The global climate crisis is inspiring scientists worldwide to redirect interest to bacteria for sustainable solutions. The native properties of the Clostridium genus of bacteria often fortuitously suit the requirements of green chemical synthesis, such as specific substrate utilisation, or natural affinity to unusual growth conditions. To improve titres and yields of desired synthetic pathways, it is necessary to improve pathway flux without overburdening the host cell. One key limitation for synthetic biology of the Clostridium genus lies in current promoter designs; the best available synthetic promoter library is constitutive and has expression levels that are toxic to the E. coli cloning host. This project aimed to develop a synthetic biology promoter toolbox based on the TcdR orthogonal expression control parts to help to mitigate this E. coli cloning host toxicity. By introducing orthogonality, it was hypothesised that it may be possible to build large combinatorial pathway libraries, and screen vast design spaces to rapidly identify optimal variants. Initial work investigated the potential for an optimised regulator expression level in E. coli cloning hosts to ‘dampen’ the expression from synthetic promoters. Several approaches were tested for this optimisation, including a ‘top-down’ and ‘bottom-up’ design. Next, parts were characterised in a Clostridium butyricum DSM 10702 TcdR knock-in. This highlighted the unpredictability of the current gold-standard for in silico RBS design when working with non-model species. Finally, the comparison between two synthetic randomised libraries, with 25% or 79% similarity to the template, was made to assess synthetic promoter functionality. Future work will be required to take this study further, however, this work provides a novel study of the development of an orthogonal promoter library in a non-model organism.