常用酿酒酵母菌株基因型教程文件

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一株产核酸酿酒酵母工程菌及其构建方法与应用

一株产核酸酿酒酵母工程菌及其构建方法与应用

一株产核酸酿酒酵母工程菌及其构建方法与应用
一株产核酸酿酒酵母工程菌是指经过基因工程技术改造的酿酒酵母菌株,具有高产核酸能力的微生物。

以下是一种构建该菌株的方法和应用:
构建方法:
1. 选择合适的酿酒酵母菌株作为基础菌株。

2. 通过PCR或其他基因克隆技术,获得目标基因(例如核酸
合成酶基因)的DNA序列。

3. 将目标基因与适当的表达载体连接。

4. 将表达载体转化到酿酒酵母菌细胞中。

5. 通过筛选、鉴定等方法,获得具有目标基因表达的酿酒酵母菌株。

应用:
1. 高产核酸酿酒酵母工程菌可用于生产核酸药物,如核酸疫苗、抗肿瘤药物等。

2. 该菌株还可用于生物反应器中进行大规模生产核酸。

3. 可通过进一步改造该菌株,使其具备其他有益特性,如耐高温、耐久性等,进一步拓展其应用领域。

4. 该菌株也可用于基础科学研究,探索核酸生物学、基因调控等领域。

第十五章:酵母菌基因工程选编

第十五章:酵母菌基因工程选编

③易进行载体DNA的导入。DNA转化技 术的不断发展优化,多数酵母菌可 以取得较高的转化率;
④培养条件简单,容易进行高密度 发酵;
⑤能将外源基因表达产物分泌到培 养基中;
⑥有类似高等真核生物的蛋白质翻 译后的修饰功能。
2.缺陷在于:
①表达效率相对低; ②酵母常有密码子偏爱性,真核基
因在其中表达时需要人工修正。
2.含有ARS的YRp和YEp质粒及其构建
①ARS为酵母菌中的自主复制序列,大 小在0.8-1.5Kb,染色体上每30-40bp 就有一个ARS元件。
②由染色体ARS构成的质粒称为YRp,而 由2μ质粒构建的杂合质粒为YEp。
③上述两类质粒在酿酒酵母中的拷贝数 最高可达200个,但是经过几代培养 后,质粒丢失率达50%-70%,主要由 于分配不均匀所致。
三.抑制超糖基化作用的突变宿主菌
许多真核生物的蛋白质在其天门冬 酰胺侧链上接有寡糖基团,常常影 响蛋白质的生物活性。整个糖单位 由糖基核心和外侧糖链两部分组成。
酵母菌普遍拥有完整的糖基化系统,酿 酒酵母细胞内的天门冬酰胺侧链糖基修 饰和加工系统对来自高等动物和人的异 源蛋白活性表达是极为有利的,但野生 型酿酒酵母对异源蛋白的糖基化反应很 难控制,呈超糖基化倾向,因此超糖基 化缺陷菌株非常重要。
②YAC载体的装载量建
①YIP 载体由大肠杆菌质粒和酵母的 DNA 片段组成,可与受体或宿主的染色体 DNA 同源重组,整合进入宿主染色体中,酵母 片段只提供选择性标志,没有复制起点。
②转化率低(只有1-10转化子/微克DNA), 但转化子遗传性稳定,多用于遗传分析。
一.广泛用于外源基因表达的酵母宿主菌
目前已广泛用于外源基因表达的研究的酵母菌包括:

