JOURNALOF FERMENTATIONAND BIOENGINEERING

Vol. 80, No. 5, 520-521. 1995

TECHNICAL NOTE
Estimating Segregational Plasmid Instability in Recombinant Cell
Cultures: A Generalized Approach
PINAKI BHATTACHARYA* AND DEBASHIS ROY
Chemical Engineering Department, Jadavpur University, Calcutta 700 032, India

Received 1 May 1995/Accepted 5 September 1995

A generalized methodology for estimation of the relative segregation rate, @), in a recombinant cell culture
has been presented that does not require the a priori assumption of a constant ‘p’. Moreover, measurement of
no other variable in addition to those conventionally monitored, viz. overall culture concentration and fraction
plasmid-bearing cells is necessary.

[Key words: recombinant cell, plasmid instability]

The fundamental rate equations describing the growth On dividing by x and substituting F=X+/S, (i.e.
kinetics of a recombinant cell culture (1) are: F=fraction of plasmid-bearing cells), Eq. 5 becomes
(dX+/dt)=(l -p)p+X+ -DX+ (1) (l/Xr)(dXr/dt)+D=F/c++(l-F)p (6)
(dAp/dt)=p,utX+ +p-X- -DX- Now, if the specific growth rates p+, /1 are Monod
(2)
functions of a single limiting feed substrate, S, (say),
where ‘p’ is the relative segregation rate, D is the dilu- then
tion rate (=0 for a batch culture), p and X denote
,U+=,&[s/(s+KJ] (7a)
respectively specific growth rate and cell concentration,
and superscripts + and - refer to the plasmid-bearing and
and plasmidless cells respectively.
Analytical integration of Eqs. 1 and 2 yielding solu- P ~ =/-l;[S/(S+&)] U’b)
tions for X+(t) and X-(t) is possible only when both with the saturation constant K, usually having the same
the following conditions are satisfied: (a) cell growth is value for both recombinant and segregant species. The
exponential, i.e. p+ and pP are constant; (b) parameter ratio of the specific growth rates is thus equal to the
‘p’ is time-invariant and independent of /-1. Often condi- ratio of the maximum specific growth rates-a constant
tion (b) is assumed a priori and the integrated form of for the system, i.e.
Eq. 1, i.e.
,Lf /,Lf’+=#L&/&=a (8)
In [X+/X&1=(1-p)p+t (la) By defining the function g[x(t)] as
(where XJ is the value of X+ at time t=O), is used to
g[x(t)] =(l/x)(dx/dt) (9)
estimate ‘p’ from the slope of the straight line obtained
by plotting In [X+/X&] versus t in accordance with Eq. Equations 6 and 1 can now be rewritten as
la. The assumption of a time-invariant ‘p’ is obviously
g(Xr)+D=$[F+(l-flu] (10)
implicit in this approach.
An alternative procedure is outlined below that is and
based on the suggestion of DiBiasio and Sardonini (2) in
g(X+)+D=pt(l-p) (11)
which segregational plasmid instability may be assumed
to be dependent on growth rate. It will be seen later With the help of these Eqs., i.e. 10 and 11, it is possible
that this premise does not result in loss of generality of to estimate ‘p’ as a function of p+ corresponding to the
the proposed methodology since it is also applicable to instants at which x and F are measured experimentally
those systems where plasmid instability is independent of from O.D. measurements and agar plating, respectively.
growth rate. It should be noted here that Eq. 8, which expresses
Overall culture concentration Xr may be defined as the fact that cy is constant, depends on the assumption
that both the recombinant and segregant species have
Xr=x++x (3) identical values of KS. However, in some recombinant
Differentiation with respect to time gives cells the uptake and assimilation of growth-limiting sub-
strate are dependent on the presence of a specific gene
(dX/dt) = (dX+/dt) + (dZ/dt) (4) product encoded on the plasmid gene, e.g. toluene con-
Substitution from (1) and (2) in (4) gives sumption (and thus KS for toluene) depends upon the
presence of Tol-plasmid in Pseudomonas. The value of
(w/dt) = 11+X+ + ,u-XP ~ DXr (5) KS for such recombinant cells is, therefore, different
from that for the corresponding segregant cells. Conse-
* Corresponding author. quently, for such host-vector systems the use of Eqs. 10

520
VOL. 80, 1995 TECHNICAL NOTES 521

parameter that determines system productivity.

