Integrative Biology

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TECHNICAL INNOVATION

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Real-time optogenetic control of intracellular

protein concentration in microbial cell cultures
Justin Melendez, Michael Patel, Benjamin L. Oakes, Ping Xu, Patrick Morton and
Megan N. McClean*
Perturbations in the concentration of a specific protein are often used to study and control biological
networks. The ability to dial-in and programmatically control the concentration of a desired protein in
cultures of cells would be transformative for applications in research and biotechnology. We developed
a culturing apparatus and feedback control scheme which, in combination with an optogenetic system,
allows us to generate defined perturbations in the intracellular concentration of a specific protein in
microbial cell culture. As light can be easily added and removed, we can control protein concentration
in culture more dynamically than would be possible with long-lived chemical inducers. Control of
protein concentration is achieved by sampling individual cells from the culture apparatus, imaging and

Received 22nd May 2013,

Accepted 22nd December 2013

quantifying protein concentration, and adjusting the inducing light appropriately. The culturing apparatus
can be operated as a chemostat, allowing us to precisely control microbial growth and providing cell
material for downstream assays. We illustrate the potential for this technology by generating fixed and

DOI: 10.1039/c3ib40102b

time-varying concentrations of a specific protein in continuous steady-state cultures of the model

www.rsc.org/ibiology

of biological networks as well as external tuning of synthetic gene circuits and bioprocesses.

organism Saccharomyces cerevisiae. We anticipate that this technology will allow for quantitative studies

Insight, innovation, integration

This manuscript presents a system for real-time feedback control of the concentration of a specific protein in cultures of microbes. An optogenetic induction
system, a microfluidic sampling device, and a culturing apparatus are integrated with computer control software to simultaneously quantify and modulate
protein concentration in cultures of the important model organism Saccharomyces cerevisiae. Using this system we are able to maintain the concentration of a
specific protein at steady-state for days, as well as make programmed perturbations such as oscillations. This technology will allow researchers to specifically
perturb a biological network of interest and collect cell material to measure multiple downstream eects. In addition, we anticipate that this technology will be
useful for controlling synthetic and natural biological networks for biotechnology applications.

1 Introduction
Properties of biological signalling and regulatory networks can be
inferred from studying their response to perturbation. Phenotypes
resulting from static network perturbations, such as gene deletion,
can reveal essential network connections. Time-varying perturbations are more informative for studying network dynamics. Indeed
an intelligent perturbation scheme can distinguish relatively similar
network models based on network output.
While experimentalists have at their disposal a wealth of
experimental technologies for measuring biological response

Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton,

NJ 08544, USA. E-mail: mmcclean@princeton.edu
Electronic supplementary information (ESI) available. See DOI: 10.1039/
c3ib40102b

366 | Integr. Biol., 2014, 6, 366--372

to perturbation, the technology for making appropriate informative perturbations has lagged behind. Typical experiments
perturb the biological network with a sudden step increase in
ligand and assay downstream responses.1,2 Such static stepshock inputs stimulate the biological network at the receptor
level, leaving many unprobed signalling steps between input and
output and do not adequately probe network dynamics. Microfluidic technology allows for the generation of time-varying perturbations; however recovery of cell material from these devices is
dicult, limiting network response assays to single-cell imaging
in combination with fluorescent reporters.35 Recent optogenetic
methods for controlling proteinprotein interactions and protein
concentration also utilize microfluidic or small culture volumes
and thus suffer similar limitations.57
Perturbations in protein concentration are often used to
interrogate biological networks. Perturbing the concentration of

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Technical Innovation

a particular protein can be used to augment the activity of a

specific biological pathway and identify the downstream transcriptional and metabolic eects.8,9 Information from such
perturbative experiments can be used to constrain and improve
modelling descriptions of biological networks. In addition,
manipulating the concentration of a protein in a synthetic or
natural biological network can be used to tune that networks
behaviour.
An ideal experimental tool would allow the experimentalist
to generate defined perturbations in the concentration of a
specific protein. This tool would allow for protein concentration to be controlled in large numbers of cells making enough
cell material available to take advantage of existing measurement technologies. This tool could also be used to externally
control protein concentration to tune the behaviour of a network of interest.
In this study, we address the limitations in existing methods
of protein concentration control by integrating an optogenetic
induction system, a cell culturing and microfluidic imaging
apparatus, and feedback control to generate programmed static
or time-varying perturbations in the intracellular concentration
of a specific protein in microbial culture. Using an optogenetic
induction system, gene expression is controlled by pulses of
light leading to increased concentration of the protein of
interest. The microbial culture device can be operated as a
chemostat to maintain steady state reproducible growth conditions. Samples of cells are rapidly removed from the culture
vessel and fed into a microfluidic device for automated protein
quantification using fluorescence imaging. A feedback scheme
is used to achieve the user-defined protein concentration. We
demonstrate the utility of this system by both clamping protein
concentration at dierent levels and generating oscillations in
protein concentration in continuous cultures of the model
organism Saccharomyces cerevisiae. This device will be an asset
to researchers seeking to interrogate, model, and control both
synthetic and natural biological networks.

