Synthetic biology thus far has relied upon the engineering of new cellular function
through the insertion and deletion of genetic information in living cells. This genetic
engineering based approach has progressed rapidly. There is now available a set of
well-characterized biological parts1
2
3 that can be used to build complex genetic circuitry within and between the living
cells4
5
6. Further, entire genomes can be edited7 and synthesized8, suggesting that fully
designed organisms with heretofore unseen capabilities are likely in the future.
Despite the wide range of technologies and target pathways exploited, the desire to
control microorganisms to date has always employed direct genetic intervention. The
limitations of these prevalent methods are due to the difficulties of engineering
living systems, including evolutionary pressures that may alter engineered pathways
over time and the potential long-term consequences of altering ecosystems with engineered
organisms. However, it may not be necessary to genetically modify living cells. Extant
life is already extremely complex, endowed with numerous sensory and metabolic pathways
tuned by billions of years of evolution to be efficiently responsive to changing intracellular
and extracellular conditions. A simple change in pH, for example, results in the up
and downregulation of nearly 1,000 genes in Escherichia coli
9. In other words, cells are already capable of sensing many different stimuli and
capable of performing many tasks. Therefore, it should be possible to exploit these
existing cellular pathways to control cellular behaviour without changing the genetic
makeup of the cells.
Here we explore this idea of engineering E. coli through alternative means by targeting
the sensory pathways of E. coli. To do so without altering the genetic content of
the bacterium, we instead construct artificial cells that could interact with natural
cells in order to evoke a behavioural response. The artificial cells in this system
function as chemical translators that sense molecules that E. coli alone cannot sense.
In response, the artificial cells release a molecule that E. coli can naturally respond
to, thereby translating an unrecognized chemical message into a recognized chemical
message. In this way, the sensory capabilities of E. coli are expanded without altering
the genetic content of the bacterium. The artificial cell is built with a phospholipid
vesicle containing isopropyl β-D-1-thiogalactopyranoside (IPTG), DNA, and transcription–translation
machinery. The DNA template codes for a previously selected riboswitch that activates
translation in response to the presence of theophylline10. The theophylline riboswitch
controls the synthesis of the pore forming protein α-hemolysin (αHL). Therefore, in
the presence, but not the absence, of theophylline a pore forms that releases entrapped
IPTG. E. coli alone does not respond to theophylline, and IPTG does not cross the
vesicle membrane of the artificial cell in the absence of the pore. The ability of
E. coli to receive the chemical message sent by the artificial cells is assessed in
two ways. First, the fluorescence of E. coli carrying a plasmid encoding a fluorescent
protein behind an IPTG-responsive, lac operator sequence is evaluated. Second, the
gene expression of untransformed E. coli is monitored by reverse transcription quantitative
PCR (RT–qPCR). To our knowledge, this is the first artificial, cell-like system capable
of translating unrecognized signals into a chemical language that natural cells can
recognize. The integration of artificial translator cells with natural cells represents
a new strategy to introduce synthetic features to a biological system while circumventing
the need for direct genetic manipulation.
Results
The theophylline-sensing device is functional in vitro
To build artificial cells that sense theophylline and in response release IPTG (Fig.
1), a theophylline-sensing genetic device was built with a T7 transcriptional promoter,
a theophylline riboswitch and a gene encoding a fusion between αHL and super folder
GFP at the carboxy terminus. If functioning properly, this arrangement should result
in the expression of protein and thus green fluorescence only in the presence of theophylline.
However, cell-free expression in the presence and absence of theophylline showed similar
levels of fluorescence (Fig. 2a). Since this same riboswitch was previously shown
to function in vitro
11, the sequence of the αHL-GFP gene was more closely examined. Multiple pairs of
potential ribosome binding sites (RBS) and start codons were identified within the
αHL portion of the gene that were in-frame with the GFP-encoding region. The theophylline
riboswitch controls translation, meaning that sequences behind the theophylline riboswitch
are always transcribed. Translation from the RBS within the riboswitch is activated
by direct binding of theophylline to the messenger RNA. Therefore, if additional sequences
outside of the riboswitch but within the αHL portion of the gene were recognized by
the ribosome, then regardless of the theophylline concentration, the expression of
truncated peptide products with fluorescently active GFP would have been possible.
