质粒=plasmid
存在于许多细菌以及酵母菌等生物中,是细胞染色体外能够自主复制的很小的环状DNA分子。
——百度百科
对于和DNA、蛋白、微生物打交道的同学来说,质粒是非常常见的东西,如定义所说,质粒是一种小型环状DNA分子。质粒常用于搭载外源基因,导入受体菌种,进行蛋白表达。小编见过或者听说过的质粒没有100 也有80,而实际用过的只有10来种。在现今Addgene质粒数据库中,已经收藏了大约45000种质粒,有的质粒是天然的,有的是人为设计的;有的适用于微生物,有的则是用于更高级细胞的;有“无限”复制的pUC系列,也有兄弟众多的pET系列。总之质粒种类繁多,变化多样,在接下来的系列文章中,小编就来和大家聊聊,一般的用于微生物表达的质粒有哪些特点。
任质粒如何变化,小编认为,有四个方面信息是我们在选择质粒之前应该考虑的:复制起点(origin of replication)、复制能力(copy number)、抗性选择(antibiotic resistance)和与其他质粒的兼容性(incompatibility)。
以pBR322为例
如下图所示,这是最早适用于大肠杆菌E.coli分子克隆的质粒,至今仍然有很多人在用pBR322及其衍生版本。pBR322(GeneBank accession: J01749.1)是由两名墨西哥科学家Boliver (B) 和Rodriguez (R) 于1977年设计的,长度在4.5kb左右,包含一个复制起点(rep)、一个Ampicilin抗性片段(AmpR)和一个tetracycline抗性片段(TetR)。
Rep复制起点序列是质粒上十分重要的一个组成部分,它诱导宿主细胞启动DNA复制机制,使得质粒本身得以复制。pBR322的复制起点序列被称为pMB1,而pMB1并不是一个能诱导超高复制能力的起点,但也可以保证每个宿主细胞包含15-20个质粒。
除了pMB1诱导复制外,不得不说的是rop(repression of primer)蛋白对复制源序列的抑制作用,大致机制如下图所示:
Rep序列产生的RNAII序列,复制机制被启动;当RNAII序列量增大时,RNAI序列与RNAII互补结合,阻止质粒被继续复制;rop蛋白稳定RNAI和RNAII互补结合体,使得复制进一步被抑制。就是在这种调控之下,pMB1-rep和rop的组合才将pBR322的复制数量控制在中等水平。
为什么要关心质粒的复制源和复制能力
Origin of replication是质粒的一个特质,就好比我们知道一个人的身高,就能够想象出这个人有多高大,同样的知道了origin就可以大致判断这个质粒的复制能力有多强。很多时候,我们刚接手一个质粒的时候,是需要知道这个质粒复制能力的,比如说,想大量产出某一质粒,那么选用高复制质粒比较好;而在表达有毒蛋白的时候,选用低复制质粒对宿主细胞伤害小一些,等等。
关于复制能力和兼容性的更多内容,咱们在下几期文章中再聊。欢迎讨论各种关于质粒的问题,小编也是略懂,交流交流才能更懂嘛。如果觉得哪些质粒的特质也是值得一提的,别忘了在下面留言哦~
质粒定义来自百度百科,图片来自网络,其余文字部分为BioEngX原创,转载请注明!
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