常用酿酒酵母菌株基因型

常用酿酒酵母菌株基因型

Commonly used strains▪▪van Dijken et al. (2000) Enzyme Microb Technol 26:706-714 - compares various characteristics of commonly used lab strains▪Winzeler et al. (2003) Genetics 163:79-89 - uses SFP (single-feature polymorphisms) analysis to study genetic identity between common lab strainsS288CGenotype:MATαSUC2 gal2 mal mel flo1 flo8-1 hap1 ho bio1 bio6Notes: Strain used in the systematic sequencing project, the sequence stored in SGD. S288C does not form pseudohyphae. In addition, since it has a mutated copy of HAP1, it is not a good strain for mitochondrial studies. It has an allelic variant of MIP1 which increases petite frequency. S288C strains are gal2- and they do not use galactose anaerobically.The S288C genome was recently resequenced at the Sanger Institute.References:Mortimer and Johnston (1986) Genetics 113:35-43.BY4743Genotype:MAT a/αhis3Δ1/his3Δ1 leu2Δ0/leu2Δ0 LYS2/lys2Δ0 met15Δ0/MET15 ura3Δ0/ura3Δ0Notes: Strain used in the systematic deletion project, generated from a cross between BY4741 and BY4742, which are derived from S288C. As S288c, these strains have an allelic variant of MIP1 which increases petite frequency. See Brachmann et al. reference for details.References:Brachmann et al. (1998) Yeast 14:115-32.FY4Genotype:MAT aNotes: Derived from S288C.References:Winston et al. (1995) Yeast 11:53-55.FY1679Genotype:MAT a/αura3-52/ura3-52 trp1Δ63/TRP1 leu2Δ1/LEU2 his3Δ200/HIS3 GAL2/GALNotes: Isogenic to S288C; used in the systematic sequencing project, the sequence stored in SGD.References:Winston et al. (1995) Yeast 11:53-55.AB972Genotype:MATα X2180-1B trp10 [rho 0]Notes: Isogenic to S288C; used in the systematic sequencing project, the sequence stored in SGD. AB972 is an ethidium bromide-induced rho- derivative of the strain X2180-1B-trp1.References:Olson MV et al. (1986) Proc. Natl. Acad. Sci. USA 83:7826-7830.A364AGenotype:MAT a ade1 ade2 ura1 his7 lys2 tyr1 gal1 SUC mal cup BIONotes: Used in the systematic sequencing project, the sequence stored in SGD.References:Hartwell (1967) J. Bacteriol. 93:1662-1670.XJ24-24aGenotype:MAT a ho HMa HMα ade6 arg4-17 trp1-1 tyr7-1 MAL2Notes: Derived from, but not isogenic to, S288CReferences:Strathern et al. (1979) Cell 18:309-319DC5Genotype:MAT a leu2-3,112 his3-11,15 can1-11Notes: Isogenic to S288C; used in the systematic sequencing project, the sequence stored in SGD.References:Broach et al. (1979) Gene 8:121-133X2180-1AGenotype:MAT a SUC2 mal mel gal2 CUP1Notes:S288c spontaneously diploidized to give rise to X2180. The haploid segregants X2180-1a and X2180-1b were obtained from sporulated X2180YNN216Genotype:MAT a/αura3-52/ura3-52 lys2-801amber/lys2-801amber ade2-101ochre/ade2-101ochreNotes: Congenic to S288C (see Sikorski and Hieter). Used to derive YSS and CY strains (see Sobel and Wolin).YPH499Genotype:MAT a ura3-52 lys2-801_amber ade2-101_ochre trp1-Δ63 his3-Δ200 leu2-Δ1Notes: Contains nonrevertible (deletion) auxotrophic mutations that can be used for selection of vectors. Notethat trp1-Δ63, unlike trp1-Δ1, does not delete adjacent GAL3 UAS sequence and retains homology to TRP1 selectable marker.gal2-, does not use galactose anaerobically. Derived from the diploid strain YNN216 (Johnston and Davis 1984; original source: M. Carlson, Columbia University), which is congenic with S288C.YPH500Genotype:MATαura3-52 lys2-801_amber ade2-101_ochre trp1-Δ63 his3-Δ200 leu2-Δ1Notes:MATα strain isogenic to YPH499 except at mating type locus. Derived from the diploid strain YNN216 (Johnston and Davis 1984; original source: M. Carlson, Columbia University), which is congenic with S288C.YPH501Genotype:MAT a/MATαura3-52/ura3-52 lys2-801_amber/lys2-801_amber ade2-101_ochre/ade2-101_ochretrp1-Δ63/trp1-Δ63 his3-Δ200/his3-Δ200 leu2-Δ1/leu2-Δ1Notes:a/α diploid isogenic to YPH499 and YPH500. Derived from the diploid strain YNN216 (Johnston and Davis 1984; original source: M. Carlson, Columbia University), which is congenic with S288C.Sigma 1278BNotes: Used in pseudohyphal growth studies. Detailed notes about the sigma strains have been kindly provided by Cora Styles.Granek and Magwene, PLoS Genet. 2010 Jan 22;6(1):e1000823, established that certain lineages of theSigma1278B background contain a nonsense mutation in RIM15, a G-to-T transversion at position 1216 that converts a Gly codon to an opal stop codon. This rim15 mutation interacts epistatically with mutations in certain other genes to affect colony morphology.Annotation of the Sigma1278b genome and information about the systematic deletion collection can be found here. SK1Genotype:MAT a/α HO gal2 cup S can1R BIONotes: Commonly used for studying sporulation or meiosis. Canavanine-resistant derivative.The SK1 genome was sequenced at the Sanger Institute.References:Kane SM and Roth J. (1974) Bacteriol. 118: 8-14CEN.PK (aka CEN.PK2)Genotype:MAT a/α ura3-52/ura3-52 trp1-289/trp1-289 leu2-3_112/leu2-3_112 his3 Δ1/his3 Δ1 MAL2-8C/MAL2-8C SUC2/SUC2Notes: CEN.PK possesses a mutation in CYR1 (A5627T corresponding to a K1876M substitution near the end of the catalytic domain in adenylate cyclase which eliminates glucose- and acidification-induced cAMP signalling and delays glucose-induced loss of stress resistance (Vanhalewyn et al., 1999; Dumortier et al., 2000).References:van Dijken et al. (2000) Enzyme Microb Technol 26:706-714W303Genotype:MAT a/MATα {leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15} [phi+]Notes: W303 also contains a bud4 mutation that causes haploids to bud with a mixture of axial and bipolar budding patterns. In addition, the original W303 strain contains the rad5-535 allele. As S288c, W303 has an allelic variantof MIP1 which increases petite frequency.The W303 genome was sequenced at the Sanger Institute.References: W303 constructed by Rodney Rothstein (see detailed notes from RR and Stephan Bartsch).bud4 info: Voth et al. (2005) Eukaryotic Cell, 4:1018-28.rad5-535 info: Fan et al. (1996) Genetics 142:749W303-1AGenotype:MAT a {leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15}Notes: W303-1A possesses a ybp1-1 mutation (I7L, F328V, K343E, N571D) which abolishes Ybp1p function, increasing sensitivity to oxidative stress.References: W303 constructed by Rodney Rothstein (see detailed notes from RR and Stephan Bartsch).ybp1-1 info: Veal et al. (2003) J. Biol. Chem. 278:30896-904.W303-1BGenotype:MATα {leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15}References: W303 constructed by Rodney Rothstein (see detailed notes from RR and Stephan Bartsch).W303-K6001Genotype:MAT a; {ade2-1, trp1-1, can1-100, leu2-3,112, his3-11,15, GAL, psi+, ho::HO::CDC6 (at HO), cdc6::hisG, ura3::URA3 GAL-ubiR-CDC6 (at URA3)}References: K6001 was developed by Bobola et al in Kim Nasmyth's lab (PMID: 8625408), and has become a common model in yeast aging research (PMID: 15489200). Its genome has been sequenced by Timmermann et al (PMID: 20729566)D273-10BGenotype:MATαmalNotes: Normal cytochrome content and respiration; low frequency of rho-. This strain and its auxotrophic derivatives were used in numerious laboratories for mitochondrial and related studies and for mutant screens. Good respirer that's relatively resistant to glucose repression.References:Sherman, F. (1963) Genetics 48:375-385.FL100Genotype:MAT aReferences:Lacroute, F. (1968) J. Bacteriol. 95:824-832.Sources: ATCC: 28383SEY6210/SEY6211Genotype:MAT a/MATαleu2-3,112/leu2-3,112 ura3-52/ura3-52 his3-Δ200/his3-Δ200 trp1-Δ901/trp1-Δ901ade2/ADE2 suc2-Δ9/suc2-Δ9 GAL/GAL LYS2/lys2-801Notes: SEY6210/SEY6211, also known as SEY6210.5, was constructed by Scott Emr and has been used in studies of autophagy, protein sorting etc. It is the product of crossing with strains from 5 different labs (Gerry Fink, Ron Davis, David Botstein, Fred Sherman, Randy Schekman). It has several selectable markers, good growth properties and good sporulation.References:Robinson et al. (1988) Mol Cell Biol 8(11):4936-48SEY6210Genotype:MATαleu2-3,112 ura3-52 his3-Δ200 trp1-Δ901 suc2-Δ9 lys2-801; GALNotes: SEY6210 is a MATalpha haploid constructed by Scott Emr and has been used in studies of autophagy, protein sorting etc. It is the product of crossing with strains from 5 different labs (Gerry Fink, Ron Davis, David Botstein, Fred Sherman, Randy Schekman). It has several selectable markers and good growth properties.References:Robinson et al. (1988) Mol Cell Biol 8(11):4936-48SEY6211Genotype:MAT a leu2-3,112 ura3-52 his3-Δ200 trp1-Δ901 ade2-101 suc2-Δ9; GALNotes: SEY6211 is a MATa haploid constructed by Scott Emr and has been used in studies of autophagy, protein sorting etc. It is the product of crossing with strains from 5 different labs (Gerry Fink, Ron Davis, David Botstein, Fred Sherman, Randy Schekman). It has several selectable markers and good growth properties.References:Robinson et al. (1988) Mol Cell Biol 8(11):4936-48JK9-3dThere are a, alpha and a/alpha diploids of JK9-3d with the following genotypes:Genotypes: JK9-3da MAT a leu2-3,112 ura3-52 rme1 trp1 his4JK9-3dα has the same genotype as JK9-3da with the exception of the MAT locusJK9-3da/α is homozygous for all markers except mating typeNotes: JK9-3d was constructed by Jeanette Kunz while in Mike Hall's lab. She made the original strain while Joe Heitman isolated isogenic strains of opposite mating type and derived the a/alpha isogenic diploid by mating type switching. It has in its background S288c, a strain from the Oshima lab, and a strain from the Herskowitz lab. It was chosen because of its robust growth and sporulation, as well as good growth on galactose (GAL+) (so that genes under control of the galactose promoter could be induced). It may also have a SUP mutation that allows translation through premature STOP codons and therefore produces functional alleles with many point mutations. References:Heitman et al. (1991a) Science 253(5022):905-9 and Heitman et al. (1991b) Proc Natl Acad Sci U S A 88(5):1948-52RM11-1aGenotype:MAT a leu2Δ ura3Δ ho::KanNotes: RM11-1a is a haploid derivative of Bb32(3), a natural isolate collected by Robert Mortimer from a California vineyard, as in Mortimer et al., 1994. It has high spore viability (80–90%) and has been extensively characterized phenotypically under a wide range of conditions. It has a significantly longer life span than typical lab yeast strains and accumulates age-associated abnormalities at a lower rate. It displays approximately 0.5–1% sequence divergence relative to S288c. More information is available at the Broad Institute website.References:Brem et al. (2002) Science 296(5568):752-5Y55Genotype:MAT a /MAT alpha HO/HO。

酵母实验-学生版

酵母实验-学生版

酵母实验-学⽣版酵母系列⼤实验设计PCR介导的酿酒酵母基因敲除1、实验⽬的学习理解酿酒酵母中PCR介导的基因⼀步敲除法的原理,掌握酵母的转化⽅法,了解酵母⽣长及遗传学特性。

2、实验原理酵母菌作为最简单的真核⽣物,能以单倍体和⼆倍体两种形式稳定存在,其中单倍体含有两种交配型,可以⾃由的在单倍体和⼆倍体之间进⾏转换,在⽣物学研究中有着得天独厚的优势。

⾸先酵母菌⽣长速度很快。

对数期⽣长的单倍体菌株在YPD(富营养天然培养基)90分钟分裂⼀代,在合成培养基中140分钟分裂⼀代。

其次,酵母的基因组很⼩,遗传背景相对简单,是⼀种很容易进⾏遗传操作的模式⽣物。

酿酒酵母的基因组只有12052 Kb,其基因组序列早在1996年测序完成,已有约6000个超过100个氨基酸的ORF被报道,只有不到5%的ORF含有内含⼦,其中有约5700个蛋⽩编码基因,分散在16条染⾊体上。