To test if such internal RBSs were present, the theophylline riboswitch and thus the
RBS preceding the αHL-GFP sequence was deleted. In vitro transcription–translation
of this construct showed the accumulation of fluorescence over time similar to the
riboswitch containing construct (Fig. 2b). Sequence analysis revealed three potential
RBS-start codon pairs within the αHL coding portion of the gene. Of these, a putative
RBS of AAAGAA was selected as the most likely candidate for giving fluorescent protein
expression based on sequence composition and spacing12. The putative internal RBS
was removed by mutation to TCTACC, resulting in a carboxy-terminal GFP tagged K30S
E31T αHL construct. Fluorescence from this mutated construct was reduced threefold,
consistent with the removal of an internal RBS (Fig. 2b). Finally, K30S E31T αHL-GFP
was placed behind the theophylline riboswitch to test the activity of the cell-free
sensing device. A clear difference was observed between protein expression in the
presence and absence of theophylline (Fig. 2c), and the fluorescence arising in the
absence of theophylline was within 20% of the construct lacking an RBS upstream of
the full gene. The data were consistent with a functioning riboswitch sensor with
background fluorescent protein expression arising from internal RBS within αHL. Therefore,
the final artificial cellular mimic described below was built with αHL lacking a GFP-tag
to avoid complications arising from the expression of truncated fluorescent protein
product.
Active αHL is produced in response to theophylline in vitro
To ensure that the cell-free expressed αHL was active as a pore, the ability of αHL
to degrade rabbit red blood cells was assessed through a standard haemolysis assay13.
Each construct was expressed in vitro at 37 °C for 6 h after which, an aliquot was
removed and added to red blood cells. Haemolysis was quantified by measuring attenuance
at 650 nm. In the presence of theophylline, 90% haemolysis was observed when the genetic
construct containing a riboswitch-controlled αHL was expressed. The cell-free expression
of the same construct in the absence of theophylline gave haemolysis levels similar
to the negative control reactions (Fig. 2d), as was expected for a functioning theophylline
riboswitch that controls the production of αHL. Control reactions with commercial
αHL-purified protein and in vitro-expressed αHL and αHL–GFP all were fully active
(Fig. 2d, Supplementary Table 2), whereas aliquots from in vitro-expressed GFP alone
and αHL with a carboxy-terminal His-tag were inactive (Supplementary Table 2). αHL
with a carboxy-terminal His-tag was previously shown to have reduced activity14. Also,
comparison of the riboswitch activity fluorescence data with the haemolysis assay
data was consistent with the production of GFP containing protein fragments from an
internal RBS without an active αHL domain. For example, the αHL–GFP construct lacking
one of the putative internal RBSs failed to produce protein with haemolysis activity
(Supplementary Table 2), despite giving rise to fluorescence during in vitro transcription–translation
(Fig. 2b).
Artificial cells can translate chemical messages for E. coli
After demonstrating that the riboswitch was able to control the in vitro expression
of αHL in response to theophylline and that the expressed αHL molecules formed functional
pores, the component parts were next assembled inside of phospholipid vesicles to
build artificial cells. Theophylline is capable of passing through the membrane of
vesicles11. Phospholipid vesicles were generated in the presence of IPTG, transcription–translation
machinery and DNA encoding αHL under the control of the theophylline riboswitch. The
vesicles were then purified by dialysis at 4 °C to remove unencapsulated molecules.
The receiver bacterial cells were mid-exponential phase E. coli BL21(DE3) pLysS carrying
a plasmid encoding GFP behind a T7 promoter and a lac operator sequence. In this commonly
exploited system, IPTG induces the expression of a chromosomal copy of T7 RNA polymerase
in E. coli BL21(DE3) and derepresses the expression of GFP from the plasmid. Background
expression is typically low with such an arrangement because of the presence of constitutively
expressed lysozyme from pLysS, a natural inhibitor of T7 RNA polymerase.
To test if the artificial cells could function as chemical translators for E. coli,
the artificial cells were incubated with E. coli BL21(DE3) pLysS carrying the GFP-encoding
plasmid at 37 °C, and the fluorescence of E. coli was evaluated by flow cytometry.
A control reaction in which theophylline was directly added to E. coli in the absence
of artificial cells failed to show green fluorescence after 3 h (Fig. 3a). Similarly,
IPTG loaded vesicles that did not contain the machinery necessary to form pores did
not induce fluorescence in E. coli. Therefore, theophylline was not able to induce
a detectable response in E. coli, and IPTG could not cross the vesicle membrane in
the absence of αHL, which was consistent with permeability measurements (Supplementary
Fig. 1). However, when E. coli was incubated with artificial cells and theophylline,
17±10% and 69±3% of the bacteria fluoresced green after 0.5 and 3 h, respectively.