2 Materials and methods

2.1

Strains and plasmids

Strains and plasmids used in this study are detailed in Tables

S1 and S2 (ESI). To test the ability of blue-light to control gene
expression and subsequent protein production we utilized
yMM1146 (Mata trp1D63 leu2D1 ura3D52) transformed with
pMM301 (PGAL1-yEVenus CEN6/ARS4 scURA3), pMM159 (pGAL4ADCIB1),10 and pMM160 (pGAL4DBD-CRY2).10 Plasmid pMM301
was constructed by cloning the engineered GAL1 promoter from
McIsaac et al.11 between NheI and XmaI sites into pRS416.12
yEVenus was cloned downstream of the Gal1 promoter between
XmaI and AscI.
For all blue-light induction and protein control experiments
in the culture apparatus we used either strain yMM1079 (Mata
trp1D63 leu2D1 ura3D52 gal1DmCherry-caURA3) or yMM1134
(Mata trp1D63 leu2D1 ura3-52 gal1DmCitrine-KanMX) transformed
with the appropriate DBD-CRY2 and AD-CIB1 plasmid constructs.

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Integrative Biology

These strains were created by replacing the native GAL1 openreading frame with mCitrine13 or mCherry14 in yMM1146
(Mata trp1D63 leu2D1 ura3D52) using standard lithium acetate
transformation.15
2.2

Media and growth conditions

Unless otherwise stated, cells were grown in synthetic complete

(SC) media16 with appropriate amino acids (tryptophan, leucine
and/or uracil) excluded to maintain plasmid constructs. For
all chemostat experiments (continuous culture experiments),
cells were grown in phosphate-limited chemostat media as
previously described.17
2.3

Culture apparatus set-up and cell sampling

A custom culturing apparatus along with custom hardware and

software was created to manage sampling, induction of the
optogenetic system, and coordinate microscopy. All aspects of
cell sampling, microscopy, image analysis, and induction feedback control were automated by this device. The chemostat
consists of a culture vessel with 27 ml working volume, an air
sparging tube to oxygenate cells and provide positive pressure
to the vessel, an inoculation port, a media port, an euent
tube, and two sampling ports for microfluidic tubing.
Hardware consists of a custom control board powered by an
ATmega328p microcontroller running the Arduino bootloader.
The control board consists of a temperature probe, a relay to
control a sampling pump, and a high-powered blue LED used
for induction (Sparkfun COM-08718) that is positioned flush
with the chemostat.
The control board communicates via serial using an FTDI
cable attached to a computer running custom Java software to
manage the microscope, image analysis, sample timing, and
feedback logic. The sample pump is connected via microfluidic
tubing in a closed loop with the chemostat such that cells are
continuously removed from the chemostat vessel, run through
a single channel microfluidic flow cell mounted on a Nikon TI
Eclipse microscope, and recycled back into the chemostat.
During protein steady state experiments the pump controlling
the microfluidic sampling loop as well as the air being sparged
into the vessel is paused to allow cells to settle on the microfluidic glass surface. After a 2 minute settling period, the
software triggers a light image and a fluorescent image to be
taken by the microscope, and the sampling pump resumes for
another 2 minutes to retrieve fresh cells from the chemostat.
Fluorescent images are segmented to identify single cells and
analyzed for fluorescent intensity. Image analysis is done using
a combination of custom software and ImageJ libraries from
the ImageJ API. The distribution of average fluorescent intensities within a sample at each timepoint is then fed into the
control scheme to determine the state of the inducing light
source. A thorough description of the control and image
processing software is available as ESI.
2.4

Microfluidic sampling device

The design for the microfluidic device consisted of a straight

channel with the dimensions 50 mm  500 mm  100 mm.

