Engineering transcriptional gene circuits in probiotics and mini SimCells for bacteria therapy

Thesis

Numerous bacteria associated with human health (probiotics and pathogens) exhibit inherent properties in terms of targeting, penetrating, proliferating and killing solid tumours through various mechanisms. To rationally redesign these microorganisms into living medicine, there is a lack of genetic software with suitable induction characteristics and compatible cellular chassis with enhanced biosafety, owing to the stringent requirements and controllability during medical practice. Consequently, the development of bacteria therapy lags behind compared to other applications offered by synthetic biology.

The goal of this thesis is to address these two significant challenges to realise the full potential of bacteria therapy. Three different transcriptional control systems were constructed: (1) an aspirin/salicylate responsive regulatory SalR:PSal system (2) a DNA damage responsive Vibrio recA SOS system and (3) a nitric oxide responsive NorR:PNorV system. The system performance was rationally re-designed and optimised based on the detailed molecular mechanism. A universal competitive binding mechanism was found during the tuning process, where the expression level of the regulator played a dominant role in system dynamics and displayed dual activator/repressor features dependent on the state of inducer binding. To enhance the biosafety and usability, these genetic software were characterised and optimised in the cellular context of a probiotic chassis strain, E. coli Nissle 1917 and tested in the non-dividing anucleate E. coli mini SimCells, which are minicells with intact transcriptional/translational machinery. An optimised purification protocol was developed with a final yield of 5 × 10^9 mini SimCells/ml and high purity (total elimination of parent cells). Optimised sensor construct loaded in mini SimCells displayed functional responsive dynamics that (1) are robust against environmental fluctuation, (2) displayed faster response time (<30 mins), and (3) maintained medically relevant sensitivity. These findingsvalidated the programmability of mini SimCells to execute both ơ54 and ơ70 based transcriptional circuits and bypass host interference caused by global resource allocation or external fluctuation.

To conclude, this thesis provides a systematic workflow of designing therapy tailored transcriptional software and its functional deployment in mini SimCells. The circuits allow both external controls using established medical methods such as chemo/radiotherapy, aspirin injection or internal triggering via in-situ disease biomarkers, pave ways for the future pre-clinical development. Furthermore, this thesis demonstrates the potential of mini SimCells and its design guideline as a minimum version of bacteria-bot systems to achieve robust and sensitive performance, making it a preferred choice in clinical practice.

Authors: Chen, X

• DOI: 10.5287/ora-4rokxzbz4
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: simcell; bacteria therapy; synthetic biology; minicell
• Subjects: Synthetic biology