I would like to ask your opinion on how the presence of a plasmid bearing
antibiotic (AB) resistance (particularly kanamycin) genes would affect cell
growth and/or productivity of transformants. The expression of kanamycin
resistance, as well as kanamycin phosphorylation which is involved in the
mechanism of resistance itself, do require energy and this should be a
common feature that growth and productivity of a transformant cannot be
properly compared to that of a wild type.
The problem is that in a certain kind of experiments imply making
changes to one characteristic by genetic manipulations that would expectedly
cause changes in another characteristic, which is not directly
related/linked to the expression of the first one. In this case, you have to
make all appropriate controls, including the transformation of the wild type
with a "blank" control plasmid. The obtained control transformant is then
compared with the wild type and with the strain obtained by transformation
of the WT with the same plasmid containing a gene of interest. However, the
control transformant is grown in the presence of antibiotic and this raises
a question of to what extent the growth rates and expression rates of a
controlled characteristic is comparable to a wild type strain (that is grown
of course in the absence of an antibiotic).
I hope I made myself clear. Despite the above might be enough to raise a
discussion and there is possibly no need to read the following example, I
would like to be more specific about details of our work.
In our lab, we have a phototrophic bacterium, a wild type (WT), with
phenotype A and B, where A is a pigment content and B is production of a
certain chemical. The goal is to have as high expression of B as possible.
Both traits are supposedly not related, but we would like to prove that A
does affect B. We obtained a stable UV mutant (M) with altered A phenotype
(Ax phenotype) which also has increased productivity of a chemical (Bx
phenotype). However, the mutation is not yet identified. To identify the
mutation we made a complementation analysis by transforming the mutant with
a plasmid (_kanamycin_ resistance) containing a wild A gene that would
supposedly restore only the wild phenotype A, and succeeded with a
transformant (MA). Consequently, the wild B phenotype was also restored.
The only thing we can do to prove that a restoration of wild B phenotype
is a result of restoration of the A phenotype is to clone the control
plasmid used in complementation and lacks A-genes (MControl transformant),
and to compare the expression of B phenotype in MA and MControl
transformants. However, we have data showing that expression of B phenotype
in MControl is lower than in MA. MControl ideally should have the same level
of B phenotype as the WT, but in practice, we cannot show this, possibly
because of the presence of antibiotic in the medium for growth of the
transformant (MControl). The expression of B phenotype in MControl is in
fact ~3 times lower than in WT.
To make a correct publication we have to explain why MControl does not have
the same level of B as WT.
The only explanation we can give for lower B in MControl is the presence
of kanamycin in the medium.
However, it is better to refer to publications describing the same
phenomenon in some other cases. I guess it is quite a general problem, so I
expect a wide response. I would like to have references showing the
comparison of WT and control transformants, and evidencing that they cannot
be properly compared because of the presence of antibiotic (not only
kanamycin) in the medium. Moreover, such references would be much
appreciated, as well as your discussion on the matter.
PPS. I thought it is important to add that there is a limited number of
expression plasmids available for phototrophic bacteria. There are ones
bearing resistance for Km, Str, Chp, and, as far as I am concerned, that's
all. In our case of Chp is not applicable, because it is light sensitive and
would decompose during long-term cultivations in the strobg light; Str is
not good, too, because allegedly photrophic bacteria can spontaneously
develop resistence to this antibiotic. Amp is not used with phototroph
because of sume limitations (don'n remember what exactly). Thus, in our work
we have to use Km.
PPS. Answers by e-mail khatipov at nibh.go.jp are also welcome.
Sorry for a big size of this posting, I really tried but I could not make it
PPS. Sorry if this is a repost, because I already tried to post this message
a couple of days before, but looks like I did not succeed then.