应于缺氧条件用于选择。关于脱氮岛的转移的进一步适应性可以在Álvarez等人的中查看。 (2014年)。简言之,收获在70℃下生长的过夜培养物,并在液体TB培养基中一起洗涤并补充5单位DNA酶I.然后,将60μl混合物铺在0.22μm硝化纤维素滤膜上,并在60℃下孵育24小时。然后将细胞分离,重悬于1ml TB培养基中,并加入厌氧生长管中的10ml TB培养基中,用硝酸钾(40mM)和适当的抗生素进行修正以供选择。将管在70℃下不搅拌孵育48小时。在电镀之前在相同条件下需要两个再接种步骤。单个菌落应该划线并测试脱氮呼吸作用,并且应该通过聚合酶链反应(PCR)证实脱氮基因的存在。
  5. 锥形瓶不需要任何特殊密封。标准盖子足以防止蒸发和保存无菌条件。

食谱

  1. TB液体介质
    0.8%胰蛋白胨(w/v)
    0.4%酵母提取物(w/v)
    50mM NaCl,pH 7.5
    水(富含碳酸气的矿泉水)*
    pH可以通过加入NaOH调节
  2. TB琼脂平板
    0.8%胰蛋白胨(w/v)
    0.4%酵母提取物(w/v)
    50mM NaCl,pH 7.5
    1.8%琼脂(A级)(w/v) 水(富含碳酸气的矿泉水)*

注意:所用的弹簧矿泉水的分析组成(mg/ml)列在下面(表1)。可以使用易于购买的其他矿泉水,例如Evian水或类似此配方的实验室溶液。

表1.来自的泉水矿物成分
Jaraba Spring 在西班牙萨拉戈萨

致谢

这项工作由Grants BIO2013-44963-R和FP7-PEOPLE-2012-IAPP第324439号资助。A. Blesa与西班牙经济和竞争力部签订了一份FPI合同。

参考文献

  1. Alvarez,L.,Bricio,C.,Blesa,A.,Hidalgo,A.和Berenguer,J.(2014)。  嗜热栖热菌的可转移反硝化能力。 Appl Environ Microbiol 80(1):19 -28。
  2. Blesa,A.,Cesar,CE,Averhoff,B。和Berenguer,J.(2015)。  spp中的非规范细胞至细胞DNA转移。对原子介导的干扰不敏感。 197(1):138-146。
  3. César,CE,Alvarez,L.,Bricio,C.,van Heerden,E.,Littauer,D。和Berenguer,J.(2011)。  极端嗜热细菌中的非常规侧向基因转移。 Int Microbiol 14(4):187-
  4. Lederberg,J.和Tatum,EL(1946)。  大肠杆菌中的基因重组 158(4016):558.
  5. Ramirez-Arcos,S.,Fernandez-Herrero,LA,Marín,I。和Berenguer,J.(1998)。  厌氧生长,通过极端嗜热菌中的Hfr 样机制水平转移的性质。 180(12): 3137-3143。
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引用:Blesa, A. and Berenguer, J. (2016). Cell-to-cell DNA Transfer among Thermus Species. Bio-protocol 6(22): e2006. DOI: 10.21769/BioProtoc.2006.
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