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Sep 2021

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A Modified Fluctuation Assay with a CAN1 Reporter in Yeast
酵母中CAN1报告基因的改良波动测定   

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Abstract

Understanding the generation of mutations is fundamental to understanding evolution and genetic disease; however, the rarity of such events makes experimentally identifying them difficult. Mutation accumulation (MA) methods have been widely used. MA lines require serial bottlenecks to fix de novo mutations, followed by whole-genome sequencing. While powerful, this method is not suitable for exploring mutation variation among different genotypes due to its poor scalability with cost and labor. Alternatively, fluctuation assays estimate mutation rate in microorganisms by utilizing a reporter gene, in which Loss-of-function (LOF) mutations can be selected for using drugs toxic to cells containing the WT allele. Traditional fluctuation assays can estimate mutation rates but not their base change compositions. Here, we describe a new protocol that adapts traditional fluctuation assay using CAN1 reporter gene in Saccharomyces cerevisiae, followed by pooled sequencing methods, to identify both the rate and spectra of mutations in different strain backgrounds.

Keywords: Mutation rate (突变率), Mutation spectrum (突变谱), Fluctuation assay (波动测定), CAN1 (CAN1), Saccharomyces cerevisiae (酿酒酵母)

Background

Mutation accumulation assays (Lynch et al., 2008, Zhu et al., 2014, Sharp et al., 2018), though useful in providing a genome-wide estimate of the mutation rate, are usually cost- and labor-intensive, and do not scale up to dozens of genotypes. This protocol utilizes traditional fluctuation assays by accumulating LOF mutations in the CAN1 reporter gene, with the addition to pool and sequence mutants. This yields not only mutation rate estimates (Lang et al., 2008) but also mutation spectrum estimates for each strain assayed, enabling comparison across different genotypes. This protocol has been used by Jiang et al. (2021), which explored mutation rates and spectra of 16 haploid natural isolates of Saccharomyces cerevisiae. Jiang et al. (2021) found a 10-fold mutation rate difference among the isolates and different mutation spectra in two mosaic beer strains, which show an excessive C>A mutations, demonstrating the usefulness of this protocol.

Materials and Reagents

Consumables:

  1. Omni plates (Nunc OmniTray with Lid, catalog number: 242811)

  2. Costar Round-bottom 96-well plates (Costar, catalog number: 3788)

  3. Costar Flat-bottom 96-well plates (Costar, catalog number: 3370)

  4. 50 mL Conical tube (VWR Scientific, catalog number: 89039-656)

  5. 50 mL Reservoirs (VWR Scientific, catalog number: 89003-382)

  6. Breath-easy sealing membrane (Sigma-Aldrich, catalog number: Z380059)

  7. Transparent Sealing Films (VWR, catalog number: 60941-078)

  8. 15 mL Conical tube (Thermo Fisher Scientific, catalog number: AM12500)

  9. Locking-lid microcentrifuge tube (Fisher Scientific, catalog number: 02-681-291)

  10. Illumina Nextera XT DNA Library Prep Kit (Illumina, catalog number: FC-131-1024, or FC-131-1096)

  11. Ampure beads (Beckman Coulter, catalog number: A63880)

  12. 6% TBE gel (Invitrogen, catalog number: EC62655BOX or EC6265BOX)

  13. Zymo PCR clean up kit (Zymo, catalog number: D4004)

  14. Phusion Hifi polymerase (NEB, catalog number: M0530L)

  15. 1.7 mL microcentrifuge tubes (VWR, catalog number: 87003-294)

  16. 2 mL locking lid microcentrifuge tubes (Thermal Fisher Scientific, catalog number: 3458PK)

  17. Toothpicks and/or 10 μL tips (USA Scientific, catalog number: 1120-3710)

  18. 200 μL pipette tips (USA Scientific, catalog number: 1120-8710)

  19. Glass culture tubes with cap

  20. 0.2 mL 8-well PCR Strip tubes (Genesee Scientific, catalog number: 22-161A) and caps (Genesee Scientific, catalog number: 22-170R)

  21. 100 mm regular Petri dish (SSE, catalog number: PMP35-01)

  22. Omni plates (VWR, catalog number: 62409-600)


Media/plates needed:

Ingredients:

  1. Yeast Extract (Difco, catalog number: 212750)

  2. Peptone (Difco, catalog number: 211677)

  3. Agar (BD Bacto, catalog number: 214030)

  4. Dextrose (Fisher Scientific, catalog number: D16-3)

  5. Yeast Nitrogen Base (Difco, catalog number: 233520)

  6. Ammonium Sulfate (Spectrum, catalog number: AM185)

  7. L-canavanine (Sigma-Aldrich, catalog number: C9758-5G)

  8. SC Amino acid mix (see Recipes)

  9. SC-Arg dropout mix (see Recipes)

  10. SC-Arg-Ser dropout mix (see Recipes)


  Media/Plates:

