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Jul 2017
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Quantitative Transformation Efficiency Assay for Bacillus subtilis
枯草芽孢杆菌转化效率的定量测定   

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Abstract

Bacillus subtilis (B. subtilis) is a model Gram-positive organism used to study a variety of physiological states and stress responses, one of which is the development of competence. Competence refers to the physiological state of a cell which allows it to be transformed naturally. Through induction of competence, the efficiency of natural transformation can be quantified by plating colony forming units (CFU) and transforming units (TFU). Here we describe a protocol for quantifying relative transformability using B. subtilis.

Keywords: Natural transformation (自然转化), Competence (感受性), Bacillus subtilis (枯草芽孢杆菌), Quantitative transformation efficiency assay (转化效率定量测定), Horizontal gene transfer (HGT) (水平基因转移), DNA uptake (DNA摄取)

Background

Natural transformation is a type of horizontal gene transfer that involves a bacterium importing exogenous DNA from the environment and recombining that DNA into its genome. This process differs from induced transformation in that naturally transformable bacteria produce proteins that facilitate the import of DNA. Modification of the cell membrane, such as through electroporation or heat shock, is typically essential for induced transformation. Natural transformation can lead to the acquisition of new genetic elements, such as antibiotic resistance or catabolic genes, and may allow bacteria to adapt rapidly to their environment. The various proteins involved with the development of competence, or the physiological state of a cell which allows it to be transformed naturally, and the mechanism of natural transformation are not yet fully understood. Quantitative analysis of transformation efficiency differences between a mutant and wild type provides insight into the contribution and potential functions of the gene in question. Beginning with the description of requirements for transformation of laboratory strains of Bacillus subtilis by Anagnostopoulos and Spizizen (1961), numerous variations on the original media and conditions for transformation, both qualitative and quantitative methods, have been described including but not limited to those by Dubnau and Davidoff-Abelson (1971) and Cutting and Vander Horn (1990). The experimental procedure described here is similar to those prior, focusing on currently available and preferred materials (such as casein hydrolysate, which has been found to produce varying efficiencies based on lot and manufacturer), and includes description of statistical analysis of the results.

Materials and Reagents

  1. Sterile pipette tips
  2. Sterile 18 mm glass tubes (Fisher Scientific, catalog number: S63300)
  3. Sterile 13 mm glass tubes (Fisher Scientific, catalog number: 14-961-27)
  4. Sterile stainless steel closures (18 mm neck size) (VWR, catalog number: 71000-330)
  5. Sterile stainless steel closures (13 mm neck size) (VWR, catalog number: 71000-322)
  6. Sterile 1.7 ml microfuge tubes (Denville Scientific, catalog number: C2170)
  7. Sterile 60 ml Luer Lock syringes (Fisher Scientific)
  8. Sterile Acrodisc® 13 mm syringe filters (0.2 µm pore size) (VWR, catalog number: 28142-340)
  9. Sterile syringe filter disc (0.2 µm pore size) (VWR, catalog number: 28145-477)
  10. 1.5 ml Cuvettes (Dot Scientific, catalog number: 2410)
  11. Aluminum foil
  12. 100 x 15 mm Petri dishes (Crystalgen, catalog number: S-3006)
  13. Sterile glass beads (diameter 0.2-0.3 mm) (Sigma-Aldrich, catalog number: G1277)
  14. Sterile streaking sticks (flat toothpicks) (VWR, catalog number: 500029-808)
  15. Bacillus subtilis (B. subtilis)
    Note: This includes the control strains and tested strains. For control strains, B. subtilis subsp. subtilis str. 168 or B. subtilis str. PY79 are typically used.
  16. LB broth (Sigma-Aldrich, catalog number: L3022-1KG)
  17. LB agar (VWR, DifcoTM, catalog number: 90003-346)
  18. NaCl (Dot Scientific Inc., catalog number: 7647-14-5)
  19. KCl (RPI, catalog number: P41025-500.0)
  20. Na2HPO4 (J.T. Baker, catalog number: 7558-79-4)
  21. KH2PO4 (VWR, catalog number: 7778-77-0)
  22. NaOH (Sigma-Aldrich, catalog number: 221465-500G)
  23. K2HPO4 (Dot Scientific Inc., catalog number: DSP41300-1000)
  24. Glucose (Amresco Inc., catalog number: 50-99-7)
  25. Sodium citrate dihydrate (Na3C6O7•2H2O) (VWR, SX00445-1 EMD)
  26. Casein hydrolysate (EMD, catalog number: 1.02245.0500)
  27. Potassium glutamate monohydrate (Sigma-Aldrich, catalog number: G1501-100G)
  28. 100 ng/µl genomic DNA with selectable marker
    Note: The marker on the donor DNA should not be present in any of the strains being tested.
  29. MgSO4 (Sigma-Aldrich, catalog number: M7506)
  30. Ammonium iron citrate (J.T. Baker, catalog number: 1185-57-5)
  31. Antibiotics (for selection)
  32. 10x phosphate buffered saline (PBS) (see Recipes)
  33. 10x competence medium (MC) (see Recipes)
  34. 1 M MgSO4 (see Recipes)
  35. 1,000x Ferric Ammonium Citrate (see Recipes)

