Chemistry: molecular biology and microbiology – Apparatus – Including measuring or testing
Reexamination Certificate
1999-03-29
2003-04-29
Beisner, William H. (Department: 1744)
Chemistry: molecular biology and microbiology
Apparatus
Including measuring or testing
C435S288700, C435S297200, C435S309200, C435S030000, C422S062000, C422S081000, C436S052000
Reexamination Certificate
active
06555360
ABSTRACT:
INTRODUCTION
There is ample evidence that growing cells are heterogeneous entities that differ from one another in their physiological states. Microbial heterogeneity may arise due to phenotypic changes associated with the cell cycle (Dien and Srienc, 1991, 1992), due to changes in the microenvironments of individual cells (Fowler and Dunlop, 1989, Dunlop and Ye, 1990), or due to mutations resulting in genotypic variations in the population (Hall, 1995). Thus, rates of growth associated parameters such as protein synthesis or substrate uptake are distributed in the population. From a biochemical engineering perspective, these population dynamics have profound implications since the overall productivity of the microbial process depends upon the contribution of each individual cell. One method to estimate such dynamics is by the use of flow cytometry.
In a process environment, rapid, repeated, and long term on line manual analysis is usually impractical, if not impossible. Therefore, a certain degree of automation is desirable, particularly for more complex analysis procedures such as flow cytometry. To date, a number of flow injection systems have been designed and widely used in microbial process control and automatic analysis (Ruzicka and Hansen, 1975, 1988; Reed, 1990; Munch et al., 1992), but most of them monitor population averaged properties. The concept of automatic flow cytometric analysis was introduced by Omann et al. (1985), who constructed a sample instruction devise fitted to a Becton Dickinson FACS cytometer. Kelley (1989) also developed a similar device. Pennings et al. (1987) developed a system based on continuous pumping cells and reagents with a peristaltic pump. Although the capabilities of these designs are rather limited, they are pioneering designs in automatic flow cytometry. Successful on line flow cytometry was demonstrated using a flow injection technique (Ruzicka, 1992).
The present invention provides for a flow injection system interfaced with a flow cytometer and a bioreactor to perform on line assessment of single cell property distributions. The versatility and performance of this system are demonstrated in several preliminary examples that show the utility of the system since it provides detailed quantitative information on growing cell populations that cannot be obtained with any other existing method.
Materials and Methods
Cell Strains, Growth Medium, Growth Conditions, and Staining Conditions
E. coli
strain K12 was used in the growth experiment involving on line monitoring of optical density. Cells were grown in 300 mL 2XGYT complex medium (Sambrook, et al., s1989) in a 1 L flask placed in a 30° C. water bath and shaken at 225 rmp. The culture was aerated at 1 vvm by a peristaltic pump through an airstone (Cole Parmer, Vernon Hills, Ill.) immersed in the medium
E.coli
BL21 cells transformed with the plasmid pRSET/S65T (a kind gift from Dr. R. Y. Tsien, Howard Hughes Medical Center, San Diego) were used in the experiment to study the Gfp fluorophore formation kinetics and batch growth dynamics. The plasmid contains an ampicillin resistance marker gene, and the Gfp gene under the control of a T7RNA polymerase promoter. Cells were grown in LB complex medium containing ampicillin (100 &mgr;g/mL). Production of T7 RNA polymerase was regulated using an IPTG inducible lacZ promoter present in the host chromosome. IPTG (200 &mgr;g/mL) was used to induce Gfp expression, and chloramphenicol (30 &mgr;g/mL) was used to inhibit the protein synthesis when needed (Sambrook et al., 1989)
Saccharomyces cerevisiae
strain YPH399a (MATa, ade2-101, leu2&Dgr;1, lys2-80, his3&Dgr;200, trpl&Dgr;63, ura3-52) cells were grown overnight on 3 mL YPD medium (Bacto yeast extract, 1% w/v, bacto peptone, 2% w/v, dextrose, 2% w/v) at 30° C. and 225 rpm in a 15 mL polystyrene test tube (Falxon). Cells were diluted to a concentration of ca. 1×10
6
cells in fresh medium, and the tube was placed on ice. Samples were automatically withdrawn into the microchamber of the flow injection system. Inside the microchamber, samples were washed with ice cold PBS, treated with chromatin denaturation solution (0,1 N HCl, 0.5% w/v Triton X-100, 1.75% w/v NaCl), washed with ice cold PBS, and strained with mithramycin A (Sigma, 30 &mgr;g/mL in PBS, 2mM MgCl
2
). Fixation and denaturation steps were 2 minutes long, washes were 1 minute lone, and cells were tainted with mithramycin for 10 minutes. Fixation, denaturation, and washing were performed by continuously pumping the appropriate reagent through the microchamber while mixing the suspended cells using a magnetic stir-bar. A source of reagent is connected to a port of the microchamber such that reagent flows through this port and through the membrane and contact the sample on the sample handling side of the membrane.
