在我们有了质粒、了解了细胞转化的方法之后,就应该开始考虑选择哪种感受态细胞进行转化。市面上的感受态细胞种类相当多,说有百余种绝对不是夸张,到底选哪一个是由你要进行的实验来决定的,不同基因型的大肠菌株自然是可以满足不同的实验目的。小编在本文简单总结常用的那几种基因型(Genotype)的大肠杆菌细胞,以及他们能满足的实验目的。至于在具体实验中应该选择哪一种基因型的细胞,小编觉得最好的方法是和导师/师兄/师姐讨论,问问他们曾经用的是什么细胞以及用的效果怎么样,再结合自己的实验目的选出最合适的细胞种类。
E.coli 的历史
1885年,Dr. Theodor Escherich发现了一种长条状的细菌,从那时起这种gram-negative 细菌就被命名为Escherichia coli。 E.coli主要出现在动物肠道中,自然界中也有许多致病性极强的E.coli。大部分市面上可以买到的大肠杆菌感受态细胞来自两种单独发现的E.coli:K-12 Strain和B Strain。K-12是在1920年从一个病人身上分离出来的,后来经改造成为大肠细胞系MG1655,也就是现在市面上DH5alpha 和 DH10b的前身(a.k.a. TOP10);B的发现历史就比较曲折了,直到1942年才有正式E.coli B strain这个命名,所以原代细胞是啥时候发现的,小编也不知道,只知道现在市面上常见的BL21就是BStrain的后代细胞。
常见的感受态细胞基因型
现在市面上的大肠细胞系都是为了特定的目的而构建的,比如:高速生长/分裂,高通量克隆,普通克隆等等等等。有许多变异在各种细胞系中都有出现,特别是一些能够提高质粒产量和DNA质量的变异,更是被广泛采用。
描述:对非特异性限制酶(Non-specific Endonuclease I)进行基因敲除(Knock-out),以减少限制酶的活性,进而降低对外源DNA的剪切。
适用方向:提高外源质粒的数量和质量。
描述:hsdR位点变异,减少细胞对未甲基化的位点的剪切。
适用方向:提高某些PCR产物的转化效率。
描述:消除甲基化对腺嘌呤(Adenine)和胞嘧啶(Cytosine)。
适用方向:帮助某些外源质粒复制,比如携带易受甲基化影响的酶切位点的外源质粒。
描述:阻止细胞识别从其他组织来的甲基化序列为异物。
适用方向:基因组DNA或者甲基化cDNA的克隆。
描述:降低DNA重组(recombination)的概率。
方向:提高质粒在细胞内的稳定性。
描述:Exonuclease V的变异,使DNA重组的概率降低。
适用方向:提高质粒在细胞内的稳定性。
描述:去除某些调控基因,使得用于脱氧核糖生成的基因得到大量表达。
适用方向:使得超长质粒得以完整复制,提高转化效率。
描述:hee=high electroporation efficiency,在电穿孔的过程中,改造过的细胞表现出更强的存活率。
适用方向:更多细胞活下来,更高的转化效率。
描述:hte=high transformation efficiency。
适用方向:调高转化效率。
描述:变异的Lacl基因,导致lac repressor的大量表达,因此更严格地调控lac promoter。
适用方向:严格调控lac promoter控制的蛋白表达。
描述:T7 RNA 聚合酶的基因被重组到大肠杆菌细胞的基因组里,用于T7 promoter调控的蛋白表达。
适用方向:T7 promoter蛋白表达系统。
描述:携带T7 lysozyme(溶菌酶),用于摧毁T7 RNA polymerase。
适用方向:调控T7 promoter蛋白表达系统。
描述:ATPase-dependent protease (蛋白酶)基因的变异,导致蛋白水解能力降低。
适用方向:减少重组蛋白的水解,提高表达产量。
描述:Outer membrane protease 基因变异,因此减少外膜蛋白酶的表达量。
适用方向:减少重组蛋白的水解,提高表达产量。
描述:dnaJ 基因失活。
适用方向:提高某些外援蛋白正确折叠(folding)。
描述:Glutathione reductase基因变异,提高生成二硫键的能力。
适用方向:提高蛋白正确折叠的概率。
以上只是众多改造细胞基因型中的一小部分,还有很多不常见的变异菌株,可适用于更加特殊的实验目的,在这里小编就不多说了。还是那句话,在你的实验中具体使用哪种菌株,和用过的有经验的人讨论讨论,一般来说就能找到自己能用的细胞种类。
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这些,介绍你是哪里找到的?
我觉得很有用,比如我们想改造其他菌株,可以在其他菌株的数据库里面找这些基因的同源基因,做敲除,然后其他菌株也可以被改造成类似DH5a或者BL21这样适合做合成生物学的菌株了。