上世纪90年代末,由数个实验室联合进⾏的酿酒酵母基因组敲除项⽬的完成是酵母遗传学研究的⾥程碑事件。

在这个项⽬中,四个基因敲除菌株库被成功构建:两种交配型的单倍体菌株库、杂合⼦⼆倍体菌株库以及⾮必需基因的纯合⼦⼆倍体菌株库。

这个项⽬⼏乎完成了全部ORF的敲除,为⽣物学研究,特别是组学研究,提供了⼗分强⼤的系统性研究⼯具,为后续基因功能的研究奠定了坚实的基础。

此外,酵母作为真核⽣物,与⾼等⽣物在很多代谢通路和蛋⽩表达调节等⽅⾯是⾼度保守的,为⾼等⽣物相关基因,例如疾病基因,功能研究具有提供了简单、易于操作的系统。

⽬前,酿酒酵母已经具有⼀套⼗分灵活快速的遗传操作体系。

酵母允许外源质粒以独⽴复制⼦游离于基因组之外存在,也允许其整合到基因组中。

但跟其他⽣物相⽐,酵母⽐较独特且强⼤的特点是外源序列的整合依赖于同源重组机制。

之前提到的基因组敲除项⽬的完成就是依赖于⾼效率的同源重组,如图-1所⽰。

⼈们利⽤PCR的⽅法,在筛选标记基因两侧引⼊待敲除基因(YFG,your favorite gene)特异性序列;随后将PCR产物通过转化传递到酵母细胞内部;在同源重组的作⽤下,⼀些细胞的对应基因位点的内源性序列被含有同源臂的外源序列直接取代,并通过选择培养基筛选出来。

酵母菌的基因工程

酵母菌的基因工程

酵母菌的转化程序
碱金属离子介导的酵母菌完整细胞的转化
酿酒酵母的完整细胞经碱金属离子(如Li+等)、PEG、热休克 处理后,也可高效吸收质粒DNA,而且具有下列特性: 吸收线型DNA的能力明显大于环状DNA,两者相差80倍 共转化现象极为罕见
酵母菌的转化程序
酵母菌电击转化法
酵母菌原生质体和完整细胞均可在电击条件下吸收质粒DNA, 但在此过程中应避免使用PEG,它对受电击的细胞具有较很大的负 作用。电击转化的优点是不依赖于受体细胞的遗传特征及培养条件 适用范围广,而且转化率可高达105 / mg DNA。
a 型启动子
a1-a2阻遏a细胞特征表达
编码a2因子的基因突变型 hmla2-102能产生a2变体,
hmla2-102
MATa
a 型启动子
它能灭活a1,同时阻遏a型
a1
酵母菌启动子的可控性
超诱导型启动子
酿酒酵母 的半乳糖 利用酶系
由GAL1 GAL7和 GAL10 基因编码 半乳糖诱导时,GAL4高效表达,GAL1、GAL1、GAL10超高效表达
转录水平
羧肽酶Y 转录水平
抑制超糖基化作用的突变宿主菌
许多真核生物的蛋白质在其天门冬酰胺侧链上接有寡糖基团, 它们常常影响蛋白质的生物活性。整个糖单位由糖基核心和外侧糖
链两部分组成。
酵母菌普遍拥有蛋白 质的糖基化系统,但野生 型酿酒酵母对异源蛋白的 糖基化反应很难控制,呈 超糖基化倾向,因此超糖 基化缺陷株非常重要。
提高重组蛋白表达产率的突变宿主菌
能导致酿酒酵母中重组蛋白产量提高或质量改善的突变类型
突变类型 生物效应 改善重组蛋白分泌 提高重组蛋白表达 提高重组蛋白表达 作用位点 钙离子依赖型的ATP酶 转录后加工 转录水平

INVSC1酿酒酵母菌株使用说明

INVSC1酿酒酵母菌株使用说明

(必要时,可适当延长培养时间)。
菌 株 传 代 :
将得到的菌株的新鲜培养物转接到适宜的固体培养基及液体培养基中(尽量 增大接种量:如用无菌吸管吸取≥50μl 新鲜培养物至固体培养基,边移动边缓 慢释放),适宜温度下培养,用以菌株的保藏、传代及制备工作菌株。 注 意 事 项 : 1、菌种活化前,将冷冻管保存在低温、清洁、干燥的环境中,长时间室温下放 置会导致菌种衰退; 2、冷冻管开封、冻干粉复溶、菌株恢复培养等操作应在无菌条件下进行; 3、一些菌种经过冷冻干燥保存后,延迟期较长,部分需连续两次继代培养才能 正常生长; 4、苛养菌的培养需采用含特定营养成分的培养基,敬请正确选择,不清楚时来 电询问; 5、某些厌氧菌的培养,自开封到接种完成,均需以无氧气体充填,以保持厌氧 状态;培养过程中亦要保持厌氧状态; 6、某些菌种,如肺炎链球菌、流感嗜血杆菌、淋病奈瑟菌等需要 5-­‐10%CO2 促 进生长; 7、如发现冷冻管盖松动、复溶液浑浊等异常情况,应停止使用对应产品。 8、部分菌种有致病性、扩散性,请专业人员在专业环境下有保护性操作。 保 藏 条 件 : -­‐20℃保存(复溶液于 2-­‐8℃保存) 保 藏 时 间 : 2-­‐10 年,应根据菌种状况及时转接
ura3-­‐52
简 介 :
INVSC1 是酿酒酵母菌株,属于真核细胞。一般的针对原核生物的抗生
素例如卡那和氨苄对酵母是无效的,因此为了防止大肠杆菌等原核生物对
酵母培养菌株污染,往往会在培养基中加入一些氨苄和卡那霉素的抗生素,
pYES2 系列载体。INVSC1 酿酒酵母可在 YPD 培养基上生长,用电转化的
方法可将质粒转入酵母细胞中,用半乳糖诱导蛋白表达。用 30%的甘油与
培 养 物 混 合 可 保 藏 菌 种 。

酵母菌的基因调控及其在酿酒过程中的应用

酵母菌的基因调控及其在酿酒过程中的应用

酵母菌的基因调控及其在酿酒过程中的应用酵母菌是一类常见的单细胞真菌,在生物学和食品学等领域具有重要的应用价值。

在酿酒过程中,酵母菌的基因调控起着至关重要的作用,它决定了酵母菌的代谢途径、营养需求、分裂周期等。

本文将介绍酵母菌的基因调控机制及其在酿酒过程中的应用。

一、酵母菌基因调控机制酵母菌的基因调控机制主要涉及到基因的转录和翻译过程。

在酵母菌内,基因的转录主要受到转录因子的控制,而转录因子的表达和活性则受到多种因素的影响。

1. 转录因子的表达调控转录因子是一类广泛存在于生物体内的蛋白质,它们具有结合到DNA上的能力,并且可以促进或者抑制基因的转录。

酵母菌内的转录因子可以被调节其表达水平,从而影响到某些基因的表达。

例如,在酿酒过程中,酵母菌需要大量产生酒精以进行发酵。

这个过程中,转录因子STB5被启动,促进了基因ADH1的转录和翻译,从而使得酵母菌能够高效生成酒精。

类似地,酵母菌还可能通过调节其它转录因子的表达来适应不同的环境条件和生长状态。

2. 翻译后调节除了在转录水平上的调控外,酵母菌的基因还可以通过翻译后事件来调节其表达和调节代谢途径。

例如,酵母菌的mRNA可能会被翻译为不同的蛋白质,这些蛋白质在细胞内发挥不同的功能。

此外,酵母菌内还存在一些调节元件,例如microRNA和siRNA 等,在翻译后对基因表达进行调节。

3. 基因组重组酵母菌还具有基因组重组的能力,这个过程有利于适应不同的环境条件。

基因组重组是指染色体内部或者染色体之间的片段交换以及染色体各个区域的移动、删除和复制等过程。

酵母菌基因组重组的频率很高,研究这个过程可以对酵母菌表达调控机制的理解提供帮助。

二、酵母菌在酿酒过程中的应用酿酒是酵母菌的一种重要应用,酵母菌内的基因表达调控起着至关重要的作用。

在酿酒前,一个合适的酵母菌菌株被选择,利用其在发酵过程中产生的酶分解葡萄糖,生产大量的酒精和二氧化碳。

1. 酵母菌的筛选酿酒中使用的酵母菌通常要在制定酵母菌的工艺流程前经过筛选。

实验十四啤酒酵母菌营养缺陷型菌株的筛选

实验十四啤酒酵母菌营养缺陷型菌株的筛选

实验十四啤酒酵母菌营养缺陷型菌株的筛选一、实验原理利用化学诱变剂诱发突变是遗传学研究和育种工作的常用手段。

化学诱变剂按其诱变机理一般可分为三类:(1)通过掺入DNA分子引起突变,(2)通过和DNA直接起化学反应后引起突变,(3)通过一对核苷酸的插入或缺失引起突变。

本实验以烷化剂亚硝基胍(nitrosoguanidine,NTG)为诱变源,它的诱变机理属于第二类。

现在一般认为烷化剂的诱变机理主要是由于对鸟嘌呤N-7位置上的烷化作用(鸟嘌呤其它位置以及其它碱基的许多位置也可能被烷化),然后被烷化的碱基同碱基结构类似物作用机制一样,通过DNA复制,引起碱基错误配对导致碱基转换或颠换造成基因突变。