When the same experiment was repeated in the absence of theophylline, 3±1% and 24±5%
of the bacteria were fluorescent after 0.5 and 3 h, respectively (Fig. 3a,b). Longer
incubations resulted in diminishing differences between the two samples suggesting
the presence of low levels of αHL expression in the absence of theophylline. Also,
the GFP response was encoded within a medium copy number plasmid. Therefore, higher
background levels of GFP were to be expected in comparison with gene expression from
the chromosome. The flow cytometry experiments were consistent with the ability of
artificial cells to translate an unrecognized chemical signal (theophylline) into
a signal (IPTG) that E. coli could respond to.
Although the artificial cells were capable of communicating with E. coli, the induction
of GFP synthesis, as observed above, exploited an engineered response. To assess whether
artificial cells could elicit a natural, chromosomally encoded response, RT–qPCR was
used to measure gene expression from the lac operon of E. coli. The lac operon is
one of the most thoroughly characterized sensory pathways15. The presence of allolactose
(or the non-hydrolyzable analogue IPTG) induces the expression of lacZ, lacY and lacA.
To facilitate detection of E. coli responding to the chemical message sent from the
artificial cells, E. coli BL21 (DE3) pLysS were grown in LB supplemented with glucose
to decrease the background expression of the lac operon and then transferred to M9
minimal media prior to incubation with artificial cells. The artificial cells were
prepared as described for the GFP induction experiments above. After incubating together
artificial cells with E. coli in the presence and absence of theophylline for 4 h,
aliquots were collected for RNA isolation. The RNA was then reverse transcribed and
lacZ, lacY, and lacA expression quantified by qPCR. The RNA isolated from bacteria
incubated with artificial cells plus theophylline showed on average over 20-fold higher
lacZYA expression than samples incubated with artificial cells alone (calculated from
AC/(AC+theo) as shown in Fig. 3c). Taken together, the data are consistent with the
ability of artificial cells to translate chemical messages and induce both engineered
and natural pathways in E. coli.
Discussion
Direct genetic engineering of living cells is not needed to control cellular behaviour.
It is possible, instead, to coerce desired activity through communication with artificial
cells. The foundation for such technologies has already been laid by both cell-free
and in vivo studies. Engineered communication paths between living cells have been
constructed to coordinate cellular activities in response to external stimuli6
16 and are being developed for therapeutic purposes17. In these systems, sender cells
often can process information and in response release molecules that affect other
cells. What has been shown herein builds on these past efforts but does so by integrating
reconstituted, non-living systems with living cells. This allows for the genetic engineering
component of the system to be moved from the living, evolving, replicating cells to
the more controllable, ephemeral artificial cells. When the artificial cells degrade,
the natural cells go back to their original state, thereby diminishing the possibility
of unintended long-term consequences. For example, rather than engineering bacteria
to search for and clean up environmental contaminants, artificial cells could be built
to sense the contaminant molecules and in response release chemoattractants that bring
natural bacteria capable of feeding on the contaminants18 to the affected site.
Several recent reports have described the engineering of seek-and-destroy bacteria
for the eradication of tumours or bacterial infections19
20
21
22. However, these methods ultimately rely on administering living bacteria to the
patient. Artificial cells could be built to carry out similar tasks if the sensor
module of the artificial cell was designed to detect the chemical conditions associated
with the ailment. For instance, rather than spraying engineered bacteria into the
lungs of cystic fibrosis patients, artificial cells could be built to detect the presence
of Pseudomonas aeruginosa biofilms through the quorum signalling molecules that are
naturally secreted by the organism, such as N-(3-oxododecanoyl)-L-homoserine lactone,
a molecule capable of crossing membranes without the aid of transporters. Subsequently,
the artificial cells could release small molecules, for example, D-amino acids23,
to disperse the biofilm and thus clear the infection. Moreover, the use of dispersion
rather than killing would decrease the probability of the bacteria developing resistance.
Similar strategies with artificial cells could be developed to substitute for engineered
probiotics that integrate with gut microbiota24 and prevent disease25
26.
There are several limitations to these first generation artificial cells. First, heterogeneity
in membrane lamellarity and in encapsulation efficiency27 results in a mixture of
artificial cells with varying degrees of activity. Microfluidic-based methods for
compartment formation and solute encapsulation would likely alleviate many of the
complications associated with vesicle-to-vesicle and batch-to-batch variability. Also,
a system fully dependent upon the permeability properties of the membrane limits the
types of molecules that can be sensed and released. The development of specific membrane-associated
sensors and transporters will likely be necessary as the complexity of artificial
cells increase. Finally, the simple release of encapsulated molecules means that release
could result from compartment degradation as opposed to an engineered response to
the detection of a specific molecule. It is, therefore, important to develop an output
that is mediated by synthesis so that compartment degradation would only result in
the release of inactive starting molecules. An example of such a system is the biological
nanofactory described by Fernandes et al.
