搜索

Construction of Chromosomal In-Frame Deletion Mutants in Rhodobacter capsulatus
在荚膜红细菌中建立染色体框内缺失突变体   

下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

Abstract

Markerless chromosomal in-frame deletion mutants are often used to avoid polar effects, conserve antibiotic resistance markers or make multiple mutations. This protocol capitalizes on the sacB gene (lethal in the presence of sucrose), for selection against a single cross-over event, on a suicide vector (contains no Rhodobacter capsulatus compatible origin of replication). The bacterial strains, media and suicide vectors in this protocol are specific for R. capsulatus, but the concepts can be applied to other species.

Materials and Reagents

  1. R. capsulatus recipient strain
  2. RCV broth
  3. RCV agar
  4. Sucrose
  5. L-malic acid (Sigma-Aldrich, catalog number: 240176 )
  6. Thiamine hydrochloride (Sigma-Aldrich, catalog number: T1270 )
  7. Escherichia coli cloning strain capable of conjugation (e.g. S17-1)
  8. Suicide plasmid, such as pZJD29A (Z. Jiang and C. E. Bauer, unpublished strain construction), that contains the sacB gene and an antibiotic resistance gene, but no Rhodobacter capsulatus compatible origin of replication. The suicide plasmid pZJD29A is a derivative of pJP5603 and has a RK6-based origin of replication making it a “universal” suicide plasmid (Penfold and Pemberton, 1992).
  9. Appropriate antibiotic (resistance specified by the suicide plasmid)
  10. RCV broth/agar (Beatty and Gest, 1981) (see Recipes)
  11. Trace element solution (in 250 ml dH2O) (see Recipes)

Equipment

  1. 30 °C shaker
  2. 30 °C incubator
  3. Test tubes
  4. Petri plates

Procedure

  1. Subclone ~1 kb of upstream sequences plus the first several codons of the gene to be deleted into pUC19 or similar vector. In the resulting plasmid, clone the last several codons of the gene to be deleted plus ~1 kb of downstream sequences. Amplify this whole segment by PCR and clone into a suicide plasmid (such as pZJD29A), which contains sacB and an antibiotic resistance gene. Be sure to design your construct so that the first and last several codons of the gene to be deleted are in-frame. This cloning is done in E. coli.
  2. Conjugate the deletion construct into R. capsulatus (refer to “Bacterial Conjugation of Rhodobacter capsulatus” protocol) (Leung and Beatty, 2013).
  3. Streak the conjugated R. capsulatus on RCV agar plate supplemented with the appropriate antibiotic (i.e. the antibiotic resistance on the suicide plasmid). Colonies that grow represent single cross-over events.
  4. Confirm single cross-over event by PCR, or streaking colonies on RCV, RCV + antibiotic, and RCV + 10% sucrose. They should grow on RCV and RCV + antibiotic, but not RCV + 10% sucrose because the sacB gene results in toxicity in the presence of sucrose and only single crossover events will maintain the sacB gene.
  5. Inoculate 10 ml of RCV (NO antibiotic) with a colony that is confirmed to have a single cross-over and incubate at 30 °C, shaking at 250 rpm for 3-5 days.
  6. Spin down 1 ml of this culture in a microcentrifuge tube, decant supernatant and resuspend cell pellet in 100 μl RCV broth.
  7. Spread cell suspension on a RCV + 10% sucrose agar plate, incubate at 30 °C for 4-7 days.
  8. Colonies that grow should be restreaked on RCV, RCV + antibiotic, and RCV + 10% sucrose. The double cross-over (i.e. chromosomal in-frame deletion mutant) should grow on RCV and RCV + 10% sucrose but not on RCV + antibiotic because the second crossover event results in the loss of the sacB gene and the antibiotic resistance gene.
  9. Confirm chromosomal in-frame deletion mutant by PCR because the second cross over can either result in WT or the KO, which will both grow on RCV and RCV + 10% sucrose but not on RCV + antibiotics.

Recipes

  1. RCV broth/agar (Beatty and Gest, 1981) (in 1 L; autoclaved)
    4 g D, L-malic acid
    1 g (NH4)2SO4
    10 mM potassium phosphate buffer
    200 mg MgSO4.7H2O
    75 mg CaCl2.2H2O
    12 mg FeSO4.7H2O
    20 mg Na2EDTA
    1 ml trace element solution
    1 mg thiamine hydrochloride
    pH adjusted to 6.8 with NaOH before autoclaving
    (for agar add 1.5% agar)
  2. Trace element solution (in 250 ml dH2O)
    0.7 g H3BO3
    398 mg MnSO4.H2O
    188 mg Na2MoO4.2H2O
    60 mg ZnSO4.7H2O
    10 mg Cu(NO3).3H2O

Acknowledgments

The development of this protocol was funded by a grant to J.T.B. from the Canadian Institutes of Health Research. This protocol was adapted from protocols developed by Beatty and Gest (1981), and Penfold and Pemberton (1992).

References

  1. Beatty, J. T. and H. Gest (1981). Generation of succinyl-coenzyme A in photosynthetic bacteria.  Arch Microbiol 129(5): 335-340.
  2. Leung M.M., Brimacombe C.A., Spiegelman G.B., and Beatty J.T. (2012) The GtaR protein negatively regulates transcription of the gtaRI operon and modulates gene transfer agent (RcGTA) expression in Rhodobacter capsulatus. Mol Microbiol  83(4):759-74.
  3. Leung, M. and Beatty, J. T. (2013). Bacterial Conjugation in Rhodobacter capsulatus. Bio-protocol 3(13): e804. 
  4. Penfold, R.J. and Pemberton, J.M (1992). An improved suicide vector for construction of chromosomal insertion mutations in bacteria. Gene 118 (1): 145-146.

