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Insertional Mutagenesis of Chlamydomonas reinhardtii
莱茵衣藻的插入突变   

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Abstract

The unicellular microalga Chlamydomonas reinhardtii (C. reinhardtii) has been used as a reference model for numerous fields of research. Principle research areas are eukaryotic flagellar structure and function, basal bodies (centrioles), cell-cell recognition, cell cycle control, chloroplast biogenesis, phototaxis, nonphotochemical quenching, and especially photosynthesis for C. reinhardtii can grow in the dark on an organic carbon (e.g. acetate), and thus provides advantages over land plants (Harris, 2001; Peers et al., 2009). C. reinhardtii has a short life cycle, a sequenced genome (Merchant et al., 2007), and a growing molecular toolbox for forward and reverse genetic studies, including transformation protocols, gene silencing (Kim and Cerutti, 2009; Molnar et al., 2009), and fluorescent protein-tag (Rasala et al., 2013). There are two commonly used methods for C. reinhardtii transformation – electroporation and glass bead agitation. Electroporation is normally restricted to strains with cell wall, as it kills cell-wall-deficient strains effectively if without careful handling of osmosis. Electroporation also requires special instruments such as electroporator and cuvettes. In contrast, glass bead agitation uses simple lab equipment. The mild shear created by agitation in the presence of glass bead allows cell-wall-deficient strains to take up DNA. If glass bead method is to be applied to cell-wall strains, cells need to be treated with autolysin (http://www.chlamy.org/methods/autolysin.html) to partially lyse the wall components. A pitfall of both methods is that the DNAs are often shortened by nuclease once entering the cells, making the downstream PCR-based genotyping of insertion site rather difficult. Here I describe an improved design of insertional mutagenesis used in (Tsai et al., 2014), and the transformation protocol using glass bead as previously described in (Kindle, 1990) with minor modification. The putative mutants can be selected by autotrophic or antibiotic resistance markers, and the disrupted loci can be mapped by methods such as plasmid rescue (Peers et al., 2009) and SiteFinding PCR (Tan et al., 2005).

Keywords: Chlamydomonas(衣藻), Insertional mutagenesis(插入突变), Transformation(转型)

Materials and Reagents

  1. 15 and 50 ml conical tubes
  2. Petri dishes (90 mm in diameter)
  3. pHYG3 plasmid (http://chlamycollection.org/plasmid/phyg3/) or plasmid carrying other selection markers
  4. C. reinhardtii cell wall-less strain such as dw15 (cw15, nit1, mt+; http://chlamycollection.org/strain/cc-4619-cw15-nit1-mt-dw15-1/)
  5. Agar (Caisson Laboratories, Phytoblend)
  6. TAP medium (Harris, 1989)
  7. Restriction enzyme for linearizing the plasmid DNA, such as PvuII (New England Biolabs)
  8. Hygromycin (Life Technologies) or other selection means
  9. TAP agar plates (see Recipes)
  10. Top agar (see Recipes)

Equipment

  1. Common bench-top vortexer
  2. Spectrophotometer for optical density (OD) measurement, Z2 Coulter Counter (Beckman Coulter) or a hemocytometer
  3. 250 ml flask
  4. Bench-top centrifuge capable of centrifugation at 1,500 x g and accommodating 50 ml conical tubes
  5. Shaker
  6. Z2 Coulter Counter or hemocytometer
  7. Glass beads 425-600 μm (Sigma-Aldrich, catalog number: G8772 )
  8. Glass tubes or round bottom plastic tubes with or without cap (~10 mm in diameter)

Procedure

  1. DNA preparation (assumes use of pHYG3)
    1. Digest pHYG3 plasmid (http://www.biologie.uni-regensburg.de/Genetik/Mages/pHyg3.html) DNA with PvuII. PvuII was chosen specifically for pHYG3 plasmid because it releases a DNA fragment that contains the hygromycin B resistance gene aph7 with short flanking sequences. Based on our experience and others, linear DNAs are often shortened on each end after entering the cells of C. reinhardtii-ranging from dozens to hundreds of nucleotides-due to the exonuclease activity. It makes it harder to map the disrupted loci because the precise sequence of the insertion is unpredictable and different in each clone. Hence, we recommend using a DNA fragment that is just enough to cover the selectable marker (i.e. promoter, coding sequence, and terminator), so if the transformed DNAs are shortened to an extent that compromises the integrity of the marker, the clones harboring them will not be pulled out of the screen. In other words, the clones in the mutant library have the insertion whose length lies between the PvuII-digested fragment and the combined length of promoter, coding sequence, and terminator.
    2. Gel-excise the shorter, 2,012-bp fragments of the pHYG3 plasmid.

  2. Growth condition
    In general, cells were grown in liquid TAP medium under continuous light (70-80 μmol/m-2 s-1) at 22 °C, with shaking at 100 rpm; or ambient room temperature (~22 °C) for solid media.

  3. Glass bead transformation
    1. Grow cultures in TAP media (50 ml in 250 ml flask) until cell density reaches 1-2 x 106 cells/ml determined by Z2 Coulter Counter or hemocytometer, or approximately 0.2~0.3 OD550 by spectrophotometer.
    2. While cells are growing, prepare hardware: add ~0.3 g (~300 µl) glass beads to test tubes, autoclave.
    3. Pellet the cells by centrifugation (1,500 x g for 2 min), and resuspend in TAP to a concentration of ~2 x 107 cells/ml.
    4. Transfer 0.3 ml of cells to the glass bead-containing test tube, add ~1 µg linearized plasmid DNA prepared earlier (see Procedure A DNA preparation), and vortex 15 sec at top speed (Note 2).
    5. Add 6 ml TAP to the test tube, carefully transfer the cells (6 ml + 0.3 ml) to a new sterile 15 ml conical tube, and shake under growth conditions overnight (Note 3) at 100 rpm.
    6. After overnight recovery, add 6 ml of top agar (microwave to melt the top agar, cool to ~40 °C before pouring into the overnight culture) into the overnight culture (1:1 mixture). Mix gently.
    7. Immediately pour the 1:1 mixture onto TAP plates (6 ml for one regular Petri dish, 2 Petri dishes needed), let set (Note 4).
    8. Wrap plates with parafilm (cut slits to allow gas exchange), and place under lights. Colonies should start appearing in 5 to 7 days on the plate surface or embedded within the top agar layer. Generally speaking, 50 ml of culture (step C1) produces approximately 500 colonies after transformation. To create a mutant library ranging the entire C. reinhardtii genome (about 18,000 genes), it would require ~100 transformation in order to get a 3 fold coverage. Use sterile toothpick or pipet tips to pick up the colonies.

Notes

  1. Transformation by glass beads is only applicable to cell wall-less strains (e.g. dw15, cw15) (Kindle, 1990). For the transformation of cell-wall strains, electroporation is more commonly used (https://www.thermofisher.com/order/catalog/product/A14258).
  2. Do not vortex the cells too vigorously otherwise DNA fragments tend to break, especially for longer fragments.
  3. If cell duplication during the overnight shaking (Procedure C, step C5) is a concern (e.g. to avoid duplicated clones for mutant screen), do 8-12 h shaking instead.
  4. Optional: Pre-warm the agar plates at 37 °C before pouring the 1:1 mixture (Procedure C, step C7).

Recipes

  1. TAP agar plates
    0.8% agar with 10 μg/ml hygromycin
  2. Top agar
    TAP medium with 0.4% agar

Acknowledgments

This work was supported by the US Air Force Office of Scientific Research [Grant FA9550-11-1-0264 (to C. B.)], by a Strategic Partnership grant from the MSU Foundation (to C. B.), and by MSU AgBioResearch (C. B.).

References

  1. Harris, E. H. (2001). Chlamydomonas as a model organism. Annu Rev Plant Physiol Plant Mol Biol 52: 363-406.
  2. Kim, E. J. and Cerutti, H. (2009). Targeted gene silencing by RNA interference in Chlamydomonas. Methods Cell Biol 93: 99-110.
  3. Kindle, K. L. (1990). High-frequency nuclear transformation of Chlamydomonas reinhardtii. Proc Natl Acad Sci U S A 87(3): 1228-1232.
  4. Hoober, J. K. (1989). The Chlamydomonas sourcebook. A comprehensive guide to biology and laboratory use. Elizabeth H. Harris. Academic Press, San Diego, CA, 1989. xiv, 780 pp., illus. $145. Science 246(4936): 1503-1504.
  5. Merchant, S. S., Prochnik, S. E., Vallon, O., Harris, E. H., Karpowicz, S. J., Witman, G. B., Terry, A., Salamov, A., Fritz-Laylin, L. K., Marechal-Drouard, L., Marshall, W. F., Qu, L. H., Nelson, D. R., Sanderfoot, A. A., Spalding, M. H., Kapitonov, V. V., Ren, Q., Ferris, P., Lindquist, E., Shapiro, H., Lucas, S. M., Grimwood, J., Schmutz, J., Cardol, P., Cerutti, H., Chanfreau, G., Chen, C. L., Cognat, V., Croft, M. T., Dent, R., Dutcher, S., Fernandez, E., Fukuzawa, H., Gonzalez-Ballester, D., Gonzalez-Halphen, D., Hallmann, A., Hanikenne, M., Hippler, M., Inwood, W., Jabbari, K., Kalanon, M., Kuras, R., Lefebvre, P. A., Lemaire, S. D., Lobanov, A. V., Lohr, M., Manuell, A., Meier, I., Mets, L., Mittag, M., Mittelmeier, T., Moroney, J. V., Moseley, J., Napoli, C., Nedelcu, A. M., Niyogi, K., Novoselov, S. V., Paulsen, I. T., Pazour, G., Purton, S., Ral, J. P., Riano-Pachon, D. M., Riekhof, W., Rymarquis, L., Schroda, M., Stern, D., Umen, J., Willows, R., Wilson, N., Zimmer, S. L., Allmer, J., Balk, J., Bisova, K., Chen, C. J., Elias, M., Gendler, K., Hauser, C., Lamb, M. R., Ledford, H., Long, J. C., Minagawa, J., Page, M. D., Pan, J., Pootakham, W., Roje, S., Rose, A., Stahlberg, E., Terauchi, A. M., Yang, P., Ball, S., Bowler, C., Dieckmann, C. L., Gladyshev, V. N., Green, P., Jorgensen, R., Mayfield, S., Mueller-Roeber, B., Rajamani, S., Sayre, R. T., Brokstein, P., Dubchak, I., Goodstein, D., Hornick, L., Huang, Y. W., Jhaveri, J., Luo, Y., Martinez, D., Ngau, W. C., Otillar, B., Poliakov, A., Porter, A., Szajkowski, L., Werner, G., Zhou, K., Grigoriev, I. V., Rokhsar, D. S. and Grossman, A. R. (2007). The Chlamydomonas genome reveals the evolution of key animal and plant functions. Science 318(5848): 245-250.
  6. Molnar, A., Bassett, A., Thuenemann, E., Schwach, F., Karkare, S., Ossowski, S., Weigel, D. and Baulcombe, D. (2009). Highly specific gene silencing by artificial microRNAs in the unicellular alga Chlamydomonas reinhardtii. Plant J 58(1): 165-174.
  7. Peers, G., Truong, T. B., Ostendorf, E., Busch, A., Elrad, D., Grossman, A. R., Hippler, M. and Niyogi, K. K. (2009). An ancient light-harvesting protein is critical for the regulation of algal photosynthesis. Nature 462(7272): 518-521.
  8. Rasala, B. A., Barrera, D. J., Ng, J., Plucinak, T. M., Rosenberg, J. N., Weeks, D. P., Oyler, G. A., Peterson, T. C., Haerizadeh, F. and Mayfield, S. P. (2013). Expanding the spectral palette of fluorescent proteins for the green microalga Chlamydomonas reinhardtii. Plant J 74(4): 545-556.
  9. Tan, G., Gao, Y., Shi, M., Zhang, X., He, S., Chen, Z. and An, C. (2005). SiteFinding-PCR: a simple and efficient PCR method for chromosome walking. Nucleic Acids Res 33(13): e122.
  10. Tsai, C. H., Warakanont, J., Takeuchi, T., Sears, B. B., Moellering, E. R. and Benning, C. (2014). The protein Compromised Hydrolysis of Triacylglycerols 7 (CHT7) acts as a repressor of cellular quiescence in Chlamydomonas. Proc Natl Acad Sci U S A 111(44): 15833-15838.