通常烷化后的碱基(G)偶然与胸腺嘧啶(T)错误配对代替胞嘧啶(C)。

NTG主要诱发GC—AT的转换。

它除有较强的诱变作用外,还能诱发邻近位置基因的并发突变,而且特别容易诱发DNA复制叉附近的基因突变,随着复制叉的移动,它的作用位置也随着移动。

NTG是一种超诱变剂,它的诱发效率可使百分之几十的细菌发生营养缺陷型突变,因此经NTG处理的细菌不必经过青霉素浓缩处理,而只要通过适当的筛选方法就能检出营养缺陷型。

一般讲,一种高效率的诱变剂,只要有一种有效的筛选方法是可以获得任何突变型的。

诱变处理所用的细胞一般为对数期细胞。

化学诱变剂的剂量一般以药物浓度表示。

一定的剂量有一定的杀菌率和诱变率,通过杀菌率和诱变率可帮助我们了解一定剂量的诱变作用。

诱变作用往往与药物处理时间和温度有关。

具有较强诱变作用,较弱杀菌作用的诱变剂(如烷化剂)可采用较低剂量(约50%的杀菌率),反之,紫外线一般采用较高杀菌作用的剂量(如90%—99.9%杀菌率)。

二、实验材料1.啤酒酵母菌单倍体26-4(来自上海酵母厂)。

2.啤酒酵母菌单倍体143—2(来自上海酵母厂)。

三、实验器具和药品1.用具:培养皿(9厘米),三角瓶(150毫升),试管,离心管,吸管(1毫升、5毫升),玻璃涂棒,玻璃珠,丝绒布,圆木柱。

自酿葡萄酒 常用酵母的型号与特性

自酿葡萄酒 常用酵母的型号与特性

酵母型号与特性一、酿造干白葡萄酒的酵母(一)、VL1(1)葡萄品种:用于各种白葡萄品种(2)酿酒特性:酒精发酵过程中生成的乙烯苯酚特别少,尤其遇到光照不足、降雨量多的年份,部分腐烂的葡萄用该菌种是最佳的首选,可限制乙烯苯酚的产生。

(3)风格:该菌种能最大限度地将葡萄含有的天然芳香物质和产地的种植土壤特点在酒中得以还原。

从而使获得的葡萄酒幽雅芬芳,丰满肥硕。

(4)酿酒类型:浓郁高端耐久存干白。

(二)、VL3(1)葡萄品种:霞多丽等。

(2)酿酒特性:该菌种可以显示和优化各种白葡萄酒的芳香潜力。

通过它对葡萄酒中香味母体的作用,其酶促结构使它显示各种特性香味。

(3)风格:具有黄杨和黑醋栗的芽等香味,浓郁丰富,令人喜爱。

(4)酿酒类型:浓郁顶级耐久存干白。

(三)、ST(1)葡萄品种:琼瑶浆等。

(2)酿酒特性:该菌种和SO2的结合潜力低,并且非常灵敏,容易使发酵停止,具有达到15%酒精发酵能力,挥发酸、硫化氢聚集较少。

(3)风格:完美和谐,口感均衡。

(4)酿酒类型:高档甜型葡萄酒。

(四)、BO213(1)葡萄品种:各种白葡萄(2)酿酒特性:适用于低温发酵的甜葡萄酒和白葡萄酒,具有抵抗高酒度的良好特性。

特别是具有再启动停止发酵的巨大潜力。

(3)酿酒类型:停止发酵的再启动。

(五)、CY3079(1)葡萄品种:霞多丽、雷司令、贵人香、白品乐等。

(2)酿酒特性:起酵缓慢、发酵平稳,挥发酸、硫化氢聚集较少,泡沫低、沉淀好,发酵结束后具有陈酿的酒泥香。

(3)风格:新鲜的黄油味、烤面包、蜂蜜、坚果、香子兰和杏仁等香味,陈酿的酒泥香伴有烤面包、榛子和木质对等香气特点。

(4)酿酒类型:浓郁顶级耐久存干白。

(六)、R-HST(1)葡萄品种:雷司令、贵人香。

(2)酿酒特性:发酵力强,在较低温度下迅速启动,既能保留葡萄品种固有的新鲜果香,又能提高结构感,也适合陈酿。

(3)风格:能展露出雷司令、贵人香等品种的典型性香气,果香浓郁、醇和爽口、酒体完整、回味延绵,陈酿后表现亦佳。

第十章 酵母基因工程-08级

第十章  酵母基因工程-08级

如果说大肠杆菌是外源基因最成熟的原 核生物表达系统, 核生物表达系统,则酵母菌是最成熟的真核 生物表达系统。 生物表达系统。
2、酵母菌表达外源基因的优势
全基因组测序,基因表达调控机理比较清楚, ① 全基因组测序,基因表达调控机理比较清楚, 遗传操作简便; 遗传操作简便; ② 具有原核细菌无法比拟的真核蛋白翻译后加工 系统; 系统; 大规模发酵历史悠久、技术成熟、工艺简单、 ③ 大规模发酵历史悠久、技术成熟、工艺简单、 成本低廉; 成本低廉; 能将外源基因表达产物分泌至培养基中; ④ 能将外源基因表达产物分泌至培养基中; 不含有特异性的病毒、不产内毒素,美国FDA FDA认 ⑤ 不含有特异性的病毒、不产内毒素,美国FDA认 定为安全的基因工程受体系统( 定为安全的基因工程受体系统(Generally Recognized As Safe GRAS); ); 酵母菌是最简单的真核模式生物。 ⑥ 酵母菌是最简单的真核模式生物。
选择标记
对应于营养缺陷型受体的野生型基因:HIS4 (组氨醇脱 对应于营养缺陷型受体的野生型基因: 氢酶基因) 精氨酸合成酶基因); 氢酶基因);ARG4(精氨酸合成酶基因 ;TRP1(色氨酸 精氨酸合成酶基因 色氨酸 合成酶基因); 尿嘧啶合成酶基因) 合成酶基因 ;URA3 (尿嘧啶合成酶基因)。 抗性选择标记:抗生素G418抗性基因和Zeocin G418抗性基因和Zeocin抗性基因 抗性选择标记:抗生素G418抗性基因和Zeocin抗性基因
2、酿酒酵母的附加型载体
pUC质粒的复制起点和amp pUC质粒的复制起点和ampr抗性基因 质粒的复制起点和 酵母菌的2μm质粒的复制区 酵母菌的2μm质粒的复制区 酵母转化的选择元件: 酵母转化的选择元件:LEU2(亮氨酸合成酶基因); (亮氨酸合成酶基因)

酿酒酵母培养基配方

酿酒酵母培养基配方

种子培养基(g/L ) : 无氨基酵母氮源(YNB, Difco) 15, 尿嘧啶0. 04, 腺嘌岭0. 04, 色氨酸0. 02,组氨酸0.02, 精氨酸0.02, 蛋氨酸0.02, 苏氨酸0.03。

常规发酵培养基(g/L ) : 大豆蛋白胨(Difco )40, 酵母粉(Difco) 40, 葡萄糖20。

合成培养基由葡萄糖(60 g/L )、氨基酸、微量矿物质和维生素构成。

氨基酸成分与种子培养基相同。

微量矿物质和维生素的构成如下:微量矿物质(g/L ) : (NH4)2SO4 20, KH2PO4 0.3, M gSO4·7H2O 0.5, EDTA 1.5, ZnSO4·7H2O0.0045, CoCl2 · 6H2O 0.003, M nCl2 · 4H2O 0.001, CuSO4 ·5H2O 0.003, CaCl2 ·2H2O 0.0045, FeSO4·7H2O 0.003, H3BO3 0.001, KI 0.001(121 °C灭菌30 m in)。

维生素(mg/L ) : 生物素0.5, 泛酸0.1, 尼克酸1.0, 肌醇25.0, 硫胺素1.0, 吡哆酸1.0, 对氨基苯甲酸20, 叶酸1, 胆碱15, 核黄素4 (过滤灭菌)。