简介

无记号染色体框内缺失突变体常用于避免极性效应,保存抗生素抗性标记或产生多个突变。 该协议利用了在sacC基因(在蔗糖存在下致死),用于针对单个交叉事件的选择,在自杀载体上(不含有红细菌Rhodobacter capsulatus 兼容性 复制起点)。 该方案中的细菌菌株,培养基和自杀载体对于R是特异性的。 capsulatus ,但这些概念可应用于其他物种

材料和试剂

  1. R。 胶囊受体菌株
  2. RCV肉汤
  3. RCV琼脂
  4. 蔗糖
  5. L-苹果酸(Sigma-Aldrich,目录号:240176)
  6. 盐酸硫胺素(Sigma-Aldrich,目录号:T1270)
  7. 能够共轭的大肠杆菌克隆菌株(例如 S17-1)
  8. 自杀质粒,例如包含sacB基因和抗生素抗性基因但没有荚膜红细菌相容来源的pZJD29A(Z.Jiang和CE Bauer,未公开的菌株构建) 的复制。 自杀质粒pZJD29A是pJP5603的衍生物,具有基于RK6的复制起点,使其成为"通用"自杀质粒(Penfold和Pemberton,1992)。
  9. 合适的抗生素(由自杀质粒指定的抗性)
  10. RCV肉汤/琼脂(Beatty和Gest,1981)(参见Recipes)
  11. 微量元素溶液(在250ml dH 2 O中)(参见配方)

设备

  1. 30℃摇床
  2. 30& C孵育器
  3. 试管
  4. 培养皿

程序

  1. 亚克隆〜1kb的上游序列加上待缺失的基因的前几个密码子到pUC19或类似载体中。在所得质粒中,克隆待删除基因的最后几个密码子加〜1kb的下游序列。通过PCR扩增整个区段并克隆到包含sacB和抗生素抗性基因的自杀质粒(例如pZJD29A)中。一定要设计你的构建,使删除的基因的第一个和最后几个密码子是框架内。这种克隆在E中完成。大肠杆菌。
  2. 将删除构建体共轭到中(请参阅"


    "细菌共轭"
  3. 连接共轭的R。补充有合适的抗生素(即自杀质粒上的抗生素抗性)的RCV琼脂平板上的荚膜囊泡。生长的菌落代表单一交换事件。
  4. 通过PCR确认单一交换事件,或在RCV,RCV +抗生素和RCV + 10%蔗糖上划线菌落。它们应当在RCV和RCV +抗生素上生长,而不是在RCV + 10%蔗糖上生长,因为sacB基因在蔗糖存在下导致毒性,并且只有单个交换事件将维持sacB em>基因
  5. 用确认具有单次交叉的菌落接种10ml RCV(NO抗生素),并在30℃下孵育,在250rpm下摇动3-5天。
  6. 在微量离心管中离心1ml该培养物,倾析上清液,并在100μlRCV肉汤中重悬细胞沉淀。
  7. 将扩散细胞悬浮液在RCV + 10%蔗糖琼脂平板上,在30℃孵育4-7天。
  8. 生长的菌落应重新划线在RCV,RCV +抗生素和RCV + 10%蔗糖上。双交换(即,染色体框内缺失突变体)应当在RCV和RCV + 10%蔗糖上生长,但不在RCV +抗生素上生长,因为第二交叉事件导致 > sacB 基因和抗生素抗性基因
  9. 通过PCR确认染色体框内缺失突变体,因为第二次交叉可以导致WT或KO,其将在RCV和RCV + 10%蔗糖上生长,而不在RCV +抗生素上生长。

食谱

  1. RCV肉汤/琼脂(Beatty和Gest,1981)(在1L中;高压灭菌) 4克D,L-苹果酸 1 g(NH 4)2 SO 2 4
    10mM磷酸钾缓冲液 200mg MgSO 4。<7h> 7H 2 O 75mg CaCl 2 2 2H 2 12mg FeSO 4 7H 2 O
    20mg Na 2 EDTA 1 ml微量元素溶液
    1mg盐酸硫胺素 在高压灭菌之前用NaOH将pH调节至6.8 (用于琼脂加1.5%琼脂)
  2. 微量元素溶液(在250ml dH 2 O中) 0.7 g H sub 3 BO sub 3
    398mg MnSO 4 。 H O
    188mg Na 2 MoO 4 2H 2 60mg ZnSO 4 7H 2 O
    10毫克Cu(NO 3)n。3 H 2 O 2。

致谢

该方案的开发由授予J.T.B.的资助。 来自加拿大健康研究所。 该协议改编自Beatty和Gest(1981)和Penfold和Pemberton(1992)开发的方案。

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. Leung M.M.,Brimacombe C.A.,Spiegelman G.B.,and Beatty J.T. (2012) GtaR蛋白负调节gtaRI操纵子的转录并调节基因转移剂(RcGTA) 在红细菌中表达 。 Mol Microbiol 83(4):759-74。
  3. Leung,M。和Beatty,J.T。(2013)。 红细胞肺泡杆菌中的细菌结合 。 生物协议 3(13):e804。
  4. 彭福尔德 和Pemberton,J.M(1992)。 用于在细菌中构建染色体插入突变的改进的自杀载体。 Gene 118(1):145-146
  • English
  • 中文翻译
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: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Leung, M. M. and Beatty, J. T. (2013). Construction of Chromosomal In-Frame Deletion Mutants in Rhodobacter capsulatus. Bio-protocol 3(4): e333. DOI: 10.21769/BioProtoc.333.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。