简介

单细胞微藻(Chlamydomonas reinhardtii)( C。reinhardtii )已被用作许多研究领域的参考模型。原理研究领域是真核鞭毛结构和功能,基底体(中心粒),细胞 - 细胞识别,细胞周期控制,叶绿体生物发生,光趋化,非光化学猝灭,特别是C的光合作用。可以在黑暗中在有机碳(例如乙酸盐)上生长,因此提供了优于陆地植物的优点(Harris,2001; Peers等人)。 2009)。 C。具有短的生命周期,测序的基因组(Merchant等人,2007),以及用于正向和反向遗传研究的增长的分子工具箱,包括转化方案,基因沉默(Kim和Cerutti,2009; Molnar等人,2009)和荧光蛋白标签(Rasala等人,2013)。有两种常用的 C方法。莱茵衣氏转化 - 电穿孔和玻璃珠搅拌。电穿孔通常限于具有细胞壁的菌株,因为如果不仔细处理渗透,其有效地杀死细胞壁缺陷菌株。电穿孔还需要特殊仪器,例如电穿孔仪和比色皿。相比之下,玻璃珠搅拌使用简单的实验室设备。在玻璃珠存在下通过搅拌产生的温和剪切允许细胞壁缺陷菌株摄取DNA。如果玻璃珠法应用于细胞壁菌株,细胞需要用自溶素处理(部分裂解壁组件。这两种方法的缺点是DNAs通常被核酸酶一旦进入细胞就被缩短,使得插入位点的下游基于PCR的基因分型相当困难。在这里,我描述了在(Tsai等人,2014)中使用的插入诱变的改进设计,以及如先前在(Kindle,1990)中所述的使用玻璃珠的转化方案,具有微小的修改。可以通过自养或抗生素抗性标记来选择推定的突变体,并且可以通过诸如质粒拯救(Peers等人,2009)和SiteFinding PCR(Tan等人)的方法来定位被破坏的基因座al。,2005)。

关键字:衣藻, 插入突变, 转型

材料和试剂

  1. 15和50ml锥形管
  2. 培养皿(直径90mm)
  3. pHYG3 质粒( http://chlamycollection.org/plasmid/phyg3/ )或携带其他选择标记的质粒
  4. C。例如 dw15 ( cw15 ,nit1,mt + ; http://chlamycollection.org/strain/cc-4619-cw15-nit1-mt-dw15- 1/
  5. 琼脂(Caisson Laboratories,Phytoblend)
  6. TAP培养基(Harris,1989)
  7. 用于线性化质粒DNA的限制酶,例如PvuII(New England Biolabs)
  8. 潮霉素(Life Technologies)或其他选择方式
  9. TAP琼脂平板(见配方)
  10. 顶级琼脂(见配方)

设备

  1. 普通台式涡流器
  2. 用于光密度(OD)测量的分光光度计,Z2 Coulter计数器(Beckman Coulter)或血细胞计数器
  3. 250 ml烧瓶
  4. 台式离心机,能够以1,500×g离心并容纳50ml锥形管
  5. 振动器
  6. Z2 Coulter计数器或血细胞计数器
  7. 玻璃珠425-600μm(Sigma-Aldrich,目录号:G8772)
  8. 玻璃管或带或不带帽(直径约10 mm)的圆底塑料管

程序

  1. DNA制备(假设使用 pHYG3 )
    1. Digest pHYG3 质粒 ( http://www.biologie.uni-regensburg.de/Genetik/Mages/pHyg3.html )DNA 与PvuII。 PvuII特异性选择pHYG3 质粒,因为它 释放含有具有短侧翼序列的潮霉素B抗性基因 aph7 的DNA片段。根据我们的经验和其他, 线性DNA通常在进入C细胞后在每端缩短。从几十到几百个核苷酸 - 由于 ?核酸外切酶活性。它使得更难以映射中断的轨迹 因为插入的精确顺序是不可预测的 在每个克隆中不同。因此,我们建议使用DNA片段 刚好足以覆盖选择标记(即启动子,编码) 序列和终止子),因此如果转化的DNA被缩短 损害标记物,克隆的完整性的程度 拥抱他们不会被拉出屏幕。换句话说, ?突变体文库中的克隆具有其长度所在的插入 在PvuII消化的片段和启动子的组合长度之间, ?编码序列和终止子
    2. 凝胶切除pHYG3 质粒的较短的,2012bp的片段。