合成培养基(g/L ):1,(NH4)2SO4 25,KH2PO4 25,MgSO47.3,CaSO4.2H2O 12,YNB 13.4,His 1,Leu 2,Trp 23,PTM1 4ml4,甘油3050%甘油补料(g/L ):1,甘油5002,12ml PTM13,His 1,Leu 2,Trp 220%半乳糖诱导(g/L ):1,半乳糖2002,6ml PTM13,His 1,Leu 2,Trp 2。

常用酿酒酵母菌株基因型教程文件

常用酿酒酵母菌株基因型教程文件

常用酿酒酵母菌株基因型Commonly used strains▪▪van Dijken et al. (2000) Enzyme Microb Technol 26:706-714 - compares various characteristics of commonly used lab strains▪Winzeler et al. (2003) Genetics 163:79-89 - uses SFP (single-feature polymorphisms) analysis to study genetic identity between common lab strainsS288CGenotype:MATαSUC2 gal2 mal mel flo1 flo8-1 hap1 ho bio1 bio6Notes: Strain used in the systematic sequencing project, the sequence stored in SGD. S288C does not form pseudohyphae. In addition, since it has a mutated copy of HAP1, it is not a good strain for mitochondrial studies. It has an allelic variant of MIP1 which increases petite frequency. S288C strains are gal2- and they do not use galactose anaerobically.The S288C genome was recently resequenced at the Sanger Institute.References:Mortimer and Johnston (1986) Genetics 113:35-43.BY4743Genotype:MAT a/αhis3Δ1/his3Δ1 leu2Δ0/leu2Δ0 LYS2/lys2Δ0 met15Δ0/MET15 ura3Δ0/ura3Δ0Notes: Strain used in the systematic deletion project, generated from a cross between BY4741 and BY4742, which are derived from S288C. As S288c, these strains have an allelic variant of MIP1 which increases petite frequency. See Brachmann et al. reference for details.References:Brachmann et al. (1998) Yeast 14:115-32.FY4Genotype:MAT aNotes: Derived from S288C.References:Winston et al. (1995) Yeast 11:53-55.FY1679Genotype:MAT a/αura3-52/ura3-52 trp1Δ63/TRP1 leu2Δ1/LEU2 his3Δ200/HIS3 GAL2/GALNotes: Isogenic to S288C; used in the systematic sequencing project, the sequence stored in SGD.References:Winston et al. (1995) Yeast 11:53-55.AB972Genotype:MATα X2180-1B trp10 [rho 0]Notes: Isogenic to S288C; used in the systematic sequencing project, the sequence stored in SGD. AB972 is an ethidium bromide-induced rho- derivative of the strain X2180-1B-trp1.References:Olson MV et al. (1986) Proc. Natl. Acad. Sci. USA 83:7826-7830.A364AGenotype:MAT a ade1 ade2 ura1 his7 lys2 tyr1 gal1 SUC mal cup BIONotes: Used in the systematic sequencing project, the sequence stored in SGD.References:Hartwell (1967) J. Bacteriol. 93:1662-1670.XJ24-24aGenotype:MAT a ho HMa HMα ade6 arg4-17 trp1-1 tyr7-1 MAL2Notes: Derived from, but not isogenic to, S288CReferences:Strathern et al. (1979) Cell 18:309-319DC5Genotype:MAT a leu2-3,112 his3-11,15 can1-11Notes: Isogenic to S288C; used in the systematic sequencing project, the sequence stored in SGD.References:Broach et al. (1979) Gene 8:121-133X2180-1AGenotype:MAT a SUC2 mal mel gal2 CUP1Notes:S288c spontaneously diploidized to give rise to X2180. The haploid segregants X2180-1a and X2180-1b were obtained from sporulated X2180YNN216Genotype:MAT a/αura3-52/ura3-52 lys2-801amber/lys2-801amber ade2-101ochre/ade2-101ochreNotes: Congenic to S288C (see Sikorski and Hieter). Used to derive YSS and CY strains (see Sobel and Wolin).YPH499Genotype:MAT a ura3-52 lys2-801_amber ade2-101_ochre trp1-Δ63 his3-Δ200 leu2-Δ1Notes: Contains nonrevertible (deletion) auxotrophic mutations that can be used for selection of vectors. Note that trp1-Δ63, unlike trp1-Δ1, does not delete adjacent GAL3 UAS sequence and retains homologyto TRP1 selectable marker.gal2-, does not use galactose anaerobically. Derived from the diploid strain YNN216 (Johnston and Davis 1984; original source: M. Carlson, Columbia University), which is congenic with S288C.YPH500Genotype:MATαura3-52 lys2-801_amber ade2-101_ochre trp1-Δ63 his3-Δ200 leu2-Δ1Notes:MATα strain isogenic to YPH499 except at mating type locus. Derived from the diploid strain YNN216 (Johnston and Davis 1984; original source: M. Carlson, Columbia University), which is congenic with S288C.YPH501Genotype:MAT a/MATαura3-52/ura3-52 lys2-801_amber/lys2-801_amber ade2-101_ochre/ade2-101_ochre trp1-Δ63/trp1-Δ63 his3-Δ200/his3-Δ200 leu2-Δ1/leu2-Δ1Notes:a/α diploid isogenic to YPH499 and YPH500. Derived from the diploid strain YNN216 (Johnston and Davis 1984; original source: M. Carlson, Columbia University), which is congenic with S288C.Sigma 1278BNotes: Used in pseudohyphal growth studies. Detailed notes about the sigma strains have been kindly provided by Cora Styles.Granek and Magwene, PLoS Genet. 2010 Jan 22;6(1):e1000823, established that certain lineages of theSigma1278B background contain a nonsense mutation in RIM15, a G-to-T transversion at position 1216 that converts a Gly codon to an opal stop codon. This rim15 mutation interacts epistatically with mutations in certain other genes to affect colony morphology.Annotation of the Sigma1278b genome and information about the systematic deletion collection can be found here. SK1Genotype:MAT a/α HO gal2 cup S can1R BIONotes: Commonly used for studying sporulation or meiosis. Canavanine-resistant derivative.The SK1 genome was sequenced at the Sanger Institute.References:Kane SM and Roth J. (1974) Bacteriol. 118: 8-14CEN.PK (aka CEN.PK2)Genotype:MAT a/α ura3-52/ura3-52 trp1-289/trp1-289 leu2-3_112/leu2-3_112 his3 Δ1/his3 Δ1 MAL2-8C/MAL2-8C SUC2/SUC2Notes: CEN.PK possesses a mutation in CYR1 (A5627T corresponding to a K1876M substitution near the end of the catalytic domain in adenylate cyclase which eliminates glucose- and acidification-induced cAMP signalling and delays glucose-induced loss of stress resistance (Vanhalewyn et al., 1999; Dumortier et al., 2000).References:van Dijken et al. (2000) Enzyme Microb Technol 26:706-714W303Genotype:MAT a/MATα {leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15} [phi+]Notes: W303 also contains a bud4 mutation that causes haploids to bud with a mixture of axial and bipolar budding patterns. In addition, the original W303 strain contains the rad5-535 allele. As S288c, W303 has an allelic variantof MIP1 which increases petite frequency.The W303 genome was sequenced at the Sanger Institute.References: W303 constructed by Rodney Rothstein (see detailed notes from RR and Stephan Bartsch).bud4 info: Voth et al. (2005) Eukaryotic Cell, 4:1018-28.rad5-535 info: Fan et al. (1996) Genetics 142:749W303-1AGenotype:MAT a {leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15}Notes: W303-1A possesses a ybp1-1 mutation (I7L, F328V, K343E, N571D) which abolishes Ybp1p function, increasing sensitivity to oxidative stress.References: W303 constructed by Rodney Rothstein (see detailed notes from RR and Stephan Bartsch).ybp1-1 info: Veal et al. (2003) J. Biol. Chem. 278:30896-904.W303-1BGenotype:MATα {leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15}References: W303 constructed by Rodney Rothstein (see detailed notes from RR and Stephan Bartsch).W303-K6001Genotype:MAT a; {ade2-1, trp1-1, can1-100, leu2-3,112, his3-11,15, GAL, psi+, ho::HO::CDC6 (at HO), cdc6::hisG, ura3::URA3 GAL-ubiR-CDC6 (at URA3)}References: K6001 was developed by Bobola et al in Kim Nasmyth's lab (PMID: 8625408), and has become a common model in yeast aging research (PMID: 15489200). Its genome has been sequenced by Timmermann et al (PMID: 20729566)D273-10BGenotype:MATαmalNotes: Normal cytochrome content and respiration; low frequency of rho-. This strain and its auxotrophic derivatives were used in numerious laboratories for mitochondrial and related studies and for mutant screens. Good respirer that's relatively resistant to glucose repression.References:Sherman, F. (1963) Genetics 48:375-385.FL100Genotype:MAT aReferences:Lacroute, F. (1968) J. Bacteriol. 95:824-832.Sources: ATCC: 28383SEY6210/SEY6211Genotype:MAT a/MATαleu2-3,112/leu2-3,112 ura3-52/ura3-52 his3-Δ200/his3-Δ200 trp1-Δ901/trp1-Δ901ade2/ADE2 suc2-Δ9/suc2-Δ9 GAL/GAL LYS2/lys2-801Notes: SEY6210/SEY6211, also known as SEY6210.5, was constructed by Scott Emr and has been used in studies of autophagy, protein sorting etc. It is the product of crossing with strains from 5 different labs (Gerry Fink, Ron Davis, David Botstein, Fred Sherman, Randy Schekman). It has several selectable markers, good growth properties and good sporulation.References:Robinson et al. (1988) Mol Cell Biol 8(11):4936-48SEY6210Genotype:MATαleu2-3,112 ura3-52 his3-Δ200 trp1-Δ901 suc2-Δ9 lys2-801; GALNotes: SEY6210 is a MATalpha haploid constructed by Scott Emr and has been used in studies of autophagy, protein sorting etc. It is the product of crossing with strains from 5 different labs (Gerry Fink, Ron Davis, David Botstein, Fred Sherman, Randy Schekman). It has several selectable markers and good growth properties. References:Robinson et al. (1988) Mol Cell Biol 8(11):4936-48SEY6211Genotype:MAT a leu2-3,112 ura3-52 his3-Δ200 trp1-Δ901 ade2-101 suc2-Δ9; GALNotes: SEY6211 is a MATa haploid constructed by Scott Emr and has been used in studies of autophagy, protein sorting etc. It is the product of crossing with strains from 5 different labs (Gerry Fink, Ron Davis, David Botstein, Fred Sherman, Randy Schekman). It has several selectable markers and good growth properties.References:Robinson et al. (1988) Mol Cell Biol 8(11):4936-48JK9-3dThere are a, alpha and a/alpha diploids of JK9-3d with the following genotypes:Genotypes: JK9-3da MAT a leu2-3,112 ura3-52 rme1 trp1 his4JK9-3dα has the same genotype as JK9-3da with the exception of the MAT locusJK9-3da/α is homozygous for all markers except mating typeNotes: JK9-3d was constructed by Jeanette Kunz while in Mike Hall's lab. She made the original strain while Joe Heitman isolated isogenic strains of opposite mating type and derived the a/alpha isogenic diploid by mating type switching. It has in its background S288c, a strain from the Oshima lab, and a strain from the Herskowitz lab. It was chosen because of its robust growth and sporulation, as well as good growth on galactose (GAL+) (so that genes under control of the galactose promoter could be induced). It may also have a SUP mutation that allows translation through premature STOP codons and therefore produces functional alleles with many point mutations. References:Heitman et al. (1991a) Science 253(5022):905-9 and Heitman et al. (1991b) Proc Natl Acad Sci U S A 88(5):1948-52RM11-1aGenotype:MAT a leu2Δ ura3Δ ho::KanNotes: RM11-1a is a haploid derivative of Bb32(3), a natural isolate collected by Robert Mortimer from a California vineyard, as in Mortimer et al., 1994. It has high spore viability (80–90%) and has been extensively characterized phenotypically under a wide range of conditions. It has a significantly longer life span than typical lab yeast strains and accumulates age-associated abnormalities at a lower rate. It displays approximately 0.5–1% sequence divergence relative to S288c. More information is available at the Broad Institute website.References:Brem et al. (2002) Science 296(5568):752-5Y55Genotype:MAT a /MAT alpha HO/HONotes: Y55 is a prototrophic, homothallic diploid strain that was originally isolated by Dennis Winge. Many auxotrophic mutant derivatives have been created by John McCusker by using ethidium bromide treatment to eliminate non-auxotrophs. Y55 background strains have been used to study the timing of meiotic recombination (Borts et al. 1984); to isolate almost all the subunits of the proteasome (McCusker and Haber 1988a, 1988b); to get mutations in PMA1 and related genes (McCusker 1986); and to do meiotic mapping and interference experiments (Malkova et al. 2004).。

