Stimuli-responsive vesicles as distributed artificial organelles for bacterial activation

Abstract

Intercellular communication is a hallmark of living systems. As such, engineering artificial cells that possess this behavior has been at the heart of activities in bottom-up synthetic biology. Communication between artificial and living cells has potential to confer novel capabilities to living organisms that could be exploited in biomedicine and biotechnology. However, most current approaches rely on the exchange of chemical signals that cannot be externally controlled. Here, we report two types of remote-controlled vesicle-based artificial organelles that translate physical inputs into chemical messages that lead to bacterial activation. Upon light or temperature stimulation, artificial cell membranes are activated, releasing signaling molecules that induce protein expression in Escherichia coli. This distributed approach differs from established methods for engineering stimuli-responsive bacteria. Here, artificial cells (as opposed to bacterial cells themselves) are the design unit. Having stimuli-responsive elements compartmentalized in artificial cells has potential applications in therapeutics, tissue engineering, and bioremediation. It will underpin the design of hybrid living/nonliving systems where temporal control over population interactions can be exerted.

Keywords: artificial cells; artificial organelles; cell signaling; membranes; synthetic biology.

Fig. 1.

Controllable activation of bacteria using light- or temperature-responsive artificial organelles. (A) Cartoon of stimuli-triggered activation. Vesicle-based artificial organelles are assembled containing a stimuli-responsive lipid within the lipid membrane. Upon external stimulation, pores open in the membrane, and the encapsulated cargo is released, thus inducing protein expression in E. coli. (B) Structure of DC_8,9PC and photopolymerization reaction upon light (254 nm) irradiation. The photopolymerization of DC_8,9PC causes the formation of pores at the grain boundaries of the lipid membrane. (C) Structure of DPPC and 16:0 lysoPC. The presence of 16:0 lysoPC in the lipid membrane promotes the formation of pores at the grain boundary between gel and liquid phase lattices when the temperature approaches the T_m.

Fig. 2.

Activation of bacterial cells using light-responsive artificial organelles. (A) YFP production upon irradiation. Fluorometry results depict how irradiated artificial cells (red, Left) achieve a twofold increase in YFP production compared with expression using nonirradiated artificial cells (blue, Right). Each data point corresponds to independent experiments. Solid lines represent the mean, error bars correspond to 1 SEM (n = 11). P values calculated using the 99.5% confidence interval (**P < 0.005). (B) Histograms of YFP expression obtained using flow cytometry. E. coli cells without added artificial cells (brown, Bottom) are used as a control for their intrinsic fluorescence. The addition of irradiated artificial cells increases the number of bacterial cells expressing YFP from ∼43% with nonirradiated artificial cells (blue, Middle) to ∼85% (red, Top).

Fig. 3.

Temperature-controlled activation of protein expression in bacteria using artificial organelles. (A) Ex situ activation. The artificial cells are heated to release the inducer before being added to the bacteria. Fluorometry results depict how heated artificial cells (red triangles, Left) achieve a 1.4-fold increase in GFP production compared with expression using nonirradiated artificial cells (blue open triangles, Right). The histograms obtained using flow cytometry show that the addition of heated artificial cells increases the number of bacterial cells expressing GFP from ∼28% with nonheated artificial cells (blue, Middle) to ∼37% (red, Top). E. coli cells alone (brown, Bottom) are used as a control for their intrinsic fluorescence. (B) In situ activation. Both artificial cells and bacteria are mixed together before the activation step. Fluorometry analysis of GFP expression of systems where the artificial cells have been activated (red stars, Left), or not (open blue stars, Right), shows a 1.3-fold increase in GFP production upon activation. Flow cytometry analysis shows that activated samples of artificial cells (red, Top) induce expression of GFP in 60% of the bacterial cells. Nonactivated samples (blue, Middle) show 50% of cells expressing GFP. Each data point corresponds to independent experiments. Box plots: solid lines represent the mean, error bars correspond to 1 SEM (n = 8). P values calculated using the 99.5% confidence interval (**P < 0.005).

Fig. 4.

Encapsulation efficiency and triggered content release from the artificial cells. (A) 254 nm light produces the photopolymerization of DC_8,9PC, which can be followed (blue) by measuring the absorbance peak appearing at 475 nm. Photopolymerized DC_8,9PC forms pores, resulting in the release of calcein (Left panel, red, normalized data using Eq. 1 from Methods). The thermoresponsive lipid vesicles release their cargo through the pores that are generated at the grain boundaries when gel and liquid phases coexist near the T_m (43 °C, Right panel, red). At lower temperatures (37 °C, Right panel, black), the lysoPC does not form pores. Error bars show 1 SD, (n = 3). (B) Encapsulation efficiency for the light-responsive composition is estimated by measuring unencapsulated IPTG with LC-MS. When purifying the artificial cells using SEC, free unencapsulated IPTG appears in the fractions that elute after the vesicles. The IPTG in the fractions eluting after the artificial cells is measured at [M + H]⁺ = 239. Error bars show 1 SEM, (n = 3). (C) The encapsulation and the purification efficiency of the temperature-sensitive artificial cells are determined using an enzymatic kit to measure rhamnose. Vesicles purified via SEC were analyzed before (23 °C, pink) and after (43 °C, cyan) stimulation.