  2. 成长条件
    通常,在22℃下在连续光(70-80μmol/m 2 -2s -1 s -1)下在液体TAP培养基中生长细胞,在100rpm振荡;或用于固体介质的环境室温(?22℃)
  3. 玻璃珠转换
    1. 在TAP培养基中生长培养物(250ml烧瓶中50ml),直到细胞密度 通过Z2 Coulter计数器确定为1-2×10 6个细胞/ml 血细胞计数器,或通过分光光度计约0.2?0.3OD 550
    2. 当细胞生长时,准备硬件:向试管中加入?0.3g(?300μl)玻璃珠,高压灭菌
    3. 通过离心(1500×g,2分钟)沉淀细胞,并在TAP中重悬至约2×10 7个细胞/ml的浓度。
    4. 转移0.3毫升细胞到含玻璃珠的试管,加入 ??1μg以前制备的线性化质粒DNA(参见程序A DNA 准备),并以最高速度涡旋15秒(注2)
    5. 加入6ml TAP到试管中,小心地将细胞(6ml + 0.3ml)转移到a 新的无菌15ml锥形管,并在生长条件下摇动 过夜(注3),转速为100rpm
    6. 过夜回收后,加入6ml ?的顶部琼脂(微波融化顶部琼脂,冷却至?40℃之前 倒入过夜培养物)过夜培养物(1:1 混合物)。轻轻混匀。
    7. 立即将1:1混合物倒在TAP上 板(对于一个常规培养皿为6ml,需要2个培养皿),放置 ?(注4)。
    8. 包装有石蜡膜(切割缝隙,允许气体 交换),并放置在灯光下。菌落应该开始出现在5 到7天,或者包埋在顶层琼脂层中。 一般来说,50ml培养物(步骤C1)大约产生 转化后500个菌落。创建一个突变文库 整个 C。 (约18,000个基因),这将需要 ?100转化以获得3倍覆盖。使用无菌 牙签或吸管尖提起殖民地。

笔记

  1. 通过玻璃珠的转化仅适用于无细胞壁菌株(例如 dw15 , cw15 )(Kindle,1990)。对于细胞壁菌株的转化,更常使用电穿孔( https://www。 thermofisher.com/order/catalog/product/A14258 )。
  2. 不要剧烈涡旋细胞,否则DNA片段会破碎,特别是对于更长的片段
  3. 如果在过夜振荡期间(方法C,步骤C5)的细胞复制是一个问题(例如,为了避免突变筛选的重复克隆),可以改为8-12小时摇动。
  4. 任选:在倾倒1:1混合物之前(步骤C,步骤C7),将琼脂板预温至37℃。

食谱

  1. TAP琼脂平板上 0.8%琼脂与10μg/ml潮霉素
  2. 顶部琼脂
    含有0.4%琼脂的TAP培养基

致谢

这项工作由美国空军科学研究办公室[批准FA9550-11-1-0264(给C.B.)],由MSU基金会(向C. B.)和MSU AgBioResearch(C.B。)的战略合作伙伴赠款支持。

参考文献

  1. Harris,E.H。(2001)。 作为模式生物。 Rev Plant Physiol Plant Mol Biol 52:363-406
  2. Kim,E.J。和Cerutti,H。(2009)。 通过衣原体中的RNA干扰进行的靶向基因沉默。 < em> Methods Cell Biol 93:99-110。
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  7. Peers,G.,Truong,T.B.,Ostendorf,E.,Busch,A.,Elrad,D.,Grossman,A.R.,Hippler,M.and Niyogi,K.K。(2009)。 古老的光收获蛋白对藻类光合作用的调节至关重要。 自然 462(7272):518-521
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  10. Tsai,C.H.,Warakanont,J.,Takeuchi,T.,Sears,B.B.,Moellering,E.R。和Benning,C.(2014)。 三酰基甘油7(CHT7)的蛋白质水解水解作用为细胞静止的抑制子,衣藻。 Proc Natl Acad Sci USA 111(44):15833-15838。
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引用:Tsai, C. and Benning, C. (2015). Insertional Mutagenesis of Chlamydomonas reinhardtii. Bio-protocol 5(24): e1680. DOI: 10.21769/BioProtoc.1680.
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