Fresh tubes of P424 and P414 plasmids made using the miniprep kit. From here, the first iteration of the build process commenced.įigure 6. The plasmids were made using New England BioLab’s Monarch® Plasmid Miniprep Kit. The first step to produce both linearized backbones was creating plasmid DNA from each of the bacterial strains. The two strains were named P424 and P414, with P424 being a high copy version and P414 a low copy version. Tomko’s lab, based out of the FSU College of Medicine. The backbones originated from bacterial strains supplied by Dr. There were two versions of the linearized backbone created. We ensured that we were revising our workflow to optimize the effectiveness of each build iteration. Failures within the build stage were taken as an opportunity to improve upon our own designs. Within the engineering cycle, our team constantly fluctuated between the build and design stages to ensure that we had the best chance of bringing our ideas to life. Photograph courtsey of Nicole.īridging the gap between imagination and creation is much simpler said than done. All vectors designed and their specific modifications.įigure 5. Given every possible variation in assembly, there were 24 vectors designed: 6 expressing CTB1 alone, and 18 co-expressing CTB1 and CTB3, with each vector utilizing different promoter and copy number variations.Ĭonfigurations For Enzyme Expression Tuningįigure 4. Given every possible variation in assembly, there were 24 vectors designed: 6 expressing CTB1 alone, and 18 co-expressing CTB1 and CTB3, with each vector utilizing different promoter and copy number variations. Overlap based assembly of enzyme sequences and their regulatory elements, with (a) modular promoters and (b) copy number variant backbones. coli for cloning, and transformation in yeast for expression.įigure 3. Workflow of our assembly: Plasmid backbone linearization and DNA fragment addition, followed by HiFi assembly, transformation in E. Assays were then implemented for enzyme and product detection, including HA epitope tagging on CTB1, 6xHis tagging on CTB3 and analytical chemistry assays for product identification and quantification inspired by existing literature (2,3,4).įigure 2. Molecular cloning was done using NEB HiFi DNA assembly, allowing DNA fragments for coding sequences and regulatory elements to be interchanged by annealing purposefully designed overlaps. Choices of plasmid copy number and promoter strength were modulated to encourage maximum production in our chassis, resulting in several combinatorial designs. beticola and sequentially inserted them into shuttle vectors for cloning in E.coli and episomal expression in S. To do this, we selected the first two enzymes in the pathway – CTB1 and CTB3 – from the genome of C. The boxed regions show the enzymes and compounds of interest in our project, as well as the sequence of metabolite synthesis and our assembly. Cercosporin biosythesis pathway following recently proposed gene cluster expansion using computational methods. With the competition timeframe and information gathered from literature in mind (enzymes required and speculated in biosynthesis, intermediate stability, and product detection protocols), we decided to transgenically express the first two enzymes (CTB1 and CTB3) in the biosynthesis pathway as a proof of concept for the yeast mediated production of cercosporin.įigure 1. beticola from their yield limiting regulatory networks and artificially control expression in a more tractable organism – Saccharomyces cerevisiae. To eliminate these restrictions, our idea was to isolate the necessary genes in C. While biosynthesis of cercosporin in organisms such as Cercospora beticola has been made extremely efficient by natural selection, its regulation has not been fine-tuned for maximum yield: Many of the genes required for biosynthesis are regulated by factors that have not been fully elucidated, and the ones we know of such as sunlight, temperature, pH, and choice of media (1) make mass production challenging. Given the complexity of the molecule and its efficient biological synthesis due to natural selection, our team sought to leverage this existing efficiency to further adapt production. This is evidenced by the increases in fungal metabolite yields in yeast attained by modifying gene regulation, swapping protein domains, fusing enzymes and co-culturing with bacteria (10,11,12,13). Our design was inspired by the potential of synthetic biology to maximimze cercosporin yield in ways that its native fungal host and chemical synthesis cannot.
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