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Integrative Biology

A transparency mask was used to create a master mold using

standard photolithography techniques and SU8-50 photoresist
(Microchem, Newton, MA). A polydimethylsiloxane (PDMS)
(Slygar 184 kit, Dow Corning, Midland, MI, USA) flow chamber
was cast onto this master mold using standard soft lithography
techniques. PDMS was used at a 1 : 9 curing agent to monomer
ratio. The PDMS was cured at 65 1C for 2 hours, cut from the
mask, and plasma bonded to a glass coverslip. The chip was
mounted on a custom made aluminium holder and connected
to the culture vessel using tubing run through dedicated
sampling ports in the chemostat.
2.5

Microscopy

All microscopy was done on an epifluorescent Nikon Eclipse-TI

inverted microscope using a Nikon 40X/0.95 NA DIC Plan Apo
objective or 100x Plan Apo oil immersion objective. The microscope is equipped with a PerfectFocus system (Nikon Instruments, Melville, NY, USA) for maintaining the correct focal
plane, a Clara CCD Camera (Andor DR328G, South Windsor, CT)
for recording fluorescent emission, and a TI-S-ER motorized
stage with encoders (Nikon MEC56100). GFP emission was
visualized at 535 nm (50 nm bandwidth) upon excitation at
470 nm (40 nm bandwidth; Chroma 49002_Nikon ETGFP filter
cube, Chroma Technologies, Bellows Falls, VT, USA). mCherry
emission was visualized at 620 nm (60 nm bandwidth) upon
excitation at 545 nm (30 nm bandwidth; Chroma 96364_Nikon
Et-DSRed filter cube).
2.6

Feedback control experiments

We chose a bangbang control scheme to control the protein

concentration. Other control schemes, such as PID control, can
be easily implemented with minor modifications to the control
software.
As a proxy for protein concentration, the desired fluorescent
intensity was set in the control software prior to beginning an
experiment. To implement a control scheme, data from the
image analysis software was read into the induction controller
library as the mean fluorescent signal for all cells at a given
sampling time point. Data from each sampling and imaging
cycle was used to determine whether the induction LED would
be on or o for the next cycle. If the average fluorescence was
above the set point during a given cycle, the LED would be
turned o for the duration of the next cycle. If the average
fluorescence was below the setpoint during a given cycle the
LED would be turned on for the next cycle.
For all protein control experiments, cells were grown in the
dark overnight to saturation in the chemostat vessel with
constant aeration and then the media pump was turned on at
the rate of 2.5 ml h 1, giving a 0.9 chemostat dilution rate. After
three days, which we found to be sucient time for our culture
to reach steady-state growth and be confirmed at such, cells
reached a steady state OD600 of 0.1, at which point we began to
control protein concentration. Cells were shielded from light in
the culturing apparatus, except when purposefully exposed to
blue-light as indicated.

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Technical Innovation

2.7

Flow cytometry

All flow cytometry was performed on a BD Biosciences LSRII

Flow Cytometer. Cell culture (250500 ml) was added to 800 ml
of PBS + 0.1% Tween and kept at 4 1C until analyzed. Samples
were sonicated with 10 bursts of 0.5 seconds each prior to
processing.

3 Results
3.1

Use of light as an inducing agent in cultures of cells

To control protein concentration, we needed a way to modulate

protein production in a culture of cells at will. We chose to use
light as an inducing agent because unlike chemical inducers11,18
it is inexpensive and, more importantly, can be instantaneously
added and removed from a culture vessel.
To implement light control of gene expression and, therefore,
protein concentration in the model organism Saccharomyces cerevisiae we used fusions of the Arabidopsis thaliana proteins cryptochrome 2 (CRY2) and its interaction partner (CIB1) to DNA-binding
and activation domains (Fig. 1a).10 CRY2 and CIB1 dimerize on
blue-light exposure, bringing the DNA-binding domain and the
activation domain together to drive expression of the desired gene.
By putting our gene of interest under the control of a promoter
activated by the chosen DNA-binding domain we can induce gene
expression and subsequent protein production with blue-light.
Though other systems for light-induced protein dimerization have
been described,1922 we chose CRY2 and CIB1 because they do
not require an exogenous chromophore and rapidly associate in
response to the presence of blue-light.10
We chose to utilize constructs with CRY2 fused to the GAL4
DNA-binding domain (GAL4DBD-CRY2) and CIB1 fused to the
GAL4 activation domain (GAL4AD-CIB1).10 This allows us to
make our gene of interest blue-light inducible by placing it
under the control of the GAL1 promoter, which contains multiple GAL4 binding sites. To test this system, we created a YFP
reporter plasmid containing yEVenus under the control of the
GAL1 promoter and verified that co-transformation of a yeast
strain with the reporter plasmid and plasmids containing
the GAL4DBD-CRY2 and GAL4AD-CIB1 constructs made YFP
production blue-light inducible (Fig. 1b).
3.2

