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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
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.
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