  1. SC-Arg media (see Recipes)

  2. SC-Arg-Ser+Can media (see Recipes)

  3. SC-Arg-Ser+Can in Omni plates (see Recipes)

  4. YPD plates (see Recipes)

Equipment

  1. Misonix Sonicator

  2. BioTek Synergy H1 Plate reader

  3. Qubit (Invitrogen)

  4. Thermo Scientific Sorvall ST 8/8R Centrifuge (for 96-well plates)

  5. 12-channel 200 μL multi-channel pipette

  6. Tabletop Microcentrifuge

  7. Bio-Rad C1000 Thermal Cycler (Bio-Rad, 1851148)

  8. Thermo Scientific mechanical convention incubator 30M (at 30°C)

  9. Invitrogen XCell SureLock Mini-Cell (for TBE gel)

  10. Thermo Scientific MaxQ 2000 open-air shaker (VWR, 47742-750)

  11. Elmeco Drum-X tissue Culture Rotator (Elmeco Engineering, L-85)

  12. Thermo Scientific Owl Easycast B1, B1A, B2 gel box systems

  13. Bio-Rad Powerpac Basic Power Supply (Bio-Rad, 1645050)

Procedure

  1. [Step 1: Fluctuation assay]

    This section of fluctuation assay protocol is adapted from Lang et al. (2018). With modification to plate onto Omni canavanine plates using multi-channel instead of regular round plates. Sterile technique is needed for all live yeast cell-related procedures (Step 1 and Step 2).

    Before you start: Count and prepare the Omni plates needed for this round of the experiment.

    Omni plates: SC-Arg-Ser+Can, dry for at least 2 days in the incubator at 30°C before use [If the plates are freshly made (within a month), dry for at least 3 days, ideally for 4 days].

    Day 1

    1. Streak out strains with pipette tips onto YPD plates. Place in the incubator at 30°C for approximately 2 days.


    Day 3
    1. Inoculate a single colony of each strain in ~3 mL of SC-Arg media in a sterile culture tube with a cap allowing for aeration in the afternoon. Place in the incubator at 30°C overnight on a roller drum.


    Day 4

    Note: One 96-well plate (round bottom) will be used for one estimate of mutation rate of the inoculated strain. Test up to six plates in one batch. If doing multiple batches (do not exceed four), test each batch approximately 1 h apart.

    1. Dilute 4 μL from overnight culture into 40 mL of SC-Arg media (1:10,000) in a 50 mL conical tube. Vortex to mix well. Pour entire mixture into a sterile reservoir. Transfer 50 μL into each well of the 96-well round-bottom plate using a 200 μL multi-channel pipette. Seal each plate with a Breath-easy membrane. Leave the plates in the incubator at 30°C with shaking (~150 RPM).


    Day 6 (48 h from setting up 96-well plate on day 4)

    1. Use the sonicator to separate cells (put one plate at a time in the sonicating water bath).

      Protocol: Amplitude: 10. Time 1min 30s. 1s ON, 1s OFF.

    2. Pulse centrifuge for 30 s (top speed reach to 4,500 RPM) of the plates.

    3. Leave two rows (24 wells) to pool and estimate overall cell numbers (use the same rows for different strains for consistency). Use the remaining six rows to estimate mutation events. Resuspend the cells and transfer the entire culture from each well using multi-channel from the remaining six rows (72 wells) onto previously dried SC-Arg-Ser+Can Omni plates. Take care not to let the individual droplets on the plates run together. If it happens, these replicates cannot be used to estimate mutation rates.

      Note: For each strain, the desired dilution should be previously tested when plating onto the canavanine plates. If plating 50 μL of culture results in large background on canavanine plates, test diluting with sterile H2O in 1:1, 1:2, or 1:3, 1:4 ratios, then transfer to ensure plating initial culture entirely onto the canavanine plates (e.g., 1:1 dilution with H2O will end up plating two rows per culture instead of one). The 1:1 dilution works for most strains tested. The dilution that results in low background growth on canavanine plates should be used for that strain.

    4. Let the Omni plates dry at room temperature. Then place in the incubator at 30°C for 48 h.

    5. Estimating overall cell numbers:

      Mix the two rows of cells into a 1.5 mL microcentrifuge tube. Then dilute them into ~1:20,000 with sterile ddH2O (usually try dilutions from 1:10,000, 1:20,000, and 1:40,000. The 1:20,000 dilution works the best for most cases), with at least two replicates per dilution. This amount should be adjusted based on the saturation density of your strain with initial test runs by plating dilutions on YPD. Plate 100 μL of the dilution on YPD plates. Incubate at 30°C and count cells after ~2 days. Optimal dilution will result in 100–200 cells per plate to count. Then estimate the total number of cells per 50 μL culture from the cell counts. If the cell count of the 100 μL from the 1:x dilution is y, the estimated actual cell count in the non-diluted culture is x*y/2, averaged from replicates.


    Day 8 (48 h from day 6, after placing Omni plates in the incubator at 30°C)

    Mutants should grow as colonies on the canavanine plates (Figure 1). Only strains with at least one or two mutants from the 72 independent cultures grown are used. Plates can be placed at 4°C for storage (up to 2–3 weeks) before next steps.



    Figure 1. Example of mutants grown on Omni plates.

    Here, eight rows of mutants were plated. When used for estimating mutation rates, only six rows are plated, and the other two rows are mixed to estimate total cell numbers.


    Then estimate mutation rates using “rsalvador” R package (Zheng, 2017). Script to estimate mutation rate in Jiang et al. (2021) can be found: https://github.com/harrispopgen/elife_CAN1_paper/tree/main/fluc.


  2. [Step 2: Pooling mutants]

    Day 1
    1. Start in the afternoon ~3 p.m., depending on how many plates to set up. Incubate for ~43 h.

    2. Add 200 μL of SC-Arg-Ser+Can selective media to the 96-well flat bottom plate (easy for plate reader to read OD). Use a 10 μL sterile pipette tip or toothpick and pick at most one independent mutant colony from each replicate for which there are independent pickable CanR colonies. Place each mutant colony into a well on the fresh 96-well plate containing SC-Arg-Ser+Can. Use the VWR transparent film for sealing after finishing picking for the whole plate. Leave at least 1–2 empty wells on the plate to calibrate the plate reader. This process takes approximately 30 min per plate. Leave the inoculated 96-well plates in the incubator at 30°C with shaking for ~43 h.

      Note: Optionally, place one plate in a plate reader to determine if all cultures grow to saturation during the incubation.


    Day 3

    Morning (~9 a.m.)

    1. For each plate of every strain, resuspend by pipetting up and down with a multi-channel pipette. Put on a new transparent seal. Low speed centrifuge if required, but avoid as it makes resuspension difficult. Measure the OD600 of each well using a plate reader, making sure the empty wells yield a close-to-zero number. The saturated culture will yield an OD600 close to 1 and should be similar among cultures of the same plate but can be different among different strains.

    2. The pooling is done by combining independent saturated cultures of mutants from the same strain into a mix, to ensure each mutant is roughly the same and relatively high frequency (~0.03) in the pool so that they can be detected by Illumina Sequencing. Exclude cultures with below average OD600 as the determination of mutational frequency assumes a close to equal number of cells in the pool. Determine which mutants to pool together based on the total number of mutants collected by the strain, up to 35–40 mutants from the same strain per pool. Record the number of mutants per pool.

    3. Pool mutants for each strain one at a time. Transfer 150 μL of saturated culture from each well with the multi-channel pipette into a reservoir. Then transfer all the liquid to a 15 mL conical tube. Vortex to mix well, and then evenly distribute into three 2 mL locking-lid microcentrifuge tubes.

    4. Spin down for 3 min to pellet the cells (stored at -20°C).


  3. [Step 3: Preparation for sequencing of CAN1]

    1. Extract genomic DNA for each pool, which usually gives a yield of ~200–500 ng genomic DNA (use 70 ng to set up each 25 μL PCR reaction if possible). This assay was developed with the Hoffman Winston protocol (Hoffman and Winston, 1987), but any genomic extraction protocol will work. For most pools, extract one tube of genomic DNA from cell pellets. Randomly selected some pools to perform two independent extractions as replicates and test consistency in the mutation calling pipeline. Keep the third tube at -20°C in case of DNA extraction failure.

    2. Use PCR to amplify the CAN1 locus from the genomic DNA. Set up three 25 μL reactions for each mutant pool. See below for the PCR protocol [use primers for CAN1 from Lang and Murray (2008), forward: ATAGTAAGCTCATTGATCCC, reverse: TCTTCAGACTTCTTAACTCC). Use 15 cycles.


      PCR protocol (with Phusion Hifi polymerase):

      1. 98°C: 3 min

        1. 98°C: 10 s

        2. 59°C: 18 s

        3. 72°C: 2 min

        4. (Go to step 2, with a total of 15 cycles)

      2. 72°C: 5 min

      3. 12°C: forever


    3. Run 10 μL of the 25 μL on the gel; if a band is present, proceed with the other two reactions for PCR cleanup with the Zymo kit.

    4. Measure DNA concentration using Qubit (High Sensitivity kit).


  4. [Step 4: Library preparation for sequencing of CAN1]

    1. Use Illumina Nextera XT DNA Library Prep Kit. Follow the protocol with small modifications.

      1. Use starting PCR product at 0.18 ng/μL (5 μL).

      2. For the tagmentation step, use 7 min for most samples; 5 min produces an average library size of ~800 bp–1 kb, and 7min produces the appropriate library size of ~500–600 bp.

      3. Use 1:1 ratio of sample: Ampure beads during clean up.

      4. Elute in 47.5 μL Resuspension Buffer (RSB) and transfer 45 μL.

    2. Use Qubit (High Sensitivity kit) to quantify the concentration of each library. Run TBE gel in Invitrogen XCell SureLock Mini-Cell to estimate rough library size (Figure 2). Convert concentration in X ng/μL to Y nM with library size K bp, using the formula Y = X/(660*K)*1000000 (according to the Illumina Handbook). Mix library together with equal mole. Send for sequencing [spike-in each pool to get 0.25 M reads from a 150-bp paired-end Illumina sequencing reads, resulting in an estimated coverage of 44,000 for each pool (CAN1 size of 1.7kb): 0.25*106*300/(1.7*103) ~ 44,000].



      Figure 2. Example of a TBE gel obtained.

      Lanes 3 and 12 are ladders, and the rest are libraries. The estimated library size is taken as the mid-point of the spread.


    3. Analyze reads using scripts found in:

      https://github.com/harrispopgen/elife_CAN1_paper/tree/main/CAN1_mut_sequencing.

Recipes

  1. SC Amino acid mix (for 1 L)

    0.019 g Adenine

    0.019 g Arginine

    0.096 g Aspartic Acid

    0.096 g Glutamic Acid

    0.019 g Histidine

    0.077 g Isoleucine

    0.077 g Leucine

    0.058 g Lysine

    0.019 g Methionine

    0.048 g Phenylalanine

    0.384 g Serine

    0.192 g Threonine

    0.077 g Tryptophan

    0.058 g Tyrosine

    0.019 g Uracil

    0.144 g Valine

    (1.402 g total)

  2. SC-Arg dropout mix

    0.019 g Adenine

    0.096 g Aspartic Acid

    0.096 g Glutamic Acid

    0.019 g Histidine

    0.077 g Isoleucine

    0.077 g Leucine

    0.058 g Lysine

    0.019 g Methionine

    0.048 g Phenylalanine

    0.384 g Serine

    0.192 g Threonine

    0.077 g Tryptophan

    0.058 g Tyrosine

    0.019 g Uracil

    0.144 g Valine

  3. SC-Arg-Ser dropout mix

    0.019 g Adenine

    0.096 g Aspartic Acid

    0.096 g Glutamic Acid

    0.019 g Histidine

    0.077 g Isoleucine

    0.077 g Leucine

    0.058 g Lysine

    0.019 g Methionine

    0.048 g Phenylalanine

    0.192 g Threonine

    0.077 g Tryptophan

    0.058 g Tyrosine

    0.019 g Uracil

    0.144 g Valine

  4. SC-Arg media (1 L)

    1.7 g Yeast Nitrogen Base

    5 g Ammonium Sulfate

    20 g Dextrose

    1.383 g SC-Arg dropout mix

  5. SC-Arg-Ser+Can media (1 L)

    1.7 g Yeast Nitrogen Base

    5 g Ammonium Sulfate

    20 g Dextrose

    0.999 g SC-Arg-Ser dropout mix

    10 mL 100× Canavanine media (60 mg/mL)

  6. SC-Arg-Ser+Can plates

    1.7 g Yeast Nitrogen Base

    5 g Ammonium Sulfate

    20 g Dextrose

    0.999 g SC-Arg-Ser dropout mix

    10 mL 100× Canavanine media (60 mg/mL)

    17 g Agar

  7. YPD plates (1 L)

    10 g Yeast Extract

    20 g Peptone

    17 g Agar

    20 g Dextrose

Acknowledgments

Pengyao Jiang was supported by a Burroughs Wellcome Fund Career Award at the Scientific Interface awarded to Kelley Harris. Anja R. Ollodart was supported by the National Human Genome Research Institute of the NIH under award T32 HG00035. The research of Maitreya J. Dunham was supported by NIH/NIGMS award P41 GM103533 and a Faculty Scholar grant from the Howard Hughes Medical Institute. The fluctuation assay part of the protocol is adapted from Lang (2018). The protocol is the experimental procedure of the publication Jiang et al. (2021), eLife.

Competing interests

There are no conflicts of interest or competing interests.

References

  1. Hoffman, C. S. and Winston, F. (1987). A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene 57(2-3): 267-272.
  2. Jiang, P., Ollodart, A. R., Sudhesh, V., Herr, A. J., Dunham, M. J. and Harris, K. (2021). A modified fluctuation assay reveals a natural mutator phenotype that drives mutation spectrum variation within Saccharomyces cerevisiae. Elife 10: e68285.
  3. Lang, G. I. (2018). Measuring Mutation Rates Using the Luria-Delbruck Fluctuation Assay. Methods Mol Biol 1672: 21-31.
  4. Lang, G. I. and Murray, A. W. (2008). Estimating the per-base-pair mutation rate in the yeast Saccharomyces cerevisiae. Genetics 178(1): 67-82.
  5. Lynch, M., Sung, W., Morris, K., Coffey, N., Landry, C. R., Dopman, E. B., Dickinson, W. J., Okamoto, K., Kulkarni, S., Hartl, D. L., et al. (2008). A genome-wide view of the spectrum of spontaneous mutations in yeast. Proc Natl Acad Sci U S A 105(27): 9272-9277.
  6. Sharp, N. P., Sandell, L., James, C. G. and Otto, S. P. (2018). The genome-wide rate and spectrum of spontaneous mutations differ between haploid and diploid yeast. Proc Natl Acad Sci U S A 115(22): E5046-E5055.
  7. Zheng, Q. (2017). rSalvador: An R Package for the Fluctuation Experiment. G3 (Bethesda) 7(12): 3849-3856.
  8. Zhu, Y. O., Siegal, M. L., Hall, D. W. and Petrov, D. A. (2014). Precise estimates of mutation rate and spectrum in yeast. Proc Natl Acad Sci U S A 111(22): E2310-2318.

简介

[摘要] 了解突变的产生是了解进化和遗传疾病的基础;然而,此类事件的罕见性使得通过实验识别它们变得困难。突变积累(MA)方法已被广泛使用。 MA 系需要一系列瓶颈来修复从头突变,然后是全基因组测序。虽然功能强大,但该方法不适合探索不同基因型之间的突变变异,因为其成本和劳动力的可扩展性较差。或者,波动分析通过利用报告基因来估计微生物中的突变率,其中可以选择功能丧失 (LOF) 突变来使用对含有 WT 等位基因的细胞有毒的药物。传统的波动分析可以估计突变率,但不能估计它们的碱基变化成分。在这里,我们描述了一种新协议,该协议使用酿酒酵母中的CAN1报告基因适应传统波动测定,然后采用汇集测序方法,以确定不同菌株背景中突变的速率和光谱。



[背景] 突变积累 分析(Lynch et al. , 2008, Zhu et al. , 2014, Sharp et al. , 2018)虽然可用于提供突变率的全基因组估计,但通常是成本和劳动密集型的,并且不扩大到几十个基因型。该协议通过在CAN1报告基因中累积 LOF 突变来利用传统的波动测定,并添加池和序列突变体。这不仅产生突变率估计值 (Lang et al ., 2008),而且产生每个所分析菌株的突变谱估计值,从而能够在不同基因型之间进行比较。该协议已被江等人使用。 (2021),探索了 16 种酿酒酵母单倍体天然分离物的突变率和光谱。江等人。 (2021) 发现两种马赛克啤酒菌株的分离株和不同突变谱之间存在 10 倍的突变率差异,显示出过度的 C>A 突变,证明了该协议的有用性。

关键字:突变率, 突变谱, 波动测定, CAN1, 酿酒酵母



材料和试剂


消耗品:
1. Omni 板(带盖的 Nunc OmniTray,目录号:242811)
2. Costar 圆底96孔板(Costar,目录号:3788)
3. Costar平底96孔板(Costar,目录号:3370)
4. 50 mL锥形管(VWR Scientific,目录号:89039-656)
5. 50 mL 储液罐(VWR Scientific,目录号:89003-382)
6. 透气密封膜(Sigma-Aldrich,目录号:Z380059)
7. 透明密封膜(VWR,目录号:60941-078)
8. 15 mL锥形管(Thermo Fisher Scientific,目录号:AM12500)
9. 锁盖微量离心管(Fisher Scientific,目录号:02-681-291)
10. Illumina Nextera XT DNA Library Prep Kit(Illumina,目录号:FC-131-1024 或 FC-131-1096)
11. 安培珠(Beckman Coulter,目录号:A63880)
12. 6% TBE 凝胶(Invitrogen,目录号:EC62655BOX 或 EC6265BOX)
13. Zymo PCR 清理试剂盒(Zymo,目录号:D4004)
14. Phusion Hifi聚合酶(NEB,目录号:M0530L)
15. 1.7 mL微量离心管(VWR,目录号:87003-294)
16. 2 mL 带锁盖微量离心管(Thermal Fisher Scientific,目录号: 3458PK)
17. 牙签和/或 10 μ L 尖端(USA Scientific,目录号:1120-3710)
18. 200 μ L 移液器吸头(USA Scientific,目录号:1120-8710)
19. 带盖玻璃培养管
20. 0.2 mL 8 孔 PCR Strip 管(Genesee Scientific,目录号:22-161A)和盖子(Genesee Scientific,目录号:22-170R)
21. 100 mm 常规培养皿(SSE,目录号: PMP35-01)
22. Omni 板(VWR,目录号: 62409-600)


需要的介质/板:
原料:
1. 酵母提取物(Difco,目录号:212750)
2. 蛋白胨(Difco,目录号:211677)
3. 琼脂(BD Bacto,目录号:214030)
4. 葡萄糖(Fisher Scientific,目录号:D16-3)
5. 酵母氮基(Difco,目录号:233520)
6. 硫酸铵(Spectrum,目录号:AM185)
7. L-刀豆氨酸(Sigma-Aldrich,目录号:C9758-5G)
8. SC 氨基酸混合物(见食谱)
9. SC-Arg 辍学混合物(见食谱)
10. SC-Arg-Ser 辍学混合物(见食谱)


媒体/板材:
1. SC-Arg 培养基(见配方)
2. SC-Arg-Ser+Can 培养基(见配方)
3. Omni 板中的 SC-Arg-Ser+Can(参见食谱)
4. YPD 板(见食谱)


设备


1. Misonix 声波器
2. BioTek Synergy H1 读板机
3. 量子比特(英杰公司)
4. Thermo Scientific Sorvall ST 8/8R 离心机(用于 96 孔板)
5. 12 道 200 μL 多道移液器
6. 台式微量离心机
7. Bio-Rad C1000 热循环仪(Bio-Rad,1851148)
8. Thermo Scientific 机械式常规培养箱 30M(30°C 时)
9. Invitrogen XCell SureLock Mini-Cell(用于 TBE 凝胶)
10. Thermo Scientific MaxQ 2000 露天摇床(VWR,47742-750)
11. Elmeco Drum-X 组织培养旋转器(Elmeco Engineering,L-85)
12. Thermo Scientific Owl Easycast B1、B1A、B2 凝胶盒系统
13. Bio-Rad Powerpac 基本电源(Bio-Rad,1645050)


程序


A. 【第一步:波动分析】
这部分波动分析协议改编自 Lang等人。 (2018 年)。通过修改以使用多通道而不是常规圆形板在 Omni canavanine 板上进行电镀。所有活酵母细胞相关程序(步骤 1 和步骤 2)都需要无菌技术。
开始之前:计算并准备本轮实验所需的 Omni 板。
°C的培养箱中干燥至少 2 天[如果板是新鲜制作的(一个月内),干燥至少 3 天,最好干燥 4 天]。


第1天
1. 用移液器吸头将菌株划线到 YPD 板上。置于 30°C 的培养箱中约 2 天。


第 3 天
2. 在无菌培养管中接种约 3 mL 的 SC-Arg 培养基中的每个菌株的单个菌落,并在下午进行通气。放置在 30°C 的培养箱中,在滚筒上过夜。


第 4 天
注意:一个 96 孔板(圆底)将用于估计接种菌株的突变率。一批测试多达六个板。如果进行多批次(不超过四个),每批次测试大约相隔 1 小时。
3. 4 μL 从过夜培养物中稀释到 40 mL 的 SC-Arg 介质(1:10,000)中。涡旋混合均匀。将整个混合物倒入无菌容器中。使用 200 μL 多通道移液器将 50 μL 转移到 96 孔圆底板的每个孔中。用透气膜密封每个板。将板放在 30°C 的培养箱中,摇动 (~150 RPM)。


第 6 天(从第 4 天设置 96 孔板起 48 小时)
4. 使用超声波仪分离细胞(一次在超声波水浴中放置一个板)。
协议:幅度:10。时间1分30秒。 1 秒开,1 秒关。
5. 脉冲离心机 30 秒(最高速度达到 4,500 RPM)的板。
6. 留下两行(24 口井)来汇集和估计总细胞数(为不同的菌株使用相同的行以保持一致性)。使用剩余的六行来估计突变事件。重悬细胞并使用多通道将每个孔中的整个培养物从其余六行(72 个孔)转移到先前干燥的 SC-Arg-Ser+Can Omni 板上。注意不要让板上的单个液滴一起运行。如果发生这种情况,这些重复不能用于估计突变率。
注意:对于每种菌株,在电镀到刀豆氨酸板上时,应预先测试所需的稀释度。如果接种 50 μL培养物导致刀豆氨酸板上的背景较大,则以1:1、1:2 或 1:3、1:4 的比例测试用无菌 H 2 O 稀释,然后转移以确保将初始培养物完全接种到刀豆氨酸板(例如,用 H 2 O 进行1:1 稀释最终会在每个培养物中铺板两行而不是一行)。 1:1 稀释适用于大多数测试菌株。导致刀豆氨酸板上背景生长低的稀释液应用于该菌株。
7. 让 Omni 板在室温下干燥。然后置于 30°C 培养箱中培养 48 小时。
8. 估计总细胞数:
将两排细胞混合到 1.5 mL 微量离心管中。然后用无菌 ddH 2 O将它们稀释到 ~1:20,000 (通常尝试从 1:10,000、1:20,000 和 1:40,000 稀释。1:20,000 稀释在大多数情况下效果最佳),每次至少重复两次稀释。应根据初始测试运行时应变的饱和密度通过在 YPD 上电镀稀释液来调整此量。在 YPD 板上稀释100 μL 。在30 °C 下孵育 并在约 2 天后计数细胞。最佳稀释度将导致每板计数 100–200 个细胞。然后根据细胞计数估计每 50 μL培养物中的细胞总数。如果1:x 稀释的 100 μL的细胞计数为 y,则未稀释培养物中估计的实际细胞计数为 x*y/2,从重复中平均。


第 8 天(从第 6 天起 48 小时,将 Omni 板放入 30 °C的培养箱后)
突变体应在刀豆板上形成菌落(图 1)。仅使用来自 72 个独立培养物的至少一或两个突变体的菌株。在下一步之前,可以将板放置在 4 °C下进行储存(最多2-3 周)。


 
图 1.在 Omni 板上生长的突变体示例。 
在这里,镀了八排突变体。当用于估计突变率时,仅镀六行,其他两行混合以估计总细胞数。


然后使用“rsalvador”R 包(Zheng , 2017)估计突变率。估计姜等人突变率的脚本。 (2021)可以找到: https ://github.com/harrispopgen/elife_CAN1_paper/tree/main/fluc 。


B. [步骤 2:汇集突变体]


第 1 天
1. 下午 3 点左右开始,具体取决于要设置的盘子数量。孵育约 43 小时。
2. 在 96 孔平底板中加入 200 μL SC-Arg-Ser+Can 选择性培养基(便于读板器读取 OD)。使用 10 μL无菌移液器吸头或牙签,从每个具有独立可采摘 CanR 菌落的复制品中挑选最多一个独立的突变菌落。将每个突变菌落放入含有 SC-Arg-Ser+Can 的新鲜 96 孔板上的孔中。整板拾取完成后使用VWR透明膜封口。在板上至少留 1–2 个空井以校准读板器。这个过程每块板大约需要 30 分钟。将接种的 96 孔板留在 30 °C的培养箱中,摇晃约 43 小时。
注意:可选地,将一个板放在读板器中,以确定是否所有培养物在孵化过程中都长到饱和。


第 3 天
早上(~上午 9 点)
3. 对于每个菌株的每个板,通过用多通道移液器上下移液来重新悬浮。贴上新的透明封条。如果需要,低速离心机,但避免,因为它使重新悬浮变得困难。使用读板器测量每个井的 OD 600 ,确保空井产生接近于零的数字。饱和培养物将产生接近 1 的 OD 600 ,并且在同一板的培养物中应该相似,但在不同菌株之间可能不同。
4. 合并是通过将来自同一菌株的突变体的独立饱和培养物组合成混合物来完成的,以确保每个突变体在池中大致相同且频率相对较高(~0.03),以便它们可以被 Illumina 测序检测到。排除具有低于平均 OD 600的培养物,因为突变频率的确定假定池中的细胞数量接近相等。根据菌株收集的突变体总数确定要汇集在一起的突变体,每个池中最多 35-40 个突变体来自同一菌株。记录每个池的突变体数量。
5. 每次为每个菌株汇集一个突变体。使用多通道移液器将 150 μL 的饱和培养物从每口井中转移到水库中。然后将所有液体转移到 15 mL 锥形管中。涡旋混匀,然后均匀分布到三个 2 mL 带锁盖微量离心管中。
6. 离心 3 分钟以沉淀细胞(储存在 -20°C)。


C. [步骤 3:准备CAN1的排序]
1. 提取每个池的基因组 DNA,这通常产生约 200–500 ng 基因组 DNA 的产量(如果可能,使用 70 ng 来设置每个 25 μL PCR 反应)。该测定法是使用 Hoffman Winston 方案开发的( Hoffman 和 Winston,1987 ),但任何基因组提取方案都可以使用。对于大多数池,从细胞沉淀中提取一管基因组 DNA。随机选择一些池来执行两个独立的提取作为复制并测试突变调用管道中的一致性。将第三管保持在 -20°C,以防 DNA 提取失败。
2. 使用 PCR从基因组 DNA中扩增CAN1位点。为每个突变池设置三个 25 μL反应。 PCR 方案见下文 [使用来自 Lang 和 Murray (2008) 的CAN1引物,正向:ATAGTAAGCTCATTGATCCC,反向:TCTTCAGACTTCTTAACTCC)。使用 15 个循环。


PCR 方案(使用 Phusion Hifi 聚合酶):
• 98°C:3分钟
o 98°C:10 秒
o 59°C:18 秒
o 72°C:2分钟
o (转至第 2 步,共 15 个循环)
• 72°C:5分钟
• 12°C:永远


3. 在凝胶上运行25 μL中的 10 μL ;如果存在条带,则使用 Zymo 试剂盒进行其他两个反应以进行 PCR 净化。
4. 使用 Qubit(高灵敏度试剂盒)测量 DNA 浓度。


D. [步骤 4: CAN1测序文库制备]
1. 使用 Illumina Nextera XT DNA Library Prep Kit。按照协议进行少量修改。
a. 使用 0.18 ng/ μL (5 μL ) 的起始 PCR 产物。
b. 对于标记步骤,对大多数样本使用 7 分钟; 5 分钟产生平均库大小 约 800 bp–1 kb,7 分钟产生约 500–600 bp 的适当文库大小。
c. 在清理过程中使用 1:1的样品:安培珠。
d. 在 47.5 μL重悬缓冲液 (RSB) 中洗脱并转移 45 μL 。
2. 使用 Qubit(高灵敏度试剂盒)量化每个文库的浓度。在 Invitrogen XCell SureLock Mini-Cell 中运行 TBE 凝胶以估计粗略的文库大小(图 2)。使用公式 Y = X/(660* K)* 1000000(根据 Illumina 手册),将 X ng/ μL中的浓度转换为具有库大小 K bp 的 Y nM 。将库与等摩尔混合在一起。发送进行测序 [spike-in each pool to get 0.25 M reads from a 150-bp pair-end Illumina 测序读数,估计每个池的覆盖率为 44,000( CAN1大小为 1.7kb):0.25*10 6 *300 /( 1.7*10 3 ) ~ 44,000]。


 
图 2.获得的 TBE 凝胶示例。 
3 号和 12 号车道是梯子,其余的是图书馆。估计的库大小被视为传播的中点。 


3. 使用以下脚本分析读取:
https://github.com/harrispopgen/elife_CAN1_paper/tree/main/CAN1_mut_sequencing 。


食谱


1. SC 氨基酸混合物(1 L)
0.019 克腺嘌呤
0.019 克精氨酸
0.096 克天冬氨酸
0.096 克谷氨酸
0.019 克组氨酸
0.077 克异亮氨酸
0.077 克亮氨酸
0.058 克赖氨酸
0.019 克蛋氨酸
0.048 克苯丙氨酸
0.384 克丝氨酸
0.192 克苏氨酸
0.077 克色氨酸
0.058 克酪氨酸
0.019 克尿嘧啶
0.144 克缬氨酸
(总共 1.402 克)
2. SC-Arg 辍学混合物
0.019 克腺嘌呤
0.096 克天冬氨酸
0.096 克谷氨酸
0.019 克组氨酸
0.077 克异亮氨酸
0.077 克亮氨酸
0.058 克赖氨酸
0.019 克蛋氨酸
0.048 克苯丙氨酸
0.384 克丝氨酸
0.192 克苏氨酸
0.077 克色氨酸
0.058 克酪氨酸
0.019 克尿嘧啶
0.144 克缬氨酸
3. SC-Arg-Ser 辍学混合物
0.019 克腺嘌呤
0.096 克天冬氨酸
0.096 克谷氨酸
0.019 克组氨酸
0.077 克异亮氨酸
0.077 克亮氨酸
0.058 克赖氨酸
0.019 克蛋氨酸
0.048 克苯丙氨酸
0.192 克苏氨酸
0.077 克色氨酸
0.058 克酪氨酸
0.019 克尿嘧啶
0.144 克缬氨酸
4. SC-Arg 培养基(1 升)
1.7克酵母氮基
5克硫酸铵
20 克葡萄糖
1.383 g SC -Arg 脱除混合物
5. SC-Arg-Ser+Can 培养基 (1 L)
1.7克酵母氮基
5克硫酸铵
20 克葡萄糖
0.999 g SC -Arg-Ser 辍学混合物
10 mL 100 × Canavanine 培养基 (60 mg/mL)
6. SC-Arg-Ser+Can 板
1.7克酵母氮基
5克硫酸铵
20 克葡萄糖
0.999 g SC -Arg-Ser 辍学混合物
10 mL 100 × Canavanine 培养基 (60 mg/mL)
17 克琼脂
7. YPD 板(1 升)
10 克酵母提取物
20 克 蛋白胨
17 克琼脂
20 克葡萄糖


致谢


姜鹏耀在科学接口上获得了授予 Kelley Harris 的 Burroughs Wellcome 基金职业奖。 Anja R. Ollodart 得到了美国国立卫生研究院国家人类基因组研究所 T32 HG00035 奖的支持。 Maitreya J. Dunham 的研究得到了 NIH/NIGMS 奖 P41 GM103533 和霍华德休斯医学研究所的教师学者资助。该协议的波动分析部分改编自Lang (2018)。该协议是出版物江等人的实验程序。 (2021),188bet体育电竞。


利益争夺


不存在利益冲突或竞争利益。


参考


1. Hoffman, CS 和 Winston, F. (1987)。从酵母中提取的 10 分钟 DNA 可有效释放用于转化大肠杆菌的自主质粒。 基因57(2-3):267-272。
2. Jiang, P.、Ollodart, AR、Sudhesh, V.、Herr, AJ、Dunham, MJ 和 Harris, K. (2021)。修改后的波动分析揭示了一种天然突变表型,该表型驱动酿酒酵母内的突变谱变异。 生命 10:e68285 。
3. 郎,GI(2018)。使用 Luria-Delbruck 波动测定法测量突变率。 方法 Mol Biol 1672:21-31。
4. Lang, GI 和 Murray, AW (2008)。估计酵母酿酒酵母中每个碱基对的突变率。 遗传学178(1):67-82。
5. Lynch,M.,Sung,W.,Morris,K.,Coffey,N.,Landry,CR,Dopman,EB,Dickinson,WJ,Okamoto,K.,Kulkarni,S.,Hartl,DL,等。 (2008 年)。酵母自发突变谱的全基因组视图。 Proc Natl Acad Sci USA 105(27): 9272-9277。
6. Sharp,NP,Sandell,L.,James,CG 和 Otto,SP(2018 年)。单倍体和二倍体酵母的全基因组速率和自发突变谱不同。 Proc Natl Acad Sci USA 115(22):E5046-E5055。
7. 郑 Q. (2017)。 rSalvador:波动实验的 R 包。 G3(贝塞斯达) 7(12):3849-3856。
8. Zhu, YO, Siegal, ML, Hall, DW 和 Petrov, DA (2014)。精确估计酵母中的突变率和谱。 Proc Natl Acad Sci USA 111(22):E2310-2318。




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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright Jiang et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Jiang, P., Ollodart, A. R. and Dunham, M. J. (2022). A Modified Fluctuation Assay with a CAN1 Reporter in Yeast. Bio-protocol 12(11): e4435. DOI: 10.21769/BioProtoc.4435.
  2. Jiang, P., Ollodart, A. R., Sudhesh, V., Herr, A. J., Dunham, M. J. and Harris, K. (2021). A modified fluctuation assay reveals a natural mutator phenotype that drives mutation spectrum variation within Saccharomyces cerevisiae. Elife 10: e68285.
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