Hi Tedmccall-ga and thanks for your question.
I'll try to give you a basic explanation of how the lac-operon works
in recombinant DNA experiments. I agree with the comment that a
degree in biology would be helpful to understanding these principles,
however, this is not the only path to understanding the principles
involved. You will, however, need to do a significant amount of
reading to understand why things like the lac-operon work the way they
do. Depending on your background, you may benefit from basic courses
in chemistry, biology, biochemistry, molecular biology, etc.
Typically, these courses include lab sections where you would receive
one-on-one guidance in how various experiments work and why, as a
compliment to the information within the courses themselves, which
would show you the detailed principles behind what you're doing, which
are critical to understand before embarking on lab work.
_________________________________
This eScience site from the California State University has a good
explanation of the lac-operon.
http://www.escience.ws/b572/L11/L11.htm
Generally, the lac operon is used in this context as a "reporter
gene," meaning that it tells us something about something we can't
see, touch, etc. It is used as a link between the very small DNA and
something we can see - it reports for us.
At the top of this page is a plasmid map for one of the many
lac-operon based bacterial plasmids used for determining if the
plasmid has successfully incorporated the DNA we're trying to insert.
Basically, the plasmid is a circular sequence of DNA that can be
inserted into bacteria, usually by brief heat shock, which opens pores
on the bacterial membrane.
Prior to this, one has isolated a relatively short sequence of DNA of
interest that we're trying to clone (isolate and duplicate). All by
itself, the short DNA sequence doesn't contain all of the DNA
information to be amplified (replicated) in bacteria or other cell
types. The cloning plasmid does contain these sequences, but doesn't
(yet) contain the DNA of interest. The cloning plasmid can be
digested (split at one point) by a restriction enzyme and the short
DNA strand inserted (ligated) in the gap. Different plasmids are used
in different ways. The one pictured at the above website actually
uses "TA cloning," which means that the plasmid comes as an open loop
with a free T and free A on either end. One tacks a T or A onto their
DNA of interest using an alteration of the PCR primer (just add a T or
A onto the end). The plasmid is then re-closed to reform the circular
structure.
The new circular plasmid that contains our short DNA segment is then
inserted into bacteria and grown up in a vat of broth, usually
overnight. The resulting soup of bacteria will contain some bacteria
that contain no plasmid at all, some that contain only the cloning
plasmid (without the short DNA strand), and some will contain the
cloning plasmid along with the inserted DNA strand. We want to find
those bacteria that contain the whole cloning plasmid plus our DNA of
interest so that we can grow only these bacteria, which will amplify
our interesting DNA along the way. How can we isolate only these
bacteria from all the rest?
That's where the lac-operon trick comes in. If we take a small sample
from our mixed soup of bacteria and spread it out on IPTG (isopropyl
beta-D-thiogalactoside) Agar plates (round plates on which bacteria
like to grow), this will spread out the bacteria over a large area.
IPTG causes "de-repression" of the lac-operon by binding to the
lac-operon repressor, allowing the expression of a beta galactosidase
enzyme. Treatment with the substrate of this enzyme (known as X-Gal),
will result in a blue color.
Look back at the plasmid map at the top of the page from the eScience
site. The point where the PCR product (our short DNA sequence of
interest) is inserted within the lacZ gene sequence. This means that
if we successfully get our short DNA into the plasmid, we will
interrupt the lacZ gene. If we don't insert any DNA, the loop will
close reforming the lacZ gene.
So, put this all together - We want the bacteria that have our small
DNA within the plasmids. This means that we want plasmids with
interrupted lacZ genes. Because the lacZ gene is interrupted, it is
non-functional and will not generate a functional enzyme for the
conversion of the X-Gal. If the X-Gal is not converted, then no blue
color will form. Also, as an aside, how do we get rid of those
bacteria that don't contain any plasmid at all? These would also not
form a blue color. Looking at the original plasmid, we see gene
sequences for "Ampicillin" and "Kanamycin." These are resistance
genes, and any bacterium with a plasmid (with or without our DNA) will
generate ampicillin and kanamycin resistance. If we add either of
these antibiotics to our bacterial broth before we do the overnight
incubation, we will kill any bacteria that don't have a plasmid. That
means that any bacteria that survive will either have the cloning
plasmid or the cloning plasmid plus our interesting DNA.
If we pick the white colonies from the plates, we'll get the bacteria
that have our small DNA inserted into the plasmid. Each colony on a
plate is derived from a single bacterium, based on how thinly we
spread our bacterial soup on the plates. We can now grow this single
colony up in larger amounts and get a new soup of bacteria, which we
now know will all contain our small DNA. We can isolate the DNA from
the bacteria using standard kits from, e.g., Qiagen. In this way a
small sample of DNA can be amplified to large quantities in a
relatively short period of time.
_________________________________
A more detailed explanation of the lac operon can be found at the
North Dakota State University site here:
http://www.ndsu.nodak.edu/instruct/mcclean/plsc431/prokaryo/prokaryo2.htm
The California State University has a general recombinant DNA page
(click on "Syllabus"), with many examples and experiments, which would
be a useful resource if you have minimal background in these
techniques:
http://www.escience.ws/b572/
A step-by-step explanation of a simple lac operon experiment can be found here:
http://www.science-projects.com/NewLac.htm
This site explains what's happening at each step in the experiment and
would be a useful exercise to go through before trying to clone and
amplify some DNA.
Invivogen has a detailed lacZ reporter assay protocol, which includes controls:
http://www.invivogen.com/PDF/lacz-repoassay_01E11.pdf
You can find an online lecture on the lac operon, which covers it's
function fairly broadly without being too complicated, here from the
University of Washington.
I hope this information was useful. Please feel free to request any clarification.
-welte-ga |
