Mammalian Transient Expression Services
Enhance the protein yield up to 100 fold
Achieve antibody titers up to 3 g/L
Researchers at the University of California San Diego have designed a cell-to-cell communication system, focusing on bacterial quorum sensing. Previous research efforts using synthetic biology depended on using self-produced small molecules that may result in temporally self-organized systems and in turn are not easily regulated. In this study, the two main principles of population control were used were inducibility and cell-to-cell communication.
Photosynthetic bacterium, Rhodopseudomonas palustris, was taken as inspiration for its signaling molecule, an organic compound found in fruits and vegetables that has been proven safe for both bacteria and human known as p-coumaric acid (pCA). The two-step pathway that yields the compound was genetically reconstructed for conversion of pCA into p-coumaroyl-HSL (pC-HSL) through production of an intermediate molecule p-coumaroyl-CoA. These genes were coded by the 4CL2nt gene originally found in the plant Nicotania tabacum. The inducible quorum sensing (IQS) system was also coupled with a fluorescent reporter protein to characterize inducibility dynamics along with a lysis gene to analyze inducible population-dependent bacterial cargo delivery. These additional genetic modifications were driven by the pLux promoter and the model organism chosen was Escherichia Coli.
Microfluidic devices were used to investigate how the IQS would behave under different concentrations of pCA. Time-lapse fluorescence microscopy was also used to observe the fluorescent expression as a representation of population dynamics. Three population behaviors were highlighted by the fluorescence microscopy. With zero inducer, asynchronous lysis was observed in conjunction with constant cell growth. Intermediate concentrations yielded sustained synchronized oscillations, while high pCA concentrations resulted in universal cell lysis.
Results from this study demonstrated the potential of functionality in a synthetic IQS circuit and the functionality of IQS by coordinating inducers as activators of circuit quiescence, both quorum-dependent and population wide constitutive expression. The system also demonstrated the ability to induce cargo release for single or multi-strain communities using temporal and spatial control between inactivated population growth, quorum enabled cyclic cargo release, and global population death.
Systems that can provide tunable behavior instead of simple steady state dynamics can elucidate greater dynamic gene expression in nature and insight into tools for designing genetic circuits. The combination of circuit activation, timed strain elimination, and non-toxicity of pCA allow this method to be suitable for application for in vivo experimentation. The study exhibited precision control over multi-strain dynamics in an IQS system, and may provide insight for further development of IQS as a synthetic biology engineering tool involving bacterial communities.