Equipment

  1. Pipettes
  2. Roller drum with disc for 18 mm tubes (New Brunswick, TC-7 style motor with disc for 18mm tubes)
    Note: A disc for 16 mm tubes may be used if a disc for 18 mm tubes is unavailable. A 16 mm tube can be used in replacement of the 13 mm tube in an 18 mm tube setup (Figure 1).
  3. Vortexer (VWR, catalog number: 58816-121)
  4. 37 °C incubator with shelves
  5. Microcentrifuge capable of 6,200 x g
  6. UV/vis Spectrometer (Thermo Fisher Scientific, model: 4001/4)
  7. Autoclave

Procedure

  1. Preparation of preparatory cultures
    1. Streak desired B. subtilis cells onto LB agar, with selective antibiotic, when appropriate.
    2. Incubate plates at 37 °C overnight or RT for 2-3 days.
    3. In a 13 mm glass tube placed within an 18 mm glass tube, inoculate 1 ml of LB culture with a single colony of B. subtilis approximately 12 h before starting preculture and grow the cells overnight at RT (Figure 1).
      Note: This setup helps with transferring liquid cultures, as some pipettes may not reach the bottom of the 18 mm glass tube.


      Figure 1. Two possible ways to set up a 13 mm tube within an 18 mm tube. In setup 1, the end of a 1 ml pipette tip is cut off and inserted into the 18 mm tube, with the 13 mm tube set on top of the tip. In setup 2, two 13 mm glass tube caps are placed on top of each other and placed into the bottom of the 18 mm tube.

  2. Transformation assay setup
    1. Grow cells rolling at 37 °C for one hour.
      Note: Roller drum should be set to 9 RPM.
    2. Transfer 1 ml of each culture to a 1.7 ml microfuge tube.
    3. Harvest cells by centrifugation (6,200 x g; 2 min, room temperature). There should be a visible pellet at the bottom of the microfuge tube.
    4. Pipette to remove supernatant and discard.
    5. Resuspend cells in 1 ml of 1x PBS.
    6. Centrifuge again (6,200 x g; 2 min, room temperature).
    7. Pipette to remove supernatant and discard.
    8. Resuspend cells in 1 ml of 1x PBS.
    9. Centrifuge again (6,200 x g; 2 min, room temperature).
    10. Discard supernatant and resuspend cells in 500 µl of freshly made 1x MC (see Recipes).
    11. Check OD600, using 150 µl of resuspended cells and 450 µl of 1x PBS (Blank with 150 µl of 1x MC and 450 µl of 1x PBS).
      Note: OD600 should be 0.2-1.2.
    12. Inoculate washed cells into a new 13 mm tube in an 18 mm tube and fill to 1 ml of 1x MC, so the culture has a starting OD600 of 0.01 to 0.05.
      Note: 1 µl of the selective antibiotic pertaining to the original strain resistance (not the donor DNA antibiotic marker) can be added to prevent any accidental contamination.
    13. Grow cells while rolling at 37 °C for 4.5 to 5 h.
    14. After 4.5 to 5 h, transfer 300 µl of each culture to a new 13 mm glass tube.
    15. Add 3 µl of genomic DNA (at a concentration of 100 ng/µl) and 3 µl of 1 M MgSO4 to each culture tube.
      Note: Include one culture without DNA added as a control for each experiment.
    16. Continue rolling cells at 37 °C for 2 h.