Moreover, the proposed methodology also provides an
indirect method for evaluation of the specific growth
rates, pLf and 1_1--,beyond the exponential growth phase,
Le. when their values are changing with time, without
necessitating measurement of the limiting substrate
concentration as required by the Monod Eqs. 7a and 7b.
If ‘p’ is indeed a function of p+, then in the exponen-
tial growth phase when ,u+ =p,$ a constant, ‘p’ may be
expected to remain constant as well. However, for a
given host-vector system, the value of pm+ is a function
of culture conditions, e.g. temperature, pH etc. The
corresponding value of ‘p’ should therefore be a function
of culture conditions too. Thus the approach described
herein can be utilised to obtain quantitative information
on the dependence of ‘p’ on culture conditions which
should be of immense importance in subsequent engineer-
ing design work.
However, the absence of any valid correlation between
woo0.G 2.0 4.0 6.0 8.0 10.0
‘p’ and p+ cannot be ruled out as an a priori. There
may indeed be systems, as indicated earlier, where the
nature of variation of ‘p’ may be perfectly random, even
Time (h)
in the exponential growth phase. Subsequently, depend-
FIG. 1. Illustration of Type 1 and Type 2 systems. 0, Sp. gr. ing on the nature of the trend observed in progressive
rate (h-l); x , p (Type 1); 0 , p (Type 2). values of ‘p’ in the course of a recombinant cell fermen-
tation, host-vector systems can broadly be categorised
and 11 to estimate ‘p’ and /*+ values is limited for those into two types, viz. Type l-where a definite correlation
conditions only in which growth-limiting substrate is in exists between ‘p’ and p’+ and Type 2-where ‘p’ cannot
excess (i.e. S>>ZQ, so that inspite of differing K, values be correlated with p+, the nature of its variation being
for the recombinant and segregant strains, Eq. 8 may apparently random. To ascertain which category a par-
still be obtained from Eqs. 7a and 7b. ticular system belongs to, experiments must be per-
A scheme for evaluation of the parameter (Y is out- formed and the data analysed in accordance with the
lined below: (i) Estimate p; from a separate experiment scheme outlined in this report. In Fig. 1 is plotted a typi-
with segregant cells only (It is worthwhile to note here cal set of ‘p’ vs. ,u+ data for both types of systems; for
that even if ‘p’ is assumed constant, it is generally not the Type 1 case it is assumed that {(l-p)/p+} is con-
possible to estimate /*+, p- and p from the same experi- stant whereas for Type 2 the ‘p’ values plotted represent
ment). (ii) In the actual experiment with recombinant a population of random numbers with a mean of 0.15
cells, measure the value of F at the start of exponential (the same as the Type 1 value of ‘p’ corresponding to
growth (t=to, say) in batch culture (i.e. D=O) by count- constant p+) and maximum variation of *50x about
ing number of colonies on agar plates with and without the mean.
a ‘marker’ reagent, and let F. denote the value of F. At Recombinant cell systems may also be characterized
this instant, p + =& and P- =I*;. Consequently, at t= to, on the basis of the effect of dilution rate variations (in
Eq. 10 reduces to the form continuous culture) on the value of ‘p’. Since productiv-
ity of a bioreactor harbouring a recombinant cell culture
s(~T)t=to=~,+Fo+~,(l --Fo) (lOa) depends substantially on the magnitude and variational
In Eq. 10a the only unknown is ,u,$ which is calculated trends of the relative segregation rate, the importance of
directly. Thus cy is obtained. such characterizations cannot be overestimated from the
Now, once (Y is known, the value of p+ can be ob- reaction engineering perspective.
tained at all the instants at which F and P have been
measured using Eq. 10. Again X+ =FS. Therefore (l- One of the authors (D. Roy) is grateful to Jadavpur University
p)p+ can also be subsequently estimated using Eq. 11. for the grant of a research fellowship.
Finally, a plot of (1 -p),u+ versus p+, or ‘p’ versus t may
be prepared depending on whether the objective is to REFERENCES
correlate ‘p’ and p”+ or simply to observe the variational
1. Imanaka, T. and Aiba, S.: A perspective on the application of
trends in the progressive values of ‘p’ in the course of genetic engineering: stability of recombinant plasmid. Ann.
the fermentation. N.Y. Acad. Sci., 369, 1-14 (1981).
Even though the method described above appears to 2. DiBiasio, D. and Sardonini, C.: Stability of continuous culture
be simple and straightforward, it is potentially useful with recombinant organisms. Ann. N.Y. Acad. Sci., 469, lll-
from the reaction engineering viewpoint since it provides 117 (1986).
a realistic estimate of the crucial plasmid instability