Subject: STUDENT POST/E coli strain 146 -> Lactose Metabolism?

From: [email-masked] (John Patrick Latino)
Date: 30 Jan 1994 01:13:15 GMT

An individual colony of E. coli 146 (deletion of gpt-lac), carrying a plasmid
with genotype lac I-, Z-, pro+, was streaked onto a minimal lactose plate
(lactose as sole carbon source). Presumably, it should not have formed any
colonies, but in my lab group's case, it did (after incubation @ 37 deg.C for
48 hours. My question is: Since our strain was incapable of utilizing lactose
because of a chromosomal deletion _and_ an amber base pair substitution in the
episome, could the growth on lactose be due to a selection for revertants in
the amber mutation?

 We also plated colonies of the same strain on different
media, including minimal glucose+X-Gal (which turns blue in the presence of 
B-galactosidase). In those cases, *white* colonies were observed. I would think
that if the colonies on the lactose plates were revertants, then revertants
would _also_ occur on the X-Gal plates, revealing blue colonies.

Any e-mailed or posted responses/input on this dilemma would be much ap-
preciated, as we have exhausted our resources (our biochem testbook was of no
use whatsoever). Thanks!   

-John Latino
-Kristina Yu

---

From: [email-masked] (Patricia Foster)
Date: 30 Jan 1994 03:25:31 GMT

Dear John and Kristina,
  You are quite right that any revertant capable of using
lactose to grow into a colony in 48 hours should be blue
on X-gal.  Therefore the logical possibilities are:

1.  The cells on the lactose plate were growing on
something else.  How big were the colonies (compared
to on glucose, for example).  How pure was your lactose?
Did you autoclave it -- it breaks down to glucose and
galactose.
2.  The colonies that you tested on Xgal were not the
same colony that you streaked on lactose.  You say you
streaked a single colony on lactose, but did you use a
different one for the Xgal?  Did you test the colonies
grown on lactose on Xgal?  Maybe the one colony was
a rare revertant, but the rest on the cells are Lac-.
Try picking a few white colonies from Xgal onto lactose.
3.  Your apparent Lac+ colony was a contaminant.  What
color was it (coli is nearly pure white on minimum
medium)?  (guess what coli smells like--
but not strongly)
Cheers,
pat

Patricia L. Foster
Boston University School of Medicine
Boston, MA USA
[email-masked]

---

From: [email-masked] (Peter Rice)
Date: 30 Jan 94 20:47:38 +0100

In article <[email-masked]>, [email-masked] (John Patrick
> would _also_ occur on the X-Gal plates, revealing blue colonies.

At a guess (it's been a while since I did this kind of lab work :-), you are
comparing streaking many bacteria (maybe 1x10^8 or 1x10^9) and seeing the
occasional revertant (back to wild-type is rare, I assume you  have amber stops
in both lacI and lacZ on the plasmid, so they will not both revert, but amber
suppressor is of course possible). As your original colony is unable to grow on
this medium you can put a large number of bacteria on to select for mutants.
This way you see a very few rare mutants in a very large population.

In the second case, the starting strain *can* grow on the medium. You are
probably adding around 100 bacteria to the plate and spreading them so each
will grow as a single colony. To see a 1/10^9 event, you would therefore
need 10 million plates.

There are more complex possibilities of course. For example, would catabolite
repression, which lac is subject to, prevent the colour reaction with glucose
in the medium (streaking wild type E.coli will tell you the answer).

Biochemistry book never were much help with these cases (I was a biochemist
originally :-) Genetics textbooks are better , but only if you can follow them.

 -----------------------------------------------------------------------------
 Peter Rice, EMBL                             | Post: Computer Group
                                              |       European Molecular
 Internet:    [email-masked]   |            Biology Laboratory
                                              |       Postfach 10-2209
 Phone:   +49-6221-387247                     |       69012 Heidelberg
 Fax:     +49-6221-387306                     |       Germany