Controlling Growth Rates of Escherichia coli

through Metabolic Loading

Meghan Tighe, Razan Alnahhas, Andrew Hirning, Matthew Bennett
1. Olin College of Engineering 2. Department of Biosciences, Rice University 3. Institute of Biosciences and Bioengineering, Rice University

Use of Microfluidic Devices

Introduction
In synthetic biology, it is very important to be able to
exercise control over the conditions in which engineered
systems are tested and grown. One particular challenge is
controlling growth rates. This study explores a method of
controlling the growth rates of multiple strains of Escherichia
coli in a complex system through metabolic loading.
Two different strains of E. coli are used, each with
different inducible fluorescent proteins. The introduction of
their respective inducers allows the corresponding cells to
begin to fluoresce, increasing their metabolic load, which in
turn decreases their growth rate. Visualization of the
changes in growth rate is also made possible through the
fluorescent proteins.
The use of microfluidic devices allows both close
monitoring of cells and control of substances added to the
media. Experiments are run overnight and the use of timelapse fluorescent microscopy allows for the tracking of
individual cells as they divide and fluoresce.

Mixers
Dial-A-Wave

The microfluidic device used for this experiment contains a critical feature
called the Dial-A-Wave (DAW). The DAW allows for more than one type of
media (in this case, media with or without inducer) to be given at specified
ratios. The DAW design is shown below.
Results confirm the hypothesis that introduction of fructose
causes heightened levels of cyan fluorescence.

Conclusions and Future Work

The two strains were designed as shown below. Each has

inducible fluorescence. Weak repressors as well as a weak
degradation tag were used, which creates a low level
constitutive fluorescence allowing for visualization and
identification of the cells.
pLac
CFP

Figure modified from Shis et. al (2014).

Acknowledgements and References

Ferry, M. S., I. A. Razinkov, and J. Hasty. "Microfluidics for Synthetic Biology: From
Design to Execution." Synthetic Biology Methods for Part/Device Characterization and
Chassis Engineering. Ed. Christopher Voigt. Vol. 497. N.p.: Elsevier, 2011. 295-372. Print.
Methods in Enzymology.

pLac
Trehalose

An experiment was run to examine induced and uninduced

fluorescence levels. The Fructose-CFP strain of cells was
used. Media with inducer was added for four hours at
intervals; it can be seen by the mCherry fluorescence of a dye
that was added to the media. CFP expression in the cells is
represented in blue in the graph below.

Cell trap

Experimental Design

Fructose

Preliminary Fluorescence Assessment

YFP

Shis, David L., Faiza Hussain, Sarah Meinhardt, Liskin Swint-Kruse, and Matthew R.
Bennett. "Modular, Multi-Input Transcriptional Logic Gating with Orthogonal LacI/GalR
Family Chimeras." ACS Synth. Biol. ACS Synthetic Biology 3.9 (2014): 645-51. Web.
This work was supported by an NSF award from the Division of Biological Infrastructure
No. 1262296.

These results show that the system works as intended

and can be used to investigate the effects of metabolic
loading on growth rates. However, the conclusions that can
be drawn for these results are limited in two regards. First,
the cells had to be cultured in minimal media to avoid
unintended induction. This greatly slowed cell growth overall
and meant that the cell trap had not filled by the time that
the experiment had begun. Also, only one strain was cultured
in this experiment. Since growth rates are largely relative, an
experiment with both strains would allow a better analysis.
Future work on this project will take a new direction.
Each cell strain will have two fluorescent proteins: one that is
induced to increase load and one that is constitutively
expressed to identify the strain of each cell. The constitutively
expressed protein will be either YFP or CFP. In each strain,
mCherry will be induced by either Arabinose or IPTG, neither
of which is present in LB. This will allow the cells to be
cultured in regular LB without being accidently induced and
will simplify the process of analyzing growth rates.