第九章酵母菌遗传

第九章酵母菌遗传

(2) ARS-结合蛋白 ORC:由6种分子质量分别为120kD、72kD、62kD、 57kD、53kD和50kD的蛋白质以复合物的形式组成,这种蛋 白质复合体叫做起始识别复合物(origin recognition complex,ORC)。相当于Dna A和 λ的O蛋白,与 A/B1 结合。 Cdc6:相当于DnaC,及λ的P蛋白,使Mcm蛋白结合到复合 体上。此对在Origin上起始复制是必须的。
端粒是真核生物线性染色体两端的特殊DNA-蛋白质复合体 结构,这种复合体结构是由 DNA重复序列和与之相结合的蛋 白质分子构成的。
在大多数生物中,端粒DNA只是由几个碱基组成的DNA重 复单位通过串联重复而形成,长度从20bp到几个kb不等。
酿酒酵母端粒DNA的长度约为300bp,其DNA重复单位为 5′C1-3A 3′G1-3T
第九章 酵母菌遗传
酵母菌属于真核微生物,其细胞内除了具有与原核生物不同 的由核膜包围的核以外,还像其他真核细胞一样,含有如下 成分:内质网、高尔基体、线粒体、液泡和过氧化物体,它 是最简单的真核生物。
酵母菌同时又具有原核生物的某些特征,如它们可以像细菌 一样在平板上迅速生长形成单菌落,也可像细菌一样进行分 裂,重要的是酿酒酵母(Saccharomyces cerevtsiae)的 基因组只是大肠杆菌的2.6倍,因此酿酒酵母已成为目前在 分子水平上研究真核生物的重要材料。
1.着丝粒(centromere) 着丝粒是真核细胞染色体DNA上的一段特殊序列。在有 丝分裂和减数分裂时,纺锤丝 (着丝粒结合蛋白)与着丝粒 结合,将染色体拉向细胞的两极 。
着丝粒有两种主要类型:
第一种:短着丝粒 (约200bp),又叫点着丝粒(point centromere),酿酒酵母的着丝粒就属于这种类型。 第二种:区域着丝粒(regional centromere),其着丝粒序 列较长(从 40kb到几个Mb),含有很多重复序列, 真菌(如脉孢菌)、果蝇、哺乳动物和人的着丝粒都 具有这种结构特征。 所有真核生物的着丝粒功能都相同,但其DNA却具有种的 特异性。

12-198 酿酒酵母EBY100菌种使用手册v1点0

12-198 酿酒酵母EBY100菌种使用手册v1点0

天净沙系列CAT#:12-198低温运输和-80℃保存酿酒酵母EBY100 Saccharomyces cerevisiae EBY100使用手册V1.0 北京天恩泽基因科技有限公司北京市海淀区上地信息路26号北京市留学人员海淀创业园中关村创业大厦506 网址:;电话:400-6765278;电邮:order@产品及特点EBY100是酿酒酵母菌株,属于真核细胞,具有氨苄和卡那霉素抗性。

质粒转化条件为电转化,应用于酵母表面展示。

此菌株必须生长在营养丰富的培养基中,例如YPD培养基,无法在minimal media中生长。

在GAL1启动子的作用下,此菌株能够表达AGA1基因。

EBY100的配套载体是pYD1载体。

此菌株的基因型如下:MATα ura3-52 trp1 leu2∆1 his3∆200pep4::HIS3 prb1∆1.6R can1 GAL (pIU211: URA3)。

