Parts

List of Parts

Here is a list of ALL the parts we used in our project!

Improvement Of A Lactose Biosensor

In our three-plasmid directed evolution system, selection for mutants with increased enzyme activity (e.g. production of a small molecule) will require a biosensor which can tether enzyme improvement to cell fitness. We therefore needed a biosensor which gave a shallow dose-response curve in response to increasing inducer concentrations and required a very high concentration of inducer to reach saturation, thereby requiring enzyme activity to improve substantially before the cell reaches the maximum possible induction of a downstream fitness-conferring gene (e.g. sorbitol 6-phosphate dehydrogenase, or chloramphenicol acetyltransferase). Saturation can be prevented by using a high-copy number plasmid, but this still requires a non-leaky promoter with a high-dynamic range between uninduced and induced states. Since the initial plan for our project was to use our system to improve the lactose synthase activity of NmlgtB (UDP-galactosyltransferase), we attempted to identify a lactose-inducible promoter with a higher dynamic range than the wild-type lacZYA promoter.

BBa_K4451000 (COMBO-lacO1-minus35cons-minus10cons-lacOsym) is a synthetic IPTG-inducible promoter, designed by Yu et al. (2021) as part of a library of 1600 lacUV5 variants (‘Pcombo’) which aimed to elucidate the combinatorial effects of RNA polymerase and operator site strengths on overall gene expression. BBa_K4451000 possesses consensus -35 and -10 RNAP binding sites, which are flanked by LacI repressor binding sites. The synthetic ‘lacOsym’ operator is found proximal to the transcription start site, whereas the second, comparatively weaker O1 operator is found further upstream. This combination of regulatory elements was found by Yu et al. to result in a high fold-change between uninduced and induced states, and relatively low leakiness when uninduced.

Team Sheffield 2022 assembled BBa_K4451000 (IDT-synthesised gBlock) into pSB1C3-GFP (BBa_I20270) in place of the constitutive promoter BBa_J23151 via NEB HiFi assembly, to create BBa_K4451017. Though preliminary experiments found that IPTG-induced cultures expressing BBa_K4451017 gave off noticeably more fluorescence than the control plasmid (BBa_K4451016) at the same optical density, we were unable to collect any quantitative data before the project deadline.