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Get a gene, put it in a plasmid, get that plasmid expressed in a host, could even be a bacterial host,right?
把一个基因导入一个质粒,让质粒在宿主中表达,这个宿主甚至可以是细菌,对吧
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Or you could ask the question, I have this restriction enzyme, at what regions on this plasmid will it cut?
你也可以这样问,我的这种限制性内切酶,会从质粒的哪个区域切开呢
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I apply ligase, and I've got the plasmid that I had before but now with my gene, colored green here,inserted.
我用连接酶,就能在质粒中把基因,这里绿色的部分,插进去
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It might be a plasmid that contains a promoter that works in lung cells and that has the cystic fibrosis gene.
这个质粒带有,可以在肺细胞表达的启动子和正常基因
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The sources are: one,the plasmid vector that we've picked, and the second is these genes that for some reason we're interested in.
第一个是我们选择的质粒载体,第二个是我们感兴趣的基因
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You take that plasmid; you put in the micro-organism you're going to make many,many copies of that plasmid.
你把一个质粒放进微生物体内,你就会得到,很多很多质粒的拷贝
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If this reforms, so the plasmid reforms back to its native state, that resistance will be recovered.
如果它自己合上了,质粒就会回到原本的状态,抗性就会恢复
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If we injected it into a muscle let's say, and your muscle cells took up this plasmid.
比如说,如果我们将其注射入肌肉内,则肌肉细胞就可以吸收这些质粒
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Now how do you find those colonies on a plate that have the plasmid that you want?
那我们如何辨别培养基上的哪些菌落,有我们需要的质粒呢
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Now to start with, the plasmid is usually shown in a diagram like this as a circle.
首先要说的是,质粒通常用图中这个环形来表示
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The first step in the process is to take our plasmid which we've selected, and to insert the gene that we want into it.
这个过程的第一步,是取得我们选择的质粒,把我们需要的基因插入到里面
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Now the plasmid started to do its thing, which is replicate and the gene gets transcribed.
那么质粒就可以开始进行复制,基因得以转录
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It's a gene that's on the plasmid, and this particular gene confers a specific biological property to cells that have the plasmid and can use it properly.
它是质粒自带的基因,如果细胞能够获得这种质粒,并正确的利用它的话,这段基因就能赋予该细胞某种生物学特性
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How would I put a gene that I'm interested in into a plasmid?
怎么把我想要的基因整合到质粒上呢
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The second issue is that the gene vector, in this case the plasmid, has to be compatible with the cells that you're trying to express the gene in.
接下来的问题是如何选择合适的基因载体,在这里用到的,质粒,必须与要用来表达基因的,受体细胞匹配
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So if you have plasmid where you know all the base pairings than you could go through that plasmid and say I want to cut it right here.
如果你已经知道某个质粒上的所有序列,你就可以决定从哪里切开质粒
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A plasmid is a highly tuned machine for making copies of itself.
质粒是一个高度调谐的自我复制的机器
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Took it out of the virus completely, cloned it into a plasmid, that plasmid was expressed in a foreign host, in this case it was expressed in yeast cells.
将基因从病毒中完全提取出来,并克隆制成质粒,该质粒在外来宿主中表达,此例中质粒在酵母细胞中表达
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One of the properties that you would like a plasmid to have is you'd like for cells to be able to replicate it, to make more copies of it.
所以你所希望质粒具备的一个属性,就是得能让细胞复制它,制造出更多的拷贝
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if you do that under the situation where you've put your plasmid into these micro-organisms then you're going to have little colonies that grow many copies of the bacterial cells.
如果你在质粒已经进入了微生物,的情况下培养它们,你就会得到许多小菌落,里面长有很多增殖出来的细菌
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Take a plasmid, cut it open, insert a gene that we want into the plasmid, and then put that plasmid in a host cell, and let the host replicate it.
取一个质粒,把它切开,把目标基因放进去,然后把这个质粒植入到宿主细胞,让宿主来进行复制
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The plasmid has some features which allow it to do that.
质粒具备一些特征,使它能做到这些
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If you selected the cells right than the only ones that have that resistance to Ampicillin are the ones that successfully got your plasmid and are using this Ampicillin resistant gene.
如果你选对了细胞,那么对氨苄青霉素有抗性的就是,成功得到了质粒并表达了基因的细胞
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Here, the plasmid is directly injected into a cell.
这里用的方法是,直接将质粒注入细胞
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This particular example of a plasmid has several regions.
这个特定的质粒样本有数个区域
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One way that you can look for that gene that you want is by making the cut in your plasmid inside of a gene that encodes for some property like resistance to an antibiotic.
一种找到成功重组质粒的方式,是在质粒控制某种特性的编码区域切割,例如,对抗生素的抗性
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Yeast cells were grown in large numbers with this plasmid inside, they expressed the plasmid and so you made Hepatitis B surface antigen not in people but in cell culture where it was not normally formed.
体内携带这种质粒的酵母细胞大量增殖,它们表达质粒,因此,乙型肝炎表面抗原不是在人体内,而是在细胞培养环境下,以非正常方式形成的
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Now one of the big advantages of plasmids,I already mentioned, is that you can take this plasmid and a plasmid is one - you can think of it as a highly tuned machine for copying itself.
质粒的一个很大优势,我提到过,是你可以用这个质粒,而质粒是一个--,你可以把质粒想成,一个高度调谐的自我复制的机器
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Then we're going to take these plasmid vectors and we're going to somehow put them in contact with cells in such a way that the cells ingest the DNA and they use it.
然后要使这些质粒载体,以一种特别的方式与细胞接触,让细胞吞入质粒载体并使之表达
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You could imagine strategies where you have multiple resistance genes on a plasmid, resistance to Ampicillin, to Penicillin, to Erythromycin for example, and you design strategies for separating out which cells are carrying the plasmid that you're interested in.
若质粒上有多种抗性基因,你就有很多筛选方法,例如对氨苄青霉素,青霉素,红霉素的抗性,而且你可以设计方法分离出,含有你感兴趣质粒的细胞
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