The precision and repeatability of in vivo biological studies is predicated upon methods for isolating a targeted subsystem from external sources of noise and variability. However, in many experimental frameworks, this is made challenging by nonstatic environments during host cell growth, as well as variability introduced by manual sampling and measurement protocols. To address these challenges, we developed Chi.Bio, a parallelised open-source platform that represents a new experimental paradigm in which all measurement and control actions can be applied to a bulk culture in situ. In addition to continuous-culturing capabilities, it incorporates tunable light outputs, spectrometry, and advanced automation features. We demonstrate its application to studies of cell growth and biofilm formation, automated in silico control of optogenetic systems, and readout of multiple orthogonal fluorescent proteins in situ. By integrating precise measurement and actuation hardware into a single low-cost platform, Chi.Bio facilitates novel experimental methods for synthetic, systems, and evolutionary biology and broadens access to cutting-edge research capabilities.

A type of chromosome-free cell called SimCells (simple cells) has been generated fromEscherichia coli,Pseudomonas putida, andRalstonia eutropha.The removal of the native chromosomes of these bacteria was achieved by double-stranded breaks made by heterologous I-CeuI endonuclease and the degradation activity of endogenous nucleases. We have shown that the cellular machinery remained functional in these chromosome-free SimCells and was able to process various genetic circuits. This includes the glycolysis pathway (composed of 10 genes) and inducible genetic circuits. It was found that the glycolysis pathway significantly extended longevity of SimCells due to its ability to regenerate ATP and NADH/NADPH. The SimCells were able to continuously express synthetic genetic circuits for 10 d after chromosome removal. As a proof of principle, we demonstrated that SimCells can be used as a safe agent (as they cannot replicate) for bacterial therapy. SimCells were used to synthesize catechol (a potent anticancer drug) from salicylic acid to inhibit lung, brain, and soft-tissue cancer cells. SimCells represent a simplified synthetic biology chassis that can be programmed to manufacture and deliver products safely without interference from the host genome.

The precise characterisation and manipulation of in vivo biological systems is critical to their study.1 However, in many experimental frameworks this is made challenging by non-static environments during cell growth,2, 3 as well as variability introduced by manual sampling and measurement protocols.4 To address these challenges we present Chi.Bio, a parallelised open-source platform that offers a new experimental paradigm in which all measurement and control actions can be applied to a bulk culture in situ. In addition to continuous-culturing capabilities (turbidostat functionality, heating, stirring) it incorporates tunable light outputs of varying wavelengths and spectrometry. We demonstrate its application to studies of cell growth and biofilm formation, automated in silico control of optogenetic systems, and readout of multiple orthogonal fluorescent proteins. By combining capabilities from many laboratory tools into a single low-cost platform, Chi.Bio facilitates novel studies in synthetic, systems, and evolutionary biology, and broadens access to cutting-edge research capabilities.

The ability to directly modify native and established biofilms has enormous potential in understanding microbial ecology and application of biofilm in 'realworld' systems. However, efficient genetic transformation of established biofilms at any scale remains challenging. In this study, we applied an ultrasoundmediated DNA delivery (UDD) technique to introduce plasmid to established non-competent biofilms in situ. Two different plasmids containing genes coding for superfolder green fluorescent protein (sfGFP) and the flavin synthesis pathway were introduced into established bacterial biofilms in microfluidic flow (transformation efficiency of 3.9 AE 0.3 × 10 -7 cells in biofilm) and microbial fuel cells (MFCs), respectively, both employing UDD. Gene expression and functional effects of genetically modified bacterial biofilms were observed, where some cells in UDD-treated Pseudomonas putida UWC1 biofilms expressed sfGFP in flow cells and UDD-treated Shewanella oneidensis MR-1 biofilms generated significantly (P < 0.05) greater (61%) bioelectricity production (21.9 AE 1.2 µA cm −2 ) in MFC than a wild-type control group (~13.6 AE 1.6 µA cm −2 ). The effects of UDD were amplified in subsequent growth under selection pressure due to antibiotic resistance and metabolism enhancement. UDD-induced gene transfer on biofilms grown in both microbial flow cells and MFC systems was successfully demonstrated, with working volumes of 0.16 cm 3 and 300 cm 3 , respectively, demonstrating a significant scale-up in operating volume. This is the first study to report on a potentially scalable direct genetic engineering method for established non-competent biofilms, which can be exploited in enhancing their capability towards environmental, industrial and medical applications.