Construction of the culturing and sampling system

Previous protein-control schemes utilized microfluidic devices

or culture dishes to maintain cells and control protein levels or
localization.5,23,24 This allowed the cells to be maintained on a
microscope stage so that the downstream eects of protein
manipulation, such as induction of fluorescent transcriptional
reporters or morphological changes, could be measured with
imaging. However, these devices do not allow cells to be
retrieved easily nor in sucient amounts to provide material
for non-imaging based assays. A recent study cultured cells
and controlled protein in a 96-well plate format.7 However, cells
had to be frequently transferred to new media to maintain
reproducible growth and sampling needed to be manually
performed.

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Technical Innovation

Integrative Biology

Fig. 1 The Arabidopsis proteins CRY2 and CIB1 are used to make transcription blue-light responsive. (a) In response to blue light, CRY2 and CIB1 bind.
When fused to appropriate DNA-binding and activation domains, transcription becomes blue-light inducible. (b) CRY2 and CIB1 constructs fused to the
GAL4 DNA-binding and GAL4 activation domains respectively. When plasmids containing these constructs are co-transformed with a PGAL1-yEVenus
reporter plasmid, yEVenus expression becomes blue-light inducible.

To circumvent these issues, we created an automated cell

culturing system (Fig. 2a). Cells are grown with constant aeration
and agitation. In this system, cells can be grown in batch or
continuous culture. When operating the device as a chemostat to
allow for continuous culture, media and euent are continuously
exchanged to keep cells at a constant growth rate. Continuous
culture allows for reproducible steady-state growth conditions,
which are an ideal background over which to introduce protein
perturbations and study network response.9,25,26 We set out to
demonstrate the utility of this technology by manipulating protein
concentration in continuous cultures of Saccharomyces cerevisiae.
Blue-light is used to illuminate the culture and induce
protein expression in cells containing the optogenetic system.
We designed an automated microfluidic sampling system
which allows us to rapidly sample cells and image them for
total fluorescence, protein localization, and cellular morphology (Fig. S1, ESI). Using this system we can rapidly induce and
quantify the expression of a fluorescently tagged protein in a
population of cells. Micro-manager27 and Java code coupled
with a custom image control board integrate control of the
individual culturing components as well as the inverted microscope used to image the cells.
We tested our ability to induce protein concentration in our
culturing apparatus by using blue-light to induce mCherry

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expression. Cells being continuously cultured in the apparatus

were exposed to blue light at 460 nm (5.75 mW intensity). We
verified using mRNA FISH that the cells in the vessel produced
mCherry transcripts within a few minutes of exposure to bluelight (Fig. S2, ESI). Furthermore, mCherry protein concentrations were able to be maintained at elevated levels by constantly
pulsing blue light (Fig. 2b). Representative samples of the
cell culture analyzed using flow cytometry indicate that,
though there is cell-to-cell variability, the shift in intracellular
protein concentration is happening within the entire population (Fig. 2c).
Blue-light has been suggested to cause a stress response in
some organisms including yeast.28 We verified that blue-light
was not causing a stress response by assaying growth rate,
gene expression and localization of the light-sensitive general
stress-response transcription factor Msn2 (Fig. S3, ESI). Under
these culturing conditions and at the highest intensity of bluelight utilized in our experiments we did not observe a stress
response.
3.3

Control of protein concentration using feedback control

In order to maintain fixed protein concentrations, as well as

generate dynamically changing protein concentrations, one needs
to be able to monitor the protein concentration in real-time and

