Periodic Reporting for period 1 - Evolving together (Directed co-evolution via engineered cell-to-cell communication)
Okres sprawozdawczy: 2021-05-01 do 2023-04-30
Evolving Together aims to tackle one of the current challenges in biotechnology which consists of the co-evolution of two target molecules and to study DNA repair. Directed evolution is a method in protein engineering that mimics the Darwinian evolution in the lab. The importance of this technique was recognized with the Nobel price in Chemistry in 2018, and has been used to evolve hundres of proteins with different applications, and has revolutionized very different fields from neuroscience, to chemical catalysis, with special relevance in main biotechnological processes. In directed evolution, the genetic variability is induced or created artificially, and the library is subject to a biochemical assay where the desired physicochemical properties of the enzyme are harnessed for its enrichment from the rest of the pool. This process normally is iterated several times until a bigger percentage of the population displays the desired evolved features. Traditionally, directed evolution targets one single protein at a time. In evolving together, we plan to co-evolve simultaneous two different proteins. To showcase this new approach, we have selected a low-affinity antigen-antibody pair, which is going to be displayed at the surface of the yeast strain Saccharomyces cerevisiae (Sc), also known as Baker’s yeast.
The development of a platform for the co-evolution of two different proteins simultaneously and a reporter system to study DNA repair in yeast will be highly desirable for many reasons. The development of this platform shed some light on other more biologically relevant processes such as cellular communication and differentiation. Moreover, antibody engineering and maturation have been the focus of many biotechnological companies, and the development of this platform will have immediate applications. Finally, the design of a genetic system that enables to visualize in a quantitive manner the different DNA-repair mechanisms will be ideal to establish new screening assays for drugs that affect the DNA repair machinery. This area of research has been propelled in the last years by the increase in research on genome engineering and editing.
The development of a platform for the co-evolution of two different proteins simultaneously and a reporter system to study DNA repair in yeast will be highly desirable for many reasons. The development of this platform shed some light on other more biologically relevant processes such as cellular communication and differentiation. Moreover, antibody engineering and maturation have been the focus of many biotechnological companies, and the development of this platform will have immediate applications. Finally, the design of a genetic system that enables to visualize in a quantitive manner the different DNA-repair mechanisms will be ideal to establish new screening assays for drugs that affect the DNA repair machinery. This area of research has been propelled in the last years by the increase in research on genome engineering and editing.
To start Evolving together, the initial constructs encoding a low-affinity antigen and antