在上期文章中(戳这里),我们了解到质粒的复制源序列,并以常用于E.coli蛋白表达的质粒pBR322为例,了解到质粒复制的调控机制。那么除了复制源序列外,还有什么因素会影响质粒在细胞内的复制量。我们就在质粒系列文章第二篇中,来聊聊质粒的复制能力和复制数量。
影响质粒复制数量的几个因素
质粒复制数量是指存在于宿主细胞内的质粒平均数量,这个复制数目可以由三个因素决定:
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Origin of replication (ori) 的种类。有的复制源序列能强力诱导宿主细胞的DNA复制机制,由ori序列的强弱而导致质粒复制数量的多少;
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质粒的长短。更长的质粒在细胞内复制的时候需要用多更多的细胞资源,对细胞自身的新陈代谢来说是一个负担;
-
细胞的培养环境。
在入手一个新质粒之前,可以先大致了解下这个质粒的复制源序列有多强,再根据自身实验的情况调整其他条件,以达到自己需求的复制数量。如下图所示,常见质粒复制源所代表的质粒大概复制能力。
如何调控质粒的复制数量(常见方法)
- 调节宿主细胞的生长温度。有一些复制源序列的激活与否是和细胞自身的生长速度有关的,当我们调节细胞生长的温度,使得细胞加速或延缓生长,是有可能调节质粒的复制数量。
- 细菌细胞氯霉素调节(Chloramphenicol amplification)。这个方法对许多带有pMB1复制源的低复制质粒有作用。氯霉素抑制细胞内蛋白的合成,因而导致细胞自身分裂能里的降低(没有蛋白了,基因组不好复制了,其他细胞复制过程也不易实现了)。同时质粒复制所需蛋白也会停止产出,但这些蛋白的寿命都一般较长,所以在细胞停止复制后,还能继续进行质粒复制,最终导致平均每个细胞中的质粒数量会增加。
- 避免巨大的质粒的使用。超长的质粒会需求更多的细胞资源来复制,增加细胞负担,降低细胞自身的复制能力,因此最后也降低了质粒的复制总量。
关于质粒兼容性的问题,我们下期再聊。如有其他常见的质粒调控方法,请在下方留言讨论哦!
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我想问一下关于松弛型质粒和严密型质粒的复制机制,想了解为什么严密型质粒的复制与受体细胞是同步的,这其中具体的过程是怎样的,越详细越好。