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Designing rPlasmid for Expression in E.Coli: Questions From A Newbie

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#1 djvan



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Posted 19 December 2013 - 06:42 AM



I'm interested in using E. Coli to express a protein of interest.


I'm currently working on designing the plasmid.  I know that the following are imperative for my plasmid:


1) Promoter, along with other host-specific elements for expression

2) My cDNA with a C-Terminal His Tag

3) Antibiotic Resistence Gene


I started looking online at expression plasmids, for instance: pTrisHis by LifeTechnologies.  With this, I would simply need to cut my cDNA and the plasmid with the same RE, add them together, and ligate.


However - the cost made me rethink my strategy.  Is it possible to isolate plasmids from your E. Coli strain (their genome is sequenced, I presume), and design an expression plasmid based off of that?  Their plasmids should already have the Promoter and other expression elements - would I just need use a restriction map and add my cDNA and Antibiotic gene?




#2 Missle



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Posted 20 December 2013 - 05:13 AM

I'm not sure if what you're proposing is feasible or not.  I wanted to mention 2 things: 1) there are many cheaper vectors out there - check out the pET family of vectors from Novagen (EMD Millipore).  They are <$200 each.  2) you know that it's a one-time cost, right?  You can perpetuate your empty vector so that you never have to buy it again.  What your proposing seems like it will cost far more in time and analytical supplies then the cost of the vector......

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