28 that synthesizes a signalling molecule from S-(5′-deoxyadenosin-5′)-L-homocysteine
via two enzymatic steps.
The absence of a living chassis opens up greater opportunities to assemble or biofabricate
various mechanisms or functions that would be difficult to implement with living cells.
For example, chemical systems housed within inorganic and peptide-based compartments
are capable of sensing the environment through, in part, the gating behaviour of the
non-lipid compartment29
30. Further, artificial cells can synthesize and release signalling molecules sensed
by living cells without exploiting genetically encoded parts31
32. The possibility of merging advances with non-genetically encoded and genetically
encoded parts may lead to the construction of artificial cells that are better able
to imitate natural cellular life33
34.
Methods
Genetic constructs
The gene encoding Staphylococcus aureus αHL was synthesized by Genscript. Super folder
GFP (BBa_I746916) was from the registry of standard biological parts ( http://parts.igem.org).
The theophylline riboswitch sequence was from Lynch and Gallivan10 and was amplified
from a previously described construct11. All genes were subcloned into pET21b (Novagen)
with NdeI and XhoI restriction sites. Mutagenesis was performed by Phusion site-directed
mutagenesis (Thermo Scientific). All constructs were confirmed by sequencing at Genechron
or Eurofins MWG Operon. Sequences of all the exploited constructs are listed in Supplementary
Table 1. All experiments were repeated at least three times. Data are reported as
averages with standard error, or representative runs are shown.
In vitro characterization of the riboswitch
Plasmids were amplified in E. coli Novablue (Novagen) and purified with Wizard Plus
SV Minipreps DNA Purification System (Promega). Plasmid DNA was phenol–chloroform
extracted, ethanol precipitated and resuspended in deionized and diethyl pyrocarbonate-treated
water. PCR products were purified with Wizard Plus SV Gel and PCR Clean-Up Systems
(Promega). Transcription–translation reactions used the PURExpress In Vitro Protein
Synthesis Kit (New England Biolabs) supplemented with 20 units of Human Placenta RNase
Inhibitor (New England Biolabs). Reactions were monitored by fluorescence with a CFX96
Touch real-time PCR (Bio-Rad) using the SYBR green filter set.
α-hemolysin activity
Each construct was expressed with the PURExpress In Vitro Protein Synthesis Kit at
37 °C in a final volume of 25 μl either in the presence or absence of 1.5 mM theophylline
for 6 h. Rabbit red blood cell (RBC) suspensions (adjusted to D=0.1 at 650 nm) were
added to a microplate where the reaction mixtures were serially diluted. Changes in
attenuance of the RBC suspension were measured at 650 nm with a microplate reader
(UVmax, Molecular Devices) for 30 min at 22 °C as reported in Laventie et al.
35 The results are reported as percentage of haemolysis or as the time necessary to
reach 50% of haemolysis.
Preparation of E. coli receiver cells
Mid-exponential E. coli BL21(DE3) pLysS transformed with a plasmid encoding super
folder GFP behind a T7 promoter and a lac operator sequence (CD101A12) were grown
in LB supplemented with 100 μg ml−1 ampicillin and 34 μg ml−1 chloramphenicol to an
optical density of 0.5 at 600 nm. A quantity of 200 μl aliquots in 10% (vol/vol) glycerol
were flash frozen with liquid nitrogen and stored at −80 °C for later use. Aliquots
were rapidly thawed and mixed with 2 ml LB supplemented with 100 μg ml−1 ampicillin
and 34 μg ml−1 chloramphenicol and incubated for 2 h at 37 °C with 220 r.p.m. shaking.
Finally, the cells were gently pelleted and resuspended in 1 ml M9 minimal media.
Preparation of artificial cells
Vesicles were prepared as previously described36
37. Briefly, 12.5 mg 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 12.5 mg
cholesterol (Avanti Polar Lipids) in chloroform were mixed in a round bottom flask.
A thin lipid film was made through rotary evaporation with a Buchi Rotovapor R-210
equipped with a Buchi Vacuum Pump V-700 for 5 h. A quantity of 2 ml DEPC-treated deionized
water was then added to the thin lipid film and vigorously vortexed. The resulting
liposome dispersion was homogenized with an IKA T10 basic homogenizer at a power setting
of 4 for 1 min. A quantity of 100 μl aliquots were frozen in liquid nitrogen or dry
ice and lyophilized overnight in a vacuum concentrator (Centritrap DNA concentrator,
Labconco) at 40 °C. The lyophilized empty liposomes were stored at −20 °C. A quantity
of 100 μl aliquots of freeze-dried liposomes were hydrated with 25 μl of 100 mM IPTG
(Sigma) dissolved in 50 mM HEPES pH7.6, 25 μl of the PURE system, 500 ng DNA and 20
units of human placenta RNase inhibitor (final volume of 50 μl), unless otherwise
noted. Solutions were gently mixed for 30 s.
