There was a professor who was teaching a little genetics and cell
biology and he taught us a little about the lac-operon. He sought of
taught us the basics of lac-operon. I just wanted to know if these
operons have anything to do with purifying a protein or anything else.
I just want to know if a operon have anything to do with proteins
purification before i take the introductory courses.

                           Ted.

Hi Ted.

I am a biology student and I have studied a bit about Lac Operons.
They are to do with proteins, and they could be used to determine the
'purity' of a strain of bacteria, for example.

Here is a brief explanation which you may find useful -

On the strand of DNA, you have sequences classified as genes, which
code for the proteins of the organism. Now, an operon is a unit on the
DNA strand which comprises of the structuraal genes (which code for
the protein), and the operator site and the promoter site, which can
control the expression of the genes.

Transcription of the structural genes is prevented if a 'repressor
molecule' binds to the operator site.

I know it is a bit difficult to understand without diagrams, but there
is a nice  diagram in the link which is below.

Now, the Lac operon is the operon that regulates lactose metablism in
E. Coli bacteria.

So the Lac operon does not directly involve anything to do with the
'purity' of the protein, but there is a way which you can check the
'purity' of the organism, if that can be said.

You know how DNA can be introduced into an organism's chromosome, for
example, adding an extra gene to a yeast or E Coli chromosome. Now how
do you check that the gene you tried to insert has inserted correctly?
You can't tell just be looking at it, because it is so small...

You can design the implant DNA to 'knock out' the lac operon - it
breaks the operon and takes up it's place in the DNA sequece. So after
the implant, you could check if the organism can survive on a mimimal
medium (petri dish) which doesn't have lactose on it.

If it does survive, that means the Lac operon is still active, and
your 'knock out' has been unsuccessful. If it doesn't survive, it
means that the Lac operon (which is needed for lactose synthesis) is
not working properly, so your 'knock out' might have worked, and the
new gene has been inserted correctly.

That is how you might be able to check the 'purity' of th yeast for example.

If you would like a more in depth understanding, with some colourful
animated pictures, please look at this website; which is simple and
to-the-point, but at the same time, quite in depth.

http://www.phschool.com/science/biology_place/biocoach/lacoperon/intro.html

Thanks.

(Please note, I am a student, I might have made a few mistakes with
the explanation. Please do not feel obliged to accept this answer if
you are not satisfied with it).

Yikes...

You know how DNA can be introduced into an organism's chromosome, for
example, adding an extra gene to a yeast or E Coli chromosome. Now how
do you check that the gene you tried to insert has inserted correctly?
You can't tell just be looking at it, because it is so small...

---> when talking about yeast and bacterial transgenesis,
incorperation of the plasmid into the genome isn't important.  Stable
copies of plasmids will be replicated along with the cellular DNA. You
can also check that its been incorperatred by PCR - design the gene
insert with novel primers and see what your PCR product is.

You can design the implant DNA to 'knock out' the lac operon - it
breaks the operon and takes up it's place in the DNA sequece. 

--> More often, a fusion gene tied to a LacZ reporter gene is
inserted.  Its not particularly useful to knock out the lac operon.

So after the implant, you could check if the organism can survive on a mimimal
medium (petri dish) which doesn't have lactose on it.

----> No, in this case the minimal medium would contain Lactose, but
no glucose.  Hence, if the plasmid has been taken up, lactose
metabolism would be intact.  If it the plasmid has not been taken up,
the organism will not be able to survive, as lactose will not be
metabolized into glucose and galactose.

If it does survive, that means the Lac operon is still active, and
your 'knock out' has been unsuccessful. 

---> See the above.  

If it doesn't survive, it
means that the Lac operon (which is needed for lactose synthesis) is
not working properly, so your 'knock out' might have worked, and the
new gene has been inserted correctly.

---> Lac operon has nothing to do with lactose synthesis, and
everything to do with lactose metabolism.  You are getting confused
with knockouts for amino acid biosynthetic operons.



------

A few other notes...

I'm not sure what sort of purification you are talking about - are you
trying to produce a pure protein, or isolate a protein from a cell
lysate, etc...

The principles of molecular/cell biology and genetics that are
described in the elegant example of the lac operon can be very useful
in cell biology.

Just to be clear with terminology, yeast and other eukaryotes do not
have operons.  Operons are all under the control of the same promoter
hence the group expression.


Breifly a review of the lac operon...


LacI --> NOT part of the operon.  This gene is under its own promoter
and encodes the regulatory protein for the lac operon (a repressor)
LacZ --> codes for beta-galactosidase which catabolizes lactose to
glucose and galactose
LacA --> Thiogalactoside transacetylase  -  fx unknown
LacY --> Lactose permease -- lactose transport

The basics LacI+Lactose --> inactive repressor, transcription can proceed.

LacI without lactose --> active repressor, blocks transcription.


LacZ is used often in molecular/cell/genetic biology as a reporter
gene.  Yeast do not contain LacZ or any functional b-galatosidase.
There is a cool substrate of b-gal called XGAl which turns blue upon
metabolism.  Using this you can couple lacZ to the promoter of a gene
of interest to check for incorperation or to study the levels of
transcription from that promoter.

Plasmid uptake is usually determined by using an antibiotic-resistance
gene present on the plasmid, but not in the host's genome. Growing the
host cells on a plate containing the antibiotic kills all
non-recipient genes. The lacz gene is used to determine whether the
plasmid contains the gene you are trying to insert. The insertion site
is in the middle of the lacz gene. So, if no insertion occurs thr lacz
remains active and can metabolise Xgal, producing a blue colony, while
successful insertion destroys the functionality of the lacz, leading
to white colonies. Both tests are usually done together by plating the
host cells onto a medium containing both the antibiotic and Xgal. You
want the white colonies that grow on this combination

Eukaryotes certainly do have operons: see  Operons in eukaryotes
Thomas Blumenthal Briefings in Functional Genomics and Proteomics,
November 2004, vol. 3, no. 3, pp. 199-211(13). Granted they are not
the most common mechanism of regulation in eukaryotes

The role of the lacA gene has been known for some time. The permease
also transports toxic thiogalactosides into the cell, and these are
detoxified by the acetylase

Whoops!

Growing the host cells on a plate containing the antibiotic kills all
non-recipient genes.

should read

Growing the host cells on a plate containing the antibiotic kills all
non-recipient cells.

polycistronic gene transcription is not the same thing as an operon -
the name is a misnomer.  While multiple genes under the control of the
same promoter may result in functionally related genes being
transcribed the control mechanism is very different from an operon.

mikewa-ga...

could you direct me to a paper that has clearly identified the in
vivo, biologically relevant substrate for GAT?  The most recent review
I remember seeing shows clear chemical characterization, mechanism,
but no natural substrate.

The reply is no, they have nothing to do.

Bacterial operons consist of several genes that are subjected to
common transcriptional regulation. All the genes in an operon are
transcribed at the same time as a single, polycistronic transcript.
This gives bacterial cells the opportunity to coordinately regulate
the synthesis of proteins that are required at the same time (for
instance, when lactose is present in the environment and can be used
as a nutrient).

As you see, operons are a regulatory trick used by bacteria, and
protein purification is a technique used by humans in the research
laboratory.