MadSci Network: Molecular Biology
Query:

Re: What are the basic requirements for constructing a cloning vector?

Date: Wed Mar 23 23:19:50 2005
Posted By: Paul Nagami, Undergraduate, Biology, California Institute of Technology
Area of science: Molecular Biology
ID: 1110955034.Mb
Message:

Your question is intriguing. Although cloning vectors clearly have to 
come from _somewhere_, practically speaking, I've usually seen people 
work with commercially available vectors rather than construct them from 
scratch.

We need to tailor our vector to our host species. If we're making the 
vector because we're dealing with a new host species, we may face even 
more difficulties.

Step one:

First, we need to make a plasmid that contains a host replicon, which is 
the origin of replication initiation (ORI) and any other sequences 
necessary for replication. This is our bare-bones requirement; a plasmid 
will replicate only with the needed ORI. There may be multiple ORIs to 
choose from.

If I recall correctly, the original isolation of an ORI was done by 
randomly cutting bacterial genomic DNA into fragments, ligating those 
fragments to DNA that carried a selection marker, and transforming host 
bacteria with the resulting circular plasmids. Bacteria that survived on 
selective medium over several generations had to be carrying plasmids 
with the marker, which implied that they were replicating the plasmid 
they received. The ligated-in sequences that conferred survival were then 
cut down until a minimal sequence needed for replication was isolated.

We know the replicon sequences for E. coli and other common hosts, but if 
we want to make a cloning vector for a new host, we'll need to either 
repeat the above experiment or find a shortcut that lets us quickly find 
the needed replicon sequences. You mentioned a bioinformatics approach in 
your letter; this might work if the genome of the host is known and it 
contains a replicon that has sequence homology to a known replicon. Then 
the candidate replicon sequences could be identified, PCR-amplified, and 
put into a marker-carrying plasmid.

Step two:

Once we have a streamlined plasmid with a selection marker (amp-
resistance, etc.) and a replicon, we need to introduce multiple cloning 
sites, which, ideally, should all be unique. Our sites should be rare 
enough that they occur only once in the entire plasmid, but common enough 
that we can work with them conveniently.

Three solutions come to mind:

1) Cut the polycloning sites out of an established vector and ligate them 
into ours.

2) Cut out our replicon region and replace the replicon region in a known 
vector. We might have to use site-directed mutagenesis to make 
restriction sites with which to do this.

The problem with these first two solutions is that our restriction sites 
might no longer be unique, since we have a different replicon. We could 
just use the ones that remain unique, though, and ignore the others.

3) Construct a new multicloning region from scratch by using 
complementary oligomers to synthesize DNA that carries a series of unique 
restriction sites. Since this region may be 70 or more bp long, we may 
need to make multiple sticky-ended fragments and stitch them together.

[Bioinformatic relevance: Several computer programs for listing or 
mapping restriction sites on plasmids are available; a quick Google 
search should give a few alternatives. This would help choose between 
possible polycloning sites to incorporate (methods 1 and 2) by letting us 
pick the ones with the largest number of unique sites.

If we make our multicloning region from scratch, then we can use a 
similar search to find the restriction sites that are absent from our 
plasmid and can therefore be made in the oligomer.]

Step three:

Now that we have a workable plasmid, we can insert useful additional 
sequences, like bacteriophage promoters or reporters. If we've used the 
second method in step 2, where we swap in our replicon, then this step 
has already been completed for us. Otherwise, we need to find sites in 
our plasmid to insert these elements. For reporters, we can just use the 
endmost restriction sites in our polycloning region. For other elements, 
we may need to use site-directed mutagenesis to make sites.

I hope that my response has been helpful; I'm pretty new at this. Please 
do not hesitate to contact me at my e-mail address below if you have any 
questions or comments.

REFERENCE:
Sambrook, Fritsch, and Maniatis. (1989) "Development of plasmid cloning 
vectors." In: Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring 
Harbor Laboratory Press, New York. pp. 1.7-1.9

Paul Nagami
nagami(AT)its.caltech.edu [address mangled to avoid spam robots.]
Undergraduate
California Institute of Technology


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