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Fig. 2 An apparatus for optical induction and feedback control of specific intracellular protein concentration in culture. (a) A schematic of the culturing
apparatus as well as the microfluidic sampling device. Cells can either be grown in batch in the culture vessel or maintained as continuous culture for
many days by running the culturing vessel as a chemostat. (b) Illumination with blue light (blue background) drives expression of the mCherry protein in
strain yMM1081 (Mat a trpD63 leu2D1 ura3D52 gal1DmCherry-caURA3 pMM159 (pGAL4AD-CIB1) pMM160 (pGAL4DBD-CRY2)) grown in the culturing
apparatus. Repeated pulses of blue light maintain protein levels at an elevated state. (c) Analysis of samples of the cell population using flow cytometry
indicates that the entire population of cells is experiencing a shift in mCherry protein concentration due to blue light induction.

adjust the blue light induction accordingly. We implemented

such real-time feedback control of the protein concentration
using our automated sampling and culturing apparatus and the
associated custom software (Fig. S4, ESI). We put yEVENUS
expression under the control of the blue-light induction expression system (Fig. 3a) and used the fast-sampling system to
image cells every four minutes and quantify total cellular
fluorescence. Based on the average fluorescent intensity of
the population as recorded by the imaging system, a bang
bang29 control scheme decides whether to turn the blue-light
on or off to illuminate the culture.

370 | Integr. Biol., 2014, 6, 366--372

Using this system, we were able to maintain distinct

fold-changes in protein concentration over the course of a
day or more in continuous cultures of Saccharomyces cerevisiae
(Fig. 3bd). Furthermore, we were able to achieve desired
protein levels whether the system was started from a low
(Fig. 3b and c) or a high (Fig. 3d) starting concentration.
We were also able to produce time-varying protein perturbations, such as oscillations (Fig. 3e) in steady-state cultures.
All control time courses were run automatically by the
apparatus and software, without user adjustments during the
experiment.

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Fig. 3 Implementation of feedback control of intracellular protein concentration in continuous cultures of Saccharomyces cerevisiae. (a) Optogenetic
control of protein concentration is implemented by creating a strain that expresses the GAL4DBD-CRY2 and VP16AD-CIB1 constructs with the protein of
interest, in this case yEVENUS, under the control of the GAL1 promoter (Mat a trp1D63 leu2D1 ura3-52 gal1DmCitrine-KanMX pMM281 (pVP16AD-CIB1)
pMM160 (pGAL4DBD-CRY2)). (bd) By programming a specific fluorescent intensity into the control software, protein concentration can be clamped at a
steady-state level (bd) for over a day in continuous cultures of S. cerevisiae. We are able to maintain the desired value whether we start from a low
(b, c) or a high (d) starting concentration. (e) Oscillations of protein concentration are also achievable over a similar timescale.

4 Conclusions
Here we have combined optogenetics, an automated culturing
apparatus, and feedback control to make programmed perturbations in intracellular protein concentration. This suggests
several types of experiments. The ability to maintain a specific
protein at a precise concentration in steady-state cultures will
allow researchers to accurately measure the relationship between
protein concentration and biological output without confounding
eects from changes in growth rate. For example, by maintaining a
specific transcription factor at dierent concentrations at
steady-state growth and sampling cells to measure gene expression response, the transfer function between transcription

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factor input and transcriptional output can be accurately

measured for multiple targets.30 One could also imagine
measuring the relationship between the concentration of a
specific enzyme and metabolic flux.
Protein concentration control can also be used for biological
network identification.11 There is already a substantial amount of
theoretical work elucidating the kinds of perturbations that are
ideal for biological network identification, much of it drawing on
theory from systems and control engineering.3133 The ability to
specifically perturb a single proteins concentration and measure
downstream signalling and transcriptional responses will allow
experimentalists to make more informative perturbations for
elucidating the kinetics and architecture of their favourite network.

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Additionally, the ability to do real-time feedback control

opens up the possibility of externally controlling synthetic
and natural networks. Feedback is notoriously hard to incorporate into engineered biological circuits yet it is often crucial for
optimal circuit function.3436 External feedback coupled continuous culture techniques will allow for rapid development
and improved function of synthetic circuits for various biotechnology applications, including metabolic engineering and
biofuel production.

Acknowledgements
We thank all members of the McClean, Noyes, and Botstein
labs for helpful comments and discussions. We thank J. Bloom
and D. Botstein for critical reading of the manuscript. We thank
D. Storton of the Princeton Microarray Facility for assistance
with gene expression experiments. We thank C. DeCoste and
C. ODonnell of the Princeton Flow Cytometry Facility for
assistance with flow cytometry. This work was supported by a
Lewis-Sigler Fellowship to MN McClean.

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