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Textbook Canon: Expressing Any Genes in Bacteria - (Aug/09/2012 )

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Yes I do :) thank you Andreea!


ascacioc on Sun Aug 12 23:33:55 2012 said:

Cloning vector vs expression vector: yes you are right; the difference is in having the promoter, rbs, terminator. I never used a cloning vector. The synthetic gene companies send the genes under the form of gene inserted in a cloning vector. Also some people working in generating DNA libraries are using cloning vectors because it is easier to handle (they are smaller, easier to transform, do not express the proteins from the genes, which make the bacteria happier).
With the origin of replication: I do not know why for expression you use the low copy number plasmids. I have just noticed that all my expression vectors have the low copy number plasmids. The reason that was given to me is that having too many copies per cell would lead to overproduction of the protein which is toxic to the cell. However, having strong promoters such as T7 has the same result. But maybe it is better not to change to many variables at once. For example, when I test which plasmid is better for my protein, I check different promoter strengths (among other things). If I would have different stregths of replication origins, I would change 2 variables in between experiments at once and I wouldn't know what is the cause for the difference.

Not following the entire discussion, but the discussion about how and low copy all depends on your needs.

High copy can indeed cause the formation of inclusion bodies in your bacteria, which could be bad, however it can also be good, because its easier to seperate inclusion bodies from the native proteins.
High copy is also not wanted if you are expressing potentially toxic proteins
High copy can also cause "stress" on your cells: all the energy goes to those plasmid/genes/proteins, this is not always what you want.
Low copy also has a more controled mechanism in many cases so that the progeny of your cells keep the plasmid.
Depending on the gene you are expressing, you might need other genes/proteins from the cell itself... a certain pathway , and you can create a bottleneck if enzym2 (you installed it, in the plasmid) is present at high concentration due to the high copy nature of your plasmid while enzym1 and 3 (from the bacteria itself) is present in lower amounts or works slower..
Also: you need to think about the energy balance of the organism: its ok to insert a certain enzym, but if this enzym used NADH you are recruiting NADH from other processes in your cell.. so you need to concider this too.
Same goes for ATP, NADP etc...
You cell has everything balanced.. as soon as you start influencing this......

I also wonder: what do you call low copy? Just 1 or?

An example of a study about this low copy vs high copy:

It all depends on your needs and most of the knowledge about these things is not found in (fundamental) research, but rather in the industry (which is often not published)

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