  3. Serial dilutions and plating
    1. Retrieve cultures from 37 °C.
    2. Create 1:10 serial dilutions of the cultures in 1x PBS to a 10-6 dilution.
    3. Plate 100 µl of desired dilutions onto LB (for CFU) and selective plates (for TFU) using glass beads (Figure 2).
      Note: A 10-6 dilution should yield 30-300 colonies for the wild-type TFU. For mutants, dilution plating is dependent on the type of defect present, so initial experiments are necessary to determine the range of dilutions required for plating.


      Figure 2. Representation of how the plating may look when comparing wild-type cells to mutants. In Row 1, the wild-type and ∆lacA strains are plated at a 10-2 dilution. The ∆comK and ∆ecsB strains are plated with undiluted culture. All plates in Row 1 contain the selectable antibiotic relevant to the marker present on the donor DNA. Row 2 is plated on LB plates with 10-6 dilutions. Each column is a different genotype. Column 1 is wild type, column 2 is ∆comK (a gene necessary for transformation), column 3 is ∆lacA (a gene not related to natural transformation), and column 4 is ∆ecsB (putatively involved with natural transformation). No colonies formed on the undiluted ∆comK selective plate, a few colonies are visible on the ∆ecsB selective plate, and colony formation was at the level of wild type for ∆lacA. CFU can also can be compared to wild type at this step.

    4. Place plates at 37 °C overnight, or leave at RT for 2-3 days.
      Note: TFU plates may need an extra day for colonies to grow due to selection.
    5. Count colonies on each plate. The colonies on each plate must be multiplied by their dilution factor to obtain CFUs and TFUs.
      Note: It may be helpful to count using a marker to mark colonies while using a tally counter to keep track of the number of colonies.

Data analysis

  1. Perform the quantitative transformation efficiency assay a minimum of three times per strain, and each time plate duplicates of each dilution. A positive control (wild-type strain) and negative control (wild type with no donor DNA added) should also be implemented during each replicate.
    Note: Each replicate is performed using different colonies from the same plate.
  2. For data analysis, transformation efficiency is calculated using the formula ηS = Cr/(CTpD), where Cr is the average number of TFU (transformants), CT is the average total number of CFU, and pD is the DNA concentration in micrograms per milliliter, from biological replicates. Each replicate is normalized to WT and run at the same time. The limit of detection of the assay is 0.5 transformants/CFU/µg of genomic DNA (as in Chilton et al., 2017; for PY79 with transforming DNA selected with 100 µg/ml spectinomycin).
  3. For statistical analysis, relative efficiency values are converted to arcsin values with the following equation to allow for normally distributed input for Dunnett’s multiple-comparison test (for comparison of all mutants together with the control): DS = [180 sin-1S/6)]/π. Dunnett's multiple-comparison test is performed on converted data with the multcomp package in the R statistical analysis software. Relative transformation efficiencies of WT and mutants are log transformed to generate normally distributed data, and t-tests comparing each mutant pairwise with corresponding controls are used to test for significant differences (as in Chilton et al., 2017).
    Note: When testing a single strain against WT, an ANOVA or t-test can be used.

Recipes

  1. 10x phosphate buffered saline (PBS) (0.5 L, pH 7.4)
    40 g NaCl
    1 g KCl
    7.26 g Na2HPO4 (anhydrous)
    1.2 g KH2PO4
    ddH2O to 0.4 L
    Dissolve solids
    Adjust pH to pH 7.4 with 50% NaOH
    Bring total volume to 0.5 L with ddH2O
    Sterilize by autoclaving
    Store at room temperature
    Dilute 1:10 in ddH2O to obtain 1x PBS
    Note: ddH2O provides consistency from batch to batch compared to dH2O.
  2. 10x competence medium (MC) (100 ml) (as in Konkol et al., 2013)
    10.7 g K2HPO4
    5.2 g KH2PO4
    20 g Dextrose anhydrous (glucose)
    0.88 g Na3C6O7•2H2O (sodium citrate dihydrate)
    1 ml 1,000x Ferric Ammonium Citrate (2.2% stock) (see below)
    1 g Casein Hydrolysate EDM
    2.2 g Potassium Glutamate monohydrate
    ddH2O to 100 ml
    Mix thoroughly; filter sterilize using 0.2 µm filter and syringe
    Store at -20 °C
    Note: To make 1 ml of fresh 1x MC, dilute 100 µl 10x MC into 900 µl ddH2O. Once the final concentration of 1 ml is obtained, add 3 µl of 1 M MgSO4.
  3. 1 M MgSO4 (0.5 L)
    60 g MgSO4 (Magnesium Sulfate Anhydrous)
    ddH2O to 0.5 L
    Sterilize by autoclaving
    Store at room temperature
  4. 1,000x Ferric Ammonium Citrate (100 ml)
    2.2 g Ammonium iron citrate
    ddH2O to 100 ml
    Filter sterilize using screw cap filter
    Wrap in aluminum foil
    Store at room temperature
    Note: This solution is light sensitive.