规格及成分成份编号热封袋包装酿酒酵母EBY100 12-198 1 mL使用手册1份运输及保存低温运输,-80℃保存,有效期一年。

自备试剂YPD培养基、TE缓冲液(pH 7.5)、乙酸锂、DTT、山梨醇、超纯水等使用方法本手册提供一个质粒转化酵母的电转化方法,供参考。

一、感受态细胞制备1.挑一环酵母菌接种于5 mL YPD培养基中,30℃、250-300 rpm培养过夜;2.取适量的过夜培养液分别接种于两瓶50 mL YPD培养基中,30℃、250-300 rpm培养约16-18小时(OD:1.3-1.5);3.于4℃,4000 g离心收集菌体,用25 mL冰浴的无菌水洗涤一次后,细胞用10 mL冰浴的无菌水重悬,可换成较小的离心管;4.加入1 mL的10×TE缓冲液(pH 7.5),摇晃均匀,再加入1 mL 10×乙酸锂,旋转摇匀,于30℃轻轻摇动45分钟;5.再加入0.4 mL 1 mol/L DTT,并同时旋转摇动,于30℃轻轻摇动15分钟;6.于4℃,4000-6000 g离心,弃上清(用枪吸),再用25 mL冰浴的无菌水洗涤后4℃离心,收集菌体;7.用2.5 mL冰冷的1 mol/L山梨醇重悬细胞,离心收集菌体,弃上清(用枪吸);8.每管用100 uL 1 mol/L的山梨醇溶解,分装于EP管中(80 uL/管),于-70℃冰箱保存。

酿酒酵母培养基配方和诱导表达方法

酿酒酵母培养基配方和诱导表达方法

第一部分酿酒酵母培养基和培养平板配方所有的液体培养基需用磷酸缓冲液配制:Na2HPO4-7H2O, L, NaH2PO4-H2O L. 称量好所需组分,先把氨基酸、氮源等添加到水中,再添加糖最后添加10X的磷酸缓冲液。

液体培养基的PH需在左右。

液体选择培养基和丰富培养基可以选择过滤除菌,但是琼脂平板培养基必须高压灭菌丰富非选择培养基2 x (YEPD or) YPD = Yeast Extract Peptone Dextrose20g酵母粉yeast extract(OXOID LP0021)40 g 蛋白胨peptone (OXOID LP0042)40g葡萄糖dextrose**(国药14434-43-7)把除葡糖糖之外的所有组分加入500ml水中溶解,定容至900ml,高压灭菌配置20%的葡萄糖过滤除菌,添加100ml。

室温保存1-2个月。

**也可以所有组分一起灭菌,但是可能会发生焦化作用,然而焦化作用不会引起任何问题。

生长选择合成培养基(Selective Growth Synthetic Media)2x (SD – CAA)1)用于EBY100菌株配套pYD 系列载体,或者二配体酵母配套pPNL20 和pPNL30 载体10 g/L 酪蛋白水解物casamino acids (-ade, -ura, -trp)(库存货号:Fluka A2427-250G)40g/L 葡萄糖dextrose(国药14434-43-7)14g/L YNB Yeast Nitrogen Base with ammonium sulfate w/out amino acids (SIGMA Y0626)g/L Na2HPO4(国药10039-32-4)g/L NaH2PO4(国药14431-43-7)442)用于YVH10 with pPNL30系列载体10 g/L 酪蛋白水解物(casamino acids (-ade, -ura, -trp)40g/L 葡萄糖dextrose14g/L YNB (Yeast Nitrogen Base with ammonium sulfate w/out amino acids )40mg/L色氨酸(tryptophan)g/L Na2HPO4g/L NaH2PO43)用于JAR with pPNL20系列载体10 g/L 酪蛋白水解物(casamino acids (-ade, -ura, -trp) (BD Bacto #223050)40g/L 葡萄糖dextrose14g/L YNB (Yeast Nitrogen Base with ammonium sulfate w/out amino acids )(BD Difco 233520)20mg/L Uracilg/L Na2HPO4g/L NaH2PO4Selective scFv (or Fab or ligand) Induction Media抗体scFv (or Fab or 配体)的选择诱导培养基2x (SG-CAA)10 g/L酪蛋白水解物casamino acids (-ade, -ura, -trp) (BD Bacto #223050)2 g/L半乳糖galactose2 g/L棉子糖raffinoseg/L葡萄糖glucose14g/L YNB Yeast Nitrogen Base with ammonium sulfate w/out amino acids (BD Difco 233520)g/L Na2HPO4g/L NaH2PO4YEPGR or YPGR = Y east E xtract P eptone Galactose/Raffinose10g 酵母粉yeast extract20 g 蛋白胨peptone2%半乳糖galactose2%葡萄糖raffinose14g/L YNB Yeast Nitrogen Base with ammonium sulfate w/out amino acids (BD Difco 233520)g/L Na2HPO4g/L NaH2PO4尿嘧啶和色氨酸母液的配置色氨酸配制成4mg/ml的100X的母液,过滤除菌尿嘧啶配制成2mg/ml的100X的母液,过滤除菌酵母培养基的配置和选择培养基的种类:应用最广泛的酿酒酵母培养基是YPD培养基(酵母粉、蛋白胨、葡萄糖),单克隆在YPD上的生长通常受尿嘧啶、精氨酸和色氨酸的限制。

