四月初六 分子实验思考题

“思考题也许会迟到,但绝不会缺席。“

三周过后,我们又迎来了新一期的分子实验思考题。

1. 我们纯化带有His标签的蛋白时,使用的镍柱有Ni-IDA和Ni-NTA,它们的区别在哪里?
2. 阅读所给的文献,回答下列问题。①使用大肠杆菌作为蛋白表达宿主的优势有哪些?②使用T7启动子表达系统时,防止蛋白本底表达的手段有哪些?③我们常用抗生素进行阳性菌的筛选,文献中还提到一种筛选系统:plasmid addiction,简述一下是什么原理,有哪几个类型。④从基因组的角度说一下BL21(DE3)作为蛋白表达宿主菌的优势。
3. 登陆 http://www.ruf.rice.edu/~bioslabs/studies/sds-page/sdsgoofs.html,观摩别人家的SDS-PAGE

1.纯化His标签蛋白需要二价的镍离子,重组蛋白组氨酸标签的咪唑环可与过渡态金属Ni2+离子形成稳定的化学配位键来特异结合,这些镍肯定不能以溶液的形式加入到样品中,我们需要某种载体来固定这些镍离子,所以就要用到一些络合剂。一般来说是通过不同官能团修饰的琼脂糖来实现这个目的。Ni-IDA和Ni-NTA是通过两种不同的对琼脂糖羟基的修饰方式来螯合镍离子。IDA指Iminodiacetic acid,亚氨基二乙酸;NTA指Iminodiacetic acid,氨三乙酸。这两种各有优劣。据称Ni-IDA与蛋白结合能力更强,有更高的柱容量。但是较之NTA上面结合的金属离子容易脱落。
2.
第一问:(i) It has unparalleled fast growth kinetics. In glucose-salts media and given the optimal environmental conditions, its doubling time is about 20 min (Sezonov et al., 2007)
(ii) High cell density cultures are easily achieved. The theoretical density limit of an E. coli liquid culture is estimated to be about 200 g dry cell weight/l or roughly 1 × 1013 viable bacteria/ml (Lee, 1996; Shiloach and Fass, 2005)
(iii) Rich complex media can be made from readily available and inexpensive components.
(iv) Transformation with exogenous DNA is fast and easy. Plasmid transformation of E. coli can be performed in as little as 5 min (Pope and Kent, 1996).
第二问:tight repression of the lac-inducible T7 RNAP gene by lacIQ, T7 RNAP inhibition by T7 lysozyme and presence of a lac operator after the T7 promoter
可以考虑根据自己的需求扩展一下?
第三问: Plasmid addiction is a phenomenon that occurs when plasmid-free cells are not able to grow or live. 比如说把E.coli基因组中的关键基因删掉,然后再质粒里放一个。这样分裂后没带质粒的就嗝屁了。这可以基于toxin/metabolism/operator repressor titration systems,前两个不用多说,这个所谓的操纵子抑制滴定系统是什么呢?可以参考Repressor titration: a novel system for selection and stable maintenance of recombinant plasmids.下面是它的摘要
The propagation of recombinant plasmids in bacterial hosts, particularly in Escherichia coli, is essential for the amplification and manipulation of cloned DNA and the production of recombinant proteins. The isolation of bacterial transformants and subsequent stable plasmid maintenance have traditionally been accomplished using plasmid-borne selectable marker genes. Here we describe a novel system that employs plasmid-mediated repressor titration to activate a chromosomal selectable marker, removing the requirement for a plasmid-borne marker gene. A modified E.coli host strain containing a conditionally essential chromosomal gene (kan) under the control of the lac operator/promoter, lac O/P, has been constructed. In the absence of an inducer (allolactose or IPTG) this strain, DH1 lackan , cannot grow on kanamycin-containing media due to the repression of kan expression by LacI protein binding to lac O/P. Transformation with a high copy-number plasmid containing the lac operator, lac O, effectively induces kan expression by titrating LacI from the operator. This strain thus allows the selection of plasmids without antibiotic resistance genes (they need only contain lac O and an origin of replication) which have clear advantages for use as gene therapy vectors. Regulation in the same way of an essential, endogenous bacterial gene will allow the production of recombinant therapeutics devoid of residual antibiotic contamination.
这个所谓的”滴定“的意思需要特别的把握一下。这段话的大概意思是说,我们把Kan抗性基因放到乳糖操纵子后面,那么lac repressor就会抑制Kan抗性基因的表达,但是这个菌体如果被转入了大量的含有lac operator的质粒,这些lac repressor就会被分配到质粒上从而解除了Kan抗性基因表达的抑制。这样做的好处就是载体上不需要出现Kan抗性基因。
第四问:BL21 cells are deficient in the Lon protease, which degrades many foreign proteins (Gottesman, 1996). Another gene missing from the genome of the ancestors of BL21 is the one coding for the outer membrane protease OmpT, whose function is to degrade extracellular proteins. The liberated amino acids are then taken up by the cell. This is problematic in the expression of a recombinant protein as, after cell lysis, OmpT may digest it (Grodberg and Dunn, 1988). In addition, plasmid loss is prevented thanks to the hsdSB mutation already present in the parental strain (B834) that gave rise to BL21. As a result, DNA methylation and degradation is disrupted. When the gene of interest is placed under a T7 promoter, then T7 RNAP should be provided. In the popular BL21(DE3) strain, the λDE3 prophage was inserted in the chromosome of BL21 and contains the T7 RNAP gene under the lacUV5 promoter, as was explained earlier.
几个原因都用红色注明了。
第三题虽然不是一个严格意义上的思考题,但是看着别人的实验搞成这个鬼样子还是蛮开心的。另外网页中提供了关于各种失败案例的原因,比较有参考价值。

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