Characterization of a synthetic bacterial self-destruction device for programmed cell death and for recombinant proteins release

Abstract

BackgroundBacterial cell lysis is a widely studied mechanism that can be achieved through the intracellular expression of phage native lytic proteins. This mechanism can be exploited for programmed cell death and for gentle cell disruption to release recombinant proteins when in vivo secretion is not feasible. Several genetic parts for cell lysis have been developed and their quantitative characterization is an essential step to enable the engineering of synthetic lytic systems with predictable behavior.ResultsHere, a BioBrick™ lysis device present in the Registry of Standard Biological Parts has been quantitatively characterized. Its activity has been measured in E. coli by assembling the device under the control of a well characterized N-3-oxohexanoyl-L-homoserine lactone (HSL) -inducible promoter and the transfer function, lysis dynamics, protein release capability and genotypic and phenotypic stability of the device have been evaluated. Finally, its modularity was tested by assembling the device to a different inducible promoter, which can be triggered by heat induction.ConclusionsThe studied device is suitable for recombinant protein release as 96% of the total amount of the intracellular proteins was successfully released into the medium. Furthermore, it has been shown that the device can be assembled to different input devices to trigger cell lysis in response to a user-defined signal. For this reason, this lysis device can be a useful tool for the rational design and construction of complex synthetic biological systems composed by biological parts with known and well characterized function. Conversely, the onset of mutants makes this device unsuitable for the programmed cell death of a bacterial population.

Modelling the effects of cell-to-cell variability on the output of interconnected gene networks in bacterial populations

“…Finally, cell-to-cell variability may not be solely explained by intrinsic and extrinsic noise, but it might depend on bistability effects in gene regulatory networks [52] or on the evolutionary context that has been found to significantly affect the quantitative behaviour of single cells in several experimental studies, by increasing the failure rate of a biological function [3,44,49,53,54]. …”

Modelling for Synthetic Biology

“…Although it is reliable, the described procedure is hard to automate and provides offline results because colonies usually appear after a 12-to 24-h incubation. An easier procedure estimates cell density via turbidity measurements with a spectrophotometer: absorbance (OD at 600 nm, OD 600 ) is widely used and provides highly consistent results with the CFU method in a specific OD 600 range [27,37]. This methodology provides real-time measurements, suitable for high-throughput assays in multiwell plates, and allows the precise estimation of cell growth rate.…”

Building Spatial Synthetic Biology with Compartments, Scaffolds, and Communities

“…This takes the shape of inducible bacterial lysis, reviewed in Rice and Bayles (2008). These strategies can involve the expression of lysozyme and pore-forming proteins in response to varied triggers (Pasotti et al 2011). However, bacteria can also be made to kill specific foreign strains of bacteria with the use of specialized protein machines.…”