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常用酿酒酵母菌株基因型Commonly used strains▪▪van Dijken et al. (2000) Enzyme Microb Technol 26:706-714 - compares various characteristics of commonly used lab strains▪Winzeler et al. (2003) Genetics 163:79-89 - uses SFP (single-feature polymorphisms) analysis to study genetic identity between common lab strainsS288CGenotype:MATαSUC2 gal2 mal mel flo1 flo8-1 hap1 ho bio1 bio6Notes: Strain used in the systematic sequencing project, the sequence stored in SGD. S288C does not form pseudohyphae. In addition, since it has a mutated copy of HAP1, it is not a good strain for mitochondrial studies. It has an allelic variant of MIP1 which increases petite frequency. S288C strains are gal2- and they do not use galactose anaerobically.The S288C genome was recently resequenced at the Sanger Institute.References:Mortimer and Johnston (1986) Genetics 113:35-43.BY4743Genotype:MAT a/αhis3Δ1/his3Δ1 leu2Δ0/leu2Δ0 LYS2/lys2Δ0 met15Δ0/MET15 ura3Δ0/ura3Δ0Notes: Strain used in the systematic deletion project, generated from a cross between BY4741 and BY4742, which are derived from S288C. As S288c, these strains have an allelic variant of MIP1 which increases petite frequency. See Brachmann et al. reference for details.References:Brachmann et al. (1998) Yeast 14:115-32.FY4Genotype:MAT aNotes: Derived from S288C.References:Winston et al. (1995) Yeast 11:53-55.FY1679Genotype:MAT a/αura3-52/ura3-52 trp1Δ63/TRP1 leu2Δ1/LEU2 his3Δ200/HIS3 GAL2/GALNotes: Isogenic to S288C; used in the systematic sequencing project, the sequence stored in SGD.References:Winston et al. (1995) Yeast 11:53-55.AB972Genotype:MATα X2180-1B trp10 [rho 0]Notes: Isogenic to S288C; used in the systematic sequencing project, the sequence stored in SGD. AB972 is an ethidium bromide-induced rho- derivative of the strain X2180-1B-trp1.References:Olson MV et al. (1986) Proc. Natl. Acad. Sci. USA 83:7826-7830.A364AGenotype:MAT a ade1 ade2 ura1 his7 lys2 tyr1 gal1 SUC mal cup BIONotes: Used in the systematic sequencing project, the sequence stored in SGD.References:Hartwell (1967) J. Bacteriol. 93:1662-1670.XJ24-24aGenotype:MAT a ho HMa HMα ade6 arg4-17 trp1-1 tyr7-1 MAL2Notes: Derived from, but not isogenic to, S288CReferences:Strathern et al. (1979) Cell 18:309-319DC5Genotype:MAT a leu2-3,112 his3-11,15 can1-11Notes: Isogenic to S288C; used in the systematic sequencing project, the sequence stored in SGD.References:Broach et al. (1979) Gene 8:121-133X2180-1AGenotype:MAT a SUC2 mal mel gal2 CUP1Notes:S288c spontaneously diploidized to give rise to X2180. The haploid segregants X2180-1a and X2180-1b were obtained from sporulated X2180YNN216Genotype:MAT a/αura3-52/ura3-52 lys2-801amber/lys2-801amber ade2-101ochre/ade2-101ochreNotes: Congenic to S288C (see Sikorski and Hieter). Used to derive YSS and CY strains (see Sobel and Wolin).YPH499Genotype:MAT a ura3-52 lys2-801_amber ade2-101_ochre trp1-Δ63 his3-Δ200 leu2-Δ1Notes: Contains nonrevertible (deletion) auxotrophic mutations that can be used for selection of vectors. Note that trp1-Δ63, unlike trp1-Δ1, does not delete adjacent GAL3 UAS sequence and retains homologyto TRP1 selectable marker.gal2-, does not use galactose anaerobically. Derived from the diploid strain YNN216 (Johnston and Davis 1984; original source: M. Carlson, Columbia University), which is congenic with S288C.YPH500Genotype:MATαura3-52 lys2-801_amber ade2-101_ochre trp1-Δ63 his3-Δ200 leu2-Δ1Notes:MATα strain isogenic to YPH499 except at mating type locus. Derived from the diploid strain YNN216 (Johnston and Davis 1984; original source: M. Carlson, Columbia University), which is congenic with S288C.YPH501Genotype:MAT a/MATαura3-52/ura3-52 lys2-801_amber/lys2-801_amber ade2-101_ochre/ade2-101_ochre trp1-Δ63/trp1-Δ63 his3-Δ200/his3-Δ200 leu2-Δ1/leu2-Δ1Notes:a/α diploid isogenic to YPH499 and YPH500. Derived from the diploid strain YNN216 (Johnston and Davis 1984; original source: M. Carlson, Columbia University), which is congenic with S288C.Sigma 1278BNotes: Used in pseudohyphal growth studies. Detailed notes about the sigma strains have been kindly provided by Cora Styles.Granek and Magwene, PLoS Genet. 2010 Jan 22;6(1):e1000823, established that certain lineages of theSigma1278B background contain a nonsense mutation in RIM15, a G-to-T transversion at position 1216 that converts a Gly codon to an opal stop codon. This rim15 mutation interacts epistatically with mutations in certain other genes to affect colony morphology.Annotation of the Sigma1278b genome and information about the systematic deletion collection can be found here. SK1Genotype:MAT a/α HO gal2 cup S can1R BIONotes: Commonly used for studying sporulation or meiosis. Canavanine-resistant derivative.The SK1 genome was sequenced at the Sanger Institute.References:Kane SM and Roth J. (1974) Bacteriol. 118: 8-14CEN.PK (aka CEN.PK2)Genotype:MAT a/α ura3-52/ura3-52 trp1-289/trp1-289 leu2-3_112/leu2-3_112 his3 Δ1/his3 Δ1 MAL2-8C/MAL2-8C SUC2/SUC2Notes: CEN.PK possesses a mutation in CYR1 (A5627T corresponding to a K1876M substitution near the end of the catalytic domain in adenylate cyclase which eliminates glucose- and acidification-induced cAMP signalling and delays glucose-induced loss of stress resistance (Vanhalewyn et al., 1999; Dumortier et al., 2000).References:van Dijken et al. (2000) Enzyme Microb Technol 26:706-714W303Genotype:MAT a/MATα {leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15} [phi+]Notes: W303 also contains a bud4 mutation that causes haploids to bud with a mixture of axial and bipolar budding patterns. In addition, the original W303 strain contains the rad5-535 allele. As S288c, W303 has an allelic variantof MIP1 which increases petite frequency.The W303 genome was sequenced at the Sanger Institute.References: W303 constructed by Rodney Rothstein (see detailed notes from RR and Stephan Bartsch).bud4 info: Voth et al. (2005) Eukaryotic Cell, 4:1018-28.rad5-535 info: Fan et al. (1996) Genetics 142:749W303-1AGenotype:MAT a {leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15}Notes: W303-1A possesses a ybp1-1 mutation (I7L, F328V, K343E, N571D) which abolishes Ybp1p function, increasing sensitivity to oxidative stress.References: W303 constructed by Rodney Rothstein (see detailed notes from RR and Stephan Bartsch).ybp1-1 info: Veal et al. (2003) J. Biol. Chem. 278:30896-904.W303-1BGenotype:MATα {leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15}References: W303 constructed by Rodney Rothstein (see detailed notes from RR and Stephan Bartsch).W303-K6001Genotype:MAT a; {ade2-1, trp1-1, can1-100, leu2-3,112, his3-11,15, GAL, psi+, ho::HO::CDC6 (at HO), cdc6::hisG, ura3::URA3 GAL-ubiR-CDC6 (at URA3)}References: K6001 was developed by Bobola et al in Kim Nasmyth's lab (PMID: 8625408), and has become a common model in yeast aging research (PMID: 15489200). Its genome has been sequenced by Timmermann et al (PMID: 20729566)D273-10BGenotype:MATαmalNotes: Normal cytochrome content and respiration; low frequency of rho-. This strain and its auxotrophic derivatives were used in numerious laboratories for mitochondrial and related studies and for mutant screens. Good respirer that's relatively resistant to glucose repression.References:Sherman, F. (1963) Genetics 48:375-385.FL100Genotype:MAT aReferences:Lacroute, F. (1968) J. Bacteriol. 95:824-832.Sources: ATCC: 28383SEY6210/SEY6211Genotype:MAT a/MATαleu2-3,112/leu2-3,112 ura3-52/ura3-52 his3-Δ200/his3-Δ200 trp1-Δ901/trp1-Δ901ade2/ADE2 suc2-Δ9/suc2-Δ9 GAL/GAL LYS2/lys2-801Notes: SEY6210/SEY6211, also known as SEY6210.5, was constructed by Scott Emr and has been used in studies of autophagy, protein sorting etc. It is the product of crossing with strains from 5 different labs (Gerry Fink, Ron Davis, David Botstein, Fred Sherman, Randy Schekman). It has several selectable markers, good growth properties and good sporulation.References:Robinson et al. (1988) Mol Cell Biol 8(11):4936-48SEY6210Genotype:MATαleu2-3,112 ura3-52 his3-Δ200 trp1-Δ901 suc2-Δ9 lys2-801; GALNotes: SEY6210 is a MATalpha haploid constructed by Scott Emr and has been used in studies of autophagy, protein sorting etc. It is the product of crossing with strains from 5 different labs (Gerry Fink, Ron Davis, David Botstein, Fred Sherman, Randy Schekman). It has several selectable markers and good growth properties. References:Robinson et al. (1988) Mol Cell Biol 8(11):4936-48SEY6211Genotype:MAT a leu2-3,112 ura3-52 his3-Δ200 trp1-Δ901 ade2-101 suc2-Δ9; GALNotes: SEY6211 is a MATa haploid constructed by Scott Emr and has been used in studies of autophagy, protein sorting etc. It is the product of crossing with strains from 5 different labs (Gerry Fink, Ron Davis, David Botstein, Fred Sherman, Randy Schekman). It has several selectable markers and good growth properties.References:Robinson et al. (1988) Mol Cell Biol 8(11):4936-48JK9-3dThere are a, alpha and a/alpha diploids of JK9-3d with the following genotypes:Genotypes: JK9-3da MAT a leu2-3,112 ura3-52 rme1 trp1 his4JK9-3dα has the same genotype as JK9-3da with the exception of the MAT locusJK9-3da/α is homozygous for all markers except mating typeNotes: JK9-3d was constructed by Jeanette Kunz while in Mike Hall's lab. She made the original strain while Joe Heitman isolated isogenic strains of opposite mating type and derived the a/alpha isogenic diploid by mating type switching. It has in its background S288c, a strain from the Oshima lab, and a strain from the Herskowitz lab. It was chosen because of its robust growth and sporulation, as well as good growth on galactose (GAL+) (so that genes under control of the galactose promoter could be induced). It may also have a SUP mutation that allows translation through premature STOP codons and therefore produces functional alleles with many point mutations. References:Heitman et al. (1991a) Science 253(5022):905-9 and Heitman et al. (1991b) Proc Natl Acad Sci U S A 88(5):1948-52RM11-1aGenotype:MAT a leu2Δ ura3Δ ho::KanNotes: RM11-1a is a haploid derivative of Bb32(3), a natural isolate collected by Robert Mortimer from a California vineyard, as in Mortimer et al., 1994. It has high spore viability (80–90%) and has been extensively characterized phenotypically under a wide range of conditions. It has a significantly longer life span than typical lab yeast strains and accumulates age-associated abnormalities at a lower rate. It displays approximately 0.5–1% sequence divergence relative to S288c. More information is available at the Broad Institute website.References:Brem et al. (2002) Science 296(5568):752-5Y55Genotype:MAT a /MAT alpha HO/HONotes: Y55 is a prototrophic, homothallic diploid strain that was originally isolated by Dennis Winge. Many auxotrophic mutant derivatives have been created by John McCusker by using ethidium bromide treatment to eliminate non-auxotrophs. Y55 background strains have been used to study the timing of meiotic recombination (Borts et al. 1984); to isolate almost all the subunits of the proteasome (McCusker and Haber 1988a, 1988b); to get mutations in PMA1 and related genes (McCusker 1986); and to do meiotic mapping and interference experiments (Malkova et al. 2004).。

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