Protocol Online logo
Top : New Forum Archives (2009-): : Molecular Cloning

Designing a Plasmid for p53 expression - I must choose between 3 given plasmids the best to express p53. (Oct/11/2009 )

I am in 2nd year university studying Plant Biotechnology. I am trying to complete an assignment for one of my courses and am finding the tasks rather beyond my knowledge. Any help would be much appreciated. I must do the following:

1) Choose a protein which can be used to treat a disease
2) Choose a plasmid to express said protein in mammalian cells
There is a choice of 3 plasmids:
Attached ImageAttached Image dc.JPG>

I must justify my choice of plasmid and then choose which components of the plasmid I wish to use to express and purify my protein.

So far I have chosen p53 as my protein
I have also chosen plasmid C - pcDNA3.1(+/-) as my plasmid. However, this choice was made because this plasmid seemed to contain all the components listed in another plasmid which is used to express p53: http://www.addgene.org/pgvec1?f=c&iden...&cmd=findpl . I do not really understand what the individual components mean or what they do. This means that I cannot really justify my choice of plasmid, nor decide if the plasmid contains any components which I do not need.

The ultimate aim of the assignment is to list all the licenses which would need to be obtained in order to use this plasmid and produce the protein, as the course is a course on Intellectual Property. I am capable of finding these licenses, however, part of the assignment requires that we show how we have minimized the number of licenses which we need to obtain. To do this I must understand the components of the plasmids and which I do and do not need.

I would love it if someone could explain to me the components of pcDNA3.1(+/-) as listed on the attachment. Or if someone could suggest I choose one of the other two plasmids. I am at quite a loss with this assignment.

I am also at at loss as to the best way to purify my protein, which I need to know in order to check the existence of any patents on the purification process.

Thank you in advance,

Bif
Attached Image

-Bif-

Look for components of the plasmid that might be patented e.g. particular promoters, antibiotic resistance genes etc.

Expression plasmids should have a promoter, a poly A site, a multiple cloning site, as well as a few other things (ori, lacZ, ampr (or similar))

-bob1-