To remove extravesicular material, the vesicles were dialyzed following a method previously
described by Zhu and Szostak38. The original membranes of 500 μl Slide-a-Lyzer dialysis
cassettes (Pierce) were exchanged with 25 mm diameter polycarbonate track-etched membranes
with a 1 μm pore size (Whatman). A quantity of 50 μl of unpurified vesicles were loaded
onto the center of the dialysis system with a 100 μl Hamilton syringe and dialyzed
against 250 ml of buffer A (50 mM HEPES, 10 mM MgCl2, 100 mM KCl, pH 7.6) with stirring.
The first four rounds of dialysis were for 10 min each. Two more rounds of dialysis
in which the buffer was changed after 30 min incubations were further performed. All
dialysis steps were carried out at 4 °C.
Artificial–natural cell communication
Purified vesicles containing DNA, the PURE system, and IPTG were incubated with E.
coli BL21(DE3) pLysS transformed with CD101A in M9 minimal media supplemented with
1 mg ml−1 of Proteinase K and 5 mM theophylline at 37 °C in a final volume of 40 μl.
Control reactions did not contain theophylline. At different time points, 1 μl was
removed and diluted 1:100 in PBS. The sample was then analysed by flow cytometry with
a FACSCanto A (BD Biosciences). The FITC filter was used for the detection of positive
cells. The incident light was at 488 nm for forward scatter (FSC), side scatter (SSC)
and fluorescence. Detection for SSC and fluorescence was at 488±10 nm and 530±30 nm,
respectively. The threshold parameters were 200 for both FSC and SSC. The PMT voltage
settings were 525 (FSC), 403 (SSC) and 600 (FITC). The flow rate was set to ‘low’.
For each sample 30,000 events were collected. Reactions were repeated three times
on three separate days. Data were analysed using FlowJo software (TreeStar, USA).
Samples were also evaluated by RT–qPCR. Here, the dialyzed vesicles and E. coli were
incubated as described above for 4 h at 37 °C. Subsequently, the total RNA was extracted
with the RNeasy Mini kit (Qiagen). A quantity of 10 μl of 500 ng of RNA was reverse
transcribed using RevertAid Reverse Transcriptase (Thermo Scientific). cDNA was quantified
with a CFX96 Touch real-time PCR (Bio-Rad) with SYBR green detection. Each sample
was diluted to 5 ng and measured in triplicate in a 96 wells plate (Bio-Rad) in a
reaction mixture containing SsoAdvanced SYBR green supermix (Bio-Rad) and 180 nM of
each primer in a 10 μl finale volume. The primers used to quantify lacZ, lacY and
lacA expression were lacZ FW: 5′-TACGATGCGCCCATCTACAC-3′, lacZ REV: 5′-AACAACCCGTCGGATTCTCC-3′,
lacY FW: 5′-GGTTTCCAGGGCGCTTATCT-3′, lacY REV: 5′-TTCATTCACCTGACGACGCA-3′, lacA FW:
5′-GCGTCACCATCGGGGATAAT-3′, lacA REV: 5′-CCACGACGTTTGGTGGAATG-3′. Gene expression
was normalized to the expression of idnT
39 with the following primers: 5′-CTGCCGTTGCGCTGTTTATT-3′ and 5′-GATTTGCTCGATGGTGCGTC-3′.
Author contributions
Design, cloning and mutagenesis of genetic constructs were done by R.L., A.C.S., J.F.,
S.P.S., M.F., and C.D.B. In vitro riboswitch activity was investigated by R.L., S.P.S.,
C.D.B., L.M., M.F. and A.C.S. αHL activity was measured by R.L., S.P.S., M.M., and
M.D.S. R.L., J.L.T., D.C., F.C. and S.P.S. ran the cell flow cytometry experiments,
and RT–qPCR was performed by R.L. and J.F. S.S.M. supervised the project. All authors
analysed and interpreted the data and contributed to the writing of the manuscript.
Additional information
How to cite this article: Lentini, R. et al. Integrating artificial with natural cells
to translate chemical messages that direct E. coli behaviour. Nat. Commun. 5:4012
doi: 10.1038/ncomms5012 (2014).
Supplementary Material
Supplementary Information
Supplementary Figure 1, Supplementary Tables 1-2 and Supplementary References