Team:Thessaloniki/Description
Project Description
Project Description
1.Motivation
Since the very first days of our brainstorming, our primary objective was to create something that could have an immediate impact on the scientific community. Among other things, the unpredictability that characterizes biological systems immediately drew our attention. Even though Synthetic Biology has come a long way in designing orthogonal and predictable systems, there are still many interfering factors, obstructing consistency and functionality. One such factor is gene/plasmid copy number. For that reason we decided to create a tool that enhances stability and robustness of synthetic biological systems. We achieve that by engineering stabilized promoters which produce the same output regardless of their biological context in order to render synthetic biological systems more predictable and reliable.
2.Why is Copy Number independence useful?
Despite the fact that copy number is, usually, regarded as being constant, many factors related with the internal and external environment of the cell contribute to copy number variation both over time and between different cells. [2]
Gene Expression is characterised by a positive correlation with the plasmid copy number. Thus, variations in copy number lead to variations in gene expression, affecting the function of the system. Particularly, in complex systems that require high precision, such as metabolic pathways and multi-input logic gates, accumulation of perturbations caused by variations in their individual parts greatly affect the functionality of said system.
Regulating the expression strength is usually achieved via the replacement of regulatory elements such as RBSs and Promoters. Still, this does not ensure a predictable behaviour over different genetic contexts, therefore re-tuning is mandatory to maintain the system’s proper function.
3.GALENE
This year we built upon the work of Shapiro et. al., [1] implementing an incoherent FeedForward Loop network motif that consists of a regulatory element which compensates for changes in copy number, thus stabilizing the promoter. We provide Galene to the iGEM Community, a genetic toolbox that enhances the stability of biological systems by disassociating transcription from gene copy number and provides translational control of protein production.
Stabilized promoters are promoter sequences that achieve expression independent of gene/plasmid copy number. They possess great potential for usage as a synthetic biology tool with many applications, ranging from part characterization to metabolic pathway and genetic circuitry fine-tuning []. Balancing gene expression in synthetic biological systems is a key parameter for their proper function. By achieving gene/plasmid copy number independence, such systems become more robust, predictable and less prone to perturbations arising from both their internal and external environment.
Through model driven feedback, we accomplish promoter stabilization using 2 distinct systems, each with its own advantages: TAL Effectors and CRISPR interference. We have, also, designed and simulated a 3rd stabilization system using Attenuator-interacting sRNA regulators.
Wanting to expand on the stabilized promoters system, we designed a theophylline Riboswitch that would allow, on top of the stabilization of a promoter, its induction and activation to the desired expression level.
- inducibility
4.Possible Applications of Stabilized Promoters
Genome Insertion
The output of genetic circuits inserted into the genome is affected both by the number of inserted copies and their distance from the Origin of Replication (ORI). [3] Stabilized promoter systems can be transferred to genome, maintaining the circuit’s level of expression, independent of the position of the inserted gene relative with the origin of replication.
Medicine
Precise control of genetic systems is of utmost importance when they are applied for therapeutic purposes. [4] Stabilized promoters can be used to introduce predictability and increase specificity in contemporary therapeutic methods, such as gene and cancer therapies, which are based upon the implementation of sophisticated synthetic biological circuits.
Toxic Protein Production
Production of highly toxicity proteins requires tight control of gene expression, so that sufficient expression can be achieved before host cells become dysfunctional. [5] Stabilized promoters can be used for sufficient expression before toxicity in the host cells comes into play.
Metabolic Engineering
The enzymes participating in metabolic pathways must be expressed in certain stoichiometric ratios, as variability in their expression could disrupt the system’s proper function. [6] Stabilized promoters can be utilized as regulatory tools for the expression of genes participating in metabolic pathways, increasing the system’s robustness and performance.
Large Scale Production
Genetic Systems used for industrial production require high fidelity to cope with the varying conditions inside a bioreactor [7] that might influence plasmid’s copy number. On those lines, stabilizing the expression of genes crucial in the production process, has the potential to increase the yield. Copy Number variation can influence the expression of fermentation-related genes in organisms used for industrial production, such as Saccharomyces cerevisiae. [8] Stabilized promoters can be a valuable tool for the control of genes related the process of fermentation and, therefore, improve the quality of the final product.