Acknowledgments

This protocol was adapted from Chilton et al. (2017). This work was funded in part by a grant from the Rita Allen Foundation.

Competing interests

The authors have no conflicts of interest or competing interests with respect to this work.

References

  1. Anagnostopoulos, C. and Spizizen, J. (1961). Requirements for transformation in Bacillus subtilis. J Bacteriol 81(5): 741-746.
  2. Chilton, S. S., Falbel, T. G., Hromada, S. and Burton, B. M. (2017). A conserved metal binding motif in the Bacillus subtilis competence protein ComFA enhances transformation. J Bacteriol 199(15): e00272-17.
  3. Cutting, S. M. and Vander Horn, P. B. (1990). Genetic Analysis. In: Harwood, C.R. and Cutting, S. M. (Eds.). Molecular Biological Methods for Bacillus. John Wiley & Sons, Ltd., West Sussex, UK. pp 27-74.
  4. Dubnau, D. and Davidoff-Abelson, R. (1971). Fate of transforming DNA following uptake by competent Bacillus subtilis. I. Formation and properties of the donor-recipient complex. J Mol Biol 56(2): 209-221.
  5. Konkol, M. A., Blair, K. M. and Kearns, D. B. (2013). Plasmid-encoded ComI inhibits competence in the ancestral 3610 strain of Bacillus subtilis. J Bacteriol 195(18): 4085-4093.

简介

枯草芽孢杆菌>( B.subtilis >)是一种模型革兰氏阳性生物,用于研究各种生理状态和应激反应,其中之一就是能力的发展。 能力是指细胞的生理状态,允许其自然转化。 通过诱导能力,可以通过电镀菌落形成单位(CFU)和转化单位(TFU)来量化自然转化的效率。 在这里,我们描述了使用 B量化相对可转换性的协议。枯草>。

【背景】 自然转化是一种水平基因转移,涉及从环境中导入外源DNA并将该DNA重组到其基因组中的细菌。该过程不同于诱导转化,因为天然可转化细菌产生促进DNA导入的蛋白质。细胞膜的修饰,例如通过电穿孔或热休克,对于诱导的转化通常是必需的。自然转化可以导致获得新的遗传元件,例如抗生素抗性或分解代谢基因,并且可以使细菌迅速适应其环境。参与能力发展的各种蛋白质,或允许其自然转化的细胞的生理状态,以及自然转化的机制尚未完全了解。对突变体和野生型之间转化效率差异的定量分析提供了对所述基因的贡献和潜在功能的深入了解。从Anagnostopoulos和Spizizen(1961)对转化枯草芽孢杆菌>实验室菌株的要求的描述开始,已经描述了原始培养基和转化条件的许多变化,包括定性和定量方法,包括但不限于Dubnau和Davidoff-Abelson(1971)以及Cutting和Vander Horn(1990)的那些。这里描述的实验程序类似于先前的实验程序,重点是目前可用的和优选的材料(例如酪蛋白水解产物,已经发现其基于批次和制造商产生不同的效率),并且包括结果的统计分析的描述。

关键字:自然转化, 感受性, 枯草芽孢杆菌, 转化效率定量测定, 水平基因转移, DNA摄取

材料和试剂

  1. 无菌移液器吸头
  2. 无菌18毫米玻璃管(Fisher Scientific,目录号:S63300)
  3. 无菌13毫米玻璃管(Fisher Scientific,目录号:14-961-27)
  4. 无菌不锈钢瓶盖(18毫米颈部尺寸)(VWR,目录号:71000-330)
  5. 无菌不锈钢瓶盖(13毫米颈部尺寸)(VWR,目录号:71000-322)
  6. 无菌1.7毫升微量离心管(Denville Scientific,目录号:C2170)
  7. 无菌60毫升Luer Lock注射器(Fisher Scientific)
  8. 无菌Acrodisc ® 13 mm注射器过滤器(0.2μm孔径)(VWR,目录号:28142-340)
  9. 无菌注射器滤盘(孔径0.2μm)(VWR,目录号:28145-477)
  10. 1.5毫升Cuvettes(Dot Scientific,目录号:2410)
  11. 铝箔
  12. 100 x 15 mm培养皿(Crystalgen,目录号:S-3006)
  13. 无菌玻璃珠(直径0.2-0.3 mm)(Sigma-Aldrich,目录号:G1277)
  14. 无菌条纹棒(扁牙签)(VWR,目录号:500029-808)
  15. Bacillus subtilis >( B. subtilis >)
    注意:这包括对照菌株和测试菌株。对于对照菌株,> B. subtilis > subsp。枯草芽孢杆菌168或> B. subtilis > str。通常使用PY79。>
  16. LB肉汤(Sigma-Aldrich,目录号:L3022-1KG)
  17. LB琼脂(VWR,Difco TM ,目录号:90003-346)
  18. NaCl(Dot Scientific Inc.,目录号:7647-14-5)
  19. KCl(RPI,目录号:P41025-500.0)
  20. Na 2 HPO 4 (J.T.Baker,目录号:7558-79-4)
  21. KH 2 PO 4 (VWR,目录号:7778-77-0)
  22. NaOH(Sigma-Aldrich,目录号:221465-500G)
  23. K 2 HPO 4 (Dot Scientific Inc.,目录号:DSP41300-1000)
  24. 葡萄糖(Amresco Inc.,目录号:50-99-7)
  25. 柠檬酸钠二水合物(Na 3 C 6 O 7 •2H 2 O)(VWR,SX00445-1 EMD) )
  26. 酪蛋白水解物(EMD,目录号:1.02245.0500)
  27. 谷氨酸钾一水合物(Sigma-Aldrich,目录号:G1501-100G)
  28. 100 ng /μl基因组DNA与选择标记
    注意:供体DNA上的标记不应存在于任何被测试的菌株中。>
  29. MgSO 4 (Sigma-Aldrich,目录号:M7506)
  30. 柠檬酸铁铵(J.T. Baker,目录号:1185-57-5)
  31. 抗生素(供选择)
  32. 10x磷酸盐缓冲盐水(PBS)(见食谱)
  33. 10x能力培养基(MC)(见食谱)
  34. 1 M MgSO 4 (见食谱)
  35. 1,000x铁柠檬酸铵(见食谱)

设备

  1. 移液器
  2. 带有圆盘的滚筒用于18毫米管(新不伦瑞克,TC-7型电机带盘,适用于18毫米管)
    注意:如果没有18毫米管的圆盘,可以使用16毫米管的圆盘。在18 mm管装置中可以使用16 mm管替换13 mm管(图1)。>
  3. Vortexer(VWR,目录号:58816-121)
  4. 37°C培养箱与架子
  5. 微量离心机能够达到6,200 x g >
  6. 紫外/可见光谱仪(Thermo Fisher Scientific,型号:4001/4)
  7. 高压灭菌器

程序

  1. 准备培养物的准备
    1. 需要条纹 B。在适当的情况下,将枯草芽孢杆菌细胞置于LB琼脂上,并使用选择性抗生素。
    2. 将板在37℃孵育过夜或室温放置2-3天。
    3. 在置于18mm玻璃管内的13mm玻璃管中,接种1ml含有 B单菌落的LB培养物。枯草芽孢杆菌>在开始预培养前约12小时,并在室温下培养细胞过夜(图1)。
      注意:此设置有助于转移液体培养物,因为有些移液器可能无法到达18 mm玻璃管的底部。>


      图1.在18 mm管内安装13 mm管的两种可能方法。 在设置1中,将1 ml移液器吸头的末端切断并插入18 mm管中,将13 mm管设置在吸头顶部。在装置2中,两个13毫米玻璃管帽彼此叠置并放入18毫米管的底部。

  2. 转化分析设置
    1. 将细胞在37°C下生长一小时。
      注意:滚筒应设置为9 RPM。>
    2. 将1ml每种培养物转移到1.7ml微量离心管中。
    3. 通过离心(6,200 x g >; 2分钟,室温)收获细胞。微量离心管底部应该有一个可见的颗粒。
    4. 用移液管移除上清液并丢弃。
    5. 将细胞重悬于1ml 1x PBS中。
    6. 再次离心(6,200 x g >; 2分钟,室温)。
    7. 用移液管移除上清液并丢弃。
    8. 将细胞重悬于1ml 1x PBS中。
    9. 再次离心(6,200 x g >; 2分钟,室温)。
    10. 弃去上清液并将细胞重悬于500μl新制的1x MC中(参见食谱)。
    11. 检查OD 600 ,使用150μl重悬细胞和450μl1xPBS(空白,150μl1xMC和450μl1xPBS)
      注意:OD > 600 > 应为0.2-1.2。>
    12. 将洗过的细胞接种到18mm管中的新的13mm管中,并填充到1ml的1x MC中,因此培养物的起始OD 600 为0.01至0.05。
      注意:可添加1μl与原始菌株抗性相关的选择性抗生素(不是供体DNA抗生素标记物),以防止任何意外污染。>
    13. 在37°C下滚动4.5至5小时,培养细胞。
    14. 4.5至5小时后,将300μl每种培养物转移至新的13mm玻璃管中。
    15. 向每个培养管中加入3μl基因组DNA(浓度为100 ng /μl)和3μl1MMgSO 4 。
      注意:包括一种没有添加DNA的培养物作为每个实验的对照。>
    16. 继续在37°C下滚动细胞2小时。

  3. 连续稀释和电镀
    1. 从37°C回收培养物。
    2. 在1x PBS中产生1:10系列稀释的培养物至10 -6 稀释液。
    3. 使用玻璃珠将100μl所需稀释液加到LB(用于CFU)和选择性板(用于TFU)上(图2)。
      注意:10 - > 6 > 稀释液应为野生型TFU产生30-300个菌落。对于突变体,稀释平板取决于存在的缺陷类型,因此需要进行初步实验以确定电镀所需的稀释范围。>


      图2.比较野生型细胞与突变体时电镀的外观。 在第1行中,野生型和Δ lacA >菌株以10 -2 稀释度接种。 Δ comK >和Δ ecsB >菌株接种未稀释的培养物。第1行中的所有平板都含有与供体DNA上存在的标记相关的可选抗生素。将第2行接种在具有10 -6 稀释液的LB平板上。每列是不同的基因型。第1列是野生型,第2列是Δ comK >(转化所需的基因),第3列是Δ lacA >(与自然转化无关的基因),第3列4是Δ ecsB >(推定参与自然转化)。在未稀释的Δ comK >选择性平板上没有形成菌落,在Δ ecsB >选择性平板上可见一些菌落,并且菌落形成处于野生型水平Δ LACA >。在此步骤中,CFU也可以与野生型进行比较。

    4. 将板置于37℃过夜,或在室温下放置2-3天。
      注意:由于选择,TFU平板可能需要额外的一天才能使菌落生长。>
    5. 在每个盘子上计数菌落。每个平板上的菌落必须乘以稀释系数才能获得CFU和TFU。
      注意:使用标记来计算菌落可能会有所帮助,同时使用计数器来跟踪菌落数。>

数据分析

  1. 每个菌株至少进行三次定量转化效率测定,每次重复每次稀释。在每次重复过程中也应实施阳性对照(野生型菌株)和阴性对照(不添加供体DNA的野生型)。
    注意:每个重复使用来自同一平板的不同菌落进行。>
  2. 对于数据分析,使用公式ηS> = Cr > /( C > T > <计算转换效率/ sub> pD >),其中 C > r > 是TFU(转化体)的平均数, C > T > 是CFU的平均总数, pD >是生物学重复中每毫升微克的DNA浓度。将每个重复标准化为WT并同时运行。测定的检测限为0.5个转化体/ CFU /μg基因组DNA(如Chilton 等人,2017;对于PY79,具有用100μg/ ml壮观霉素选择的转化DNA)。
  3. 对于统计分析,相对效率值用以下等式转换为arcsin值,以允许Dunnett多重比较测试的正态分布输入(用于比较所有突变体和对照):D S = [180 sin -1 (η S > / 6)] /π。 Dunnett的多重比较测试是使用R统计分析软件中的multcomp软件包对转换后的数据进行的。对WT和突变体的相对转化效率进行对数转换以产生正态分布的数据,并且 t > - 将每个突变体成对地与相应的对照进行比较的试验用于测试显着差异(如在Chilton 等中。>,2017)。
    注意:当针对WT测试单一菌株时,可以使用ANOVA或t检验。>

食谱

  1. 10x磷酸盐缓冲盐水(PBS)(0.5L,pH7.4)
    40克NaCl
    1克KCl
    7.26g Na 2 HPO 4 (无水)
    1.2克KH 2 PO 4
    ddH 2 O至0.4 L
    溶解固体
    用50%NaOH将pH调节至pH 7.4 用ddH 2 O使总体积达到0.5L 通过高压灭菌灭菌
    在室温下储存
    在ddH 2 O中稀释1:10以获得1x PBS
    注意:与dH相比,ddH > 2 > O提供批次之间的一致性> 2 0 >
  2. 10x能力培养基(MC)(100 ml)(如Konkol et al。>,2013)
    10.7g K 2 HPO 4
    5.2克KH 2 PO 4
    20克无水葡萄糖(葡萄糖)
    0.88g Na 3 C 6 O 7 •2H 2 O(柠檬酸钠二水合物)
    1毫升1,000倍铁柠檬酸铵(2.2%库存)(见下文)
    1克酪蛋白水解物EDM
    2.2克谷氨酸钾一水合物
    ddH 2 O至100毫升
    彻底混合;使用0.2μm过滤器和注射器过滤消毒
    储存在-20°C
    注意:要制备1 ml新鲜的1x MC,将100μl10xMC稀释到900μlddH> 2 > O中。一旦获得1ml的最终浓度,加入3μl1MMgSO 4 > >
  3. 1 M MgSO 4 (0.5 L)
    60g MgSO 4 (无水硫酸镁)
    ddH 2 O至0.5 L
    通过高压灭菌灭菌
    在室温下储存
  4. 1,000x柠檬酸铁铵(100 ml)
    2.2克柠檬酸铁铵
    ddH 2 O至100毫升
    使用螺旋盖过滤器过滤消毒
    用铝箔包裹
    在室温下储存
    注意:此解决方案对光敏感。>

致谢

该协议改编自Chilton et al。>(2017)。这项工作的部分资金来自丽塔艾伦基金会的资助。

利益争夺

作者在这项工作中没有利益冲突或利益冲突。

参考

  1. Anagnostopoulos,C。和Spizizen,J。(1961)。 Bacillus subtilis >的转化要求。 J Bacteriol > 81(5):741-746。
  2. Chilton,S。S.,Falbel,T.G。,Hromada,S。和Burton,B。M.(2017)。 枯草芽孢杆菌>能力蛋白ComFA中的保守金属结合基序增强转化。 J Bacteriol > 199(15):e00272-17。
  3. Cutting,S.M。和Vander Horn,P.B。(1990)。遗传分析。在:Harwood,C.R。和Cutting,S。M.(编辑)。 芽孢杆菌的分子生物学方法。> John Wiley&amp; Sons,Ltd.,West Sussex,UK。第27-74页。
  4. Dubnau,D。和Davidoff-Abelson,R。(1971)。 主管 Bacillus subtilis >吸收后转化DNA的命运。 I.供体 - 受体复合物的形成和性质。 J Mol Biol > 56(2):209-221。
  5. Konkol,M。A.,Blair,K。M。和Kearns,D。B.(2013)。 质粒编码的ComI抑制枯草芽孢杆菌>祖先3610株的能力。 J Bacteriol > 195(18):4085-4093。
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引用:Loyo, C. L. and Burton, B. M. (2018). Quantitative Transformation Efficiency Assay for Bacillus subtilis. Bio-protocol 8(23): e3109. DOI: 10.21769/BioProtoc.3109.
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