FI-FCM System
The equipment used to construct the flow injection system is listed in Table 1. The components were interfaced to a personal computer using DT 2805 and DAS 1601 data acquisition and system control boards through DI/O and D/A subsystems. Labtech Notebook software (Laboratories Technology Corporation, Wilmington, Mass.) was used to control both boards.
FIG. 1
shows a schematic overview of this FI-FCM system which consists of three subsystems: (i) sample delivery, (ii) sample handling, and (iii) sample injection and analysis.
A sample delivery loop transferred the cell culture from a bioreactor to the flow injection system. A static degassing unit was designed to release air bubbles trapped in the sample (
FIG. 2
a
), and sample was continuously re circulated in this loop. During sampling periods, the cell culture with air bubbles was allowed to accumulate in the glass tube, and a weak vacuum was applied using a peristaltic pump. Due to a combination of static hydraulic buoyancy force and vacuum air bubbles were rapidly eliminated from the medium through a 0.45 &mgr;m inline filter. The degassed sample was then fed into the microchamber of the sample handling subsystem for further processing. The sample residence time in the delivery loop was minimized since the environmental conditions (in particular, the aeration) in the tubing are not the same as those in the bioreactor,. However, care was taken to avoid shear induced damage of the sample that might result from the use of very high flow rates in the tubing. Hence, Cole Parmer Masterflex silicon tubing (size #13, I.D. 0.75 mm) and a flow rate of 5 mL/min (0.19 m/sec) were used, resulting in a residence time of 10.5 sec.
The sample handling consists of a 10 position switching valve (#1, Table 1) connected with a precise peristaltic pump (#4 Table 1) to select up to 10 different streams, and a two way injection valve (#2 Table 1) incorporated with a microchamber to load and infect samples (
FIG. 2
b,c
). The key component in sample handling is the microchamber which has been designed to allow on line sample dilution and staining (
FIG. 2
d
). The unit essentially represents a stirred tank reactor with three ports that serve as inlets and outlets. Since dilution, staining processes, and other enzymatic reactions are basically mixing process, they can be easily carried out in the microchamber in a predictable manner.
Port A and B are directly connected to the microchamber, while Port C is connected to the microchamber through an inline filter. With this inline filter, fluids can flow through the microchamber freely but cells are retained inside. To load sample into the microchamber outlet C is blocked (
FIG. 2
d
), sample is pumped through microchamber from port A to Port B. To perform on line dilution of cell samples, water is pumped through the microchamber from Port A to Port B at a predetermined flow rate F for a certain time t, such that the sample is diluted by a factor D given by
D
(
t
)=
C
(
t
)/
C
0
=e
(
F/V
)
t
(1)
where V is the volume of the microchamber and C
0
is the initial cell concentration. In practice, the volume term is modified to account for the dead volume of the connection tubing. To perfor
Natarajan Arvind
Srienc Friedrich
Zhao Rui
Beisner William H.
Friederichs Law Firm PLC
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