Team:USP-Brazil/Description
Project Inspiration and Description
Description
We idealize a genetic circuit that makes a switch between two states of gene expression, based on a single signal, the blue light. The output of the circuit that senses blue light (T3 sigma fragment) from RGB system from Voigt and associatesis the input of our circuit. In order to have a real memory stored in the cells and not only a specific response to distinct inputs, we use two principles: 1) Utilize invertases for storing the states of memory in the cells; 2) Utilize two types of repressor, each one repressing one another acting as a repressor loop. The system was built in E. coli and works based on DNA-protein repression loops, recombinases capable to invert the DNA orientation of a promoter region and a light-responsive genetic circuit. In addition, fluorescent proteins such as GFP, RFP and YFP function as output for our circuit. We also aim to compare the efficiency between the promoter of the light-induced system and chemical-ones, by evaluating strength and leak.
Our inspiration
Our inspiration came from our own environment: Budget is a bottleneck for a lot of different projects, from academics to the industry and in Brazil this is a reality more often than it should. Sometimes not only the costs of the project itself – substrates, catalyzers- but also the management of it. Manufacturing has costs that involve different resources that must be considered. In the recent years, different countries around the world are facing the fourth industrial revolution (or 4.0 industry) which is characterized by a set of technologies that allow the fusion of the physical, digital and biological world. Brazil ranks 69th in the Global Innovation Index, with industry productivity falling in the last 3 years. Still, in the report "Readiness for the Future of Production Report 2018" (WEF) it shows that the country is in the 47th position in the industry's production vectors. In order to think of a possible solution for this initial problem, we thought about automation, however, it's still expensive. At the same time, trying to integrate bioproduction (made in bacteria) with electrical signals is not trivial and can take a long time to turn into a real world useful product. So, we consider that light could be a possible solution. This would be contributing as well to the sustainable development of the Brazilian economy, coming in line with 4.0 industry that still faces several obstacles. In fact, the fine-tuning of gene expression that will be provided by light control, make production strategies more viable and versatile, opening up many possibilities of production that aren't possible with the use of chemical induction (standard use in Biotech industries). That way, our idea is to use led light as a production vector of Biotechnology industries and for that we are going to: optimize the light inductors plasmids, improve their control by light and facilitate their use and application by making a bacteria strain with a light inducible circuit that we call “gene switch”.
Our goals
In order to solve this problem we aim to use RGB light inducer as a innovative project strategy, focusing in three main work fronts: Characterize and optimize light promoters; Build a programmable genetic circuit using this induction; Create E. coli strains, ready to receive a light-inducible plasmid and start production right in time. Our first part of the project is based on the comparison between promoters that use chemical inductors versus promoters that use light as an inductor (obtained from Synthetic Biology Center, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA, in Christopher A. Voigt lab). In this section we aim to compare strengths and leak for further optimization of the light-responsible promoters. In the second part we are going to build a circuit that control gene expression in bacteria by a single light pulse. This circuit is based on the action of recombinases, inhibition loop and the light-promoter.
References
Our Government site: http://www.industria40.gov.br and the papers we consulted: Fernandez-Rodriguez, Jesus, et al. "Engineering RGB color vision into Escherichia coli." Nature chemical biology 13.7 (2017): 706. Fernandez-Rodriguez, Jesus, et al. "Memory and combinatorial logic based on DNA inversions: dynamics and evolutionary stability." ACS synthetic biology 4.12 (2015): 1361-1372 Ham, Timothy S., et al. "A tightly regulated inducible expression system utilizing the fim inversion recombination switch." Biotechnology and Bioengineering 94.1 (2006): 1-4 Potapov, Vladimir, et al. "Comprehensive profiling of four base overhang ligation fidelity by T4 DNA Ligase and application to DNA assembly." ACS synthetic biology 7.11 (2018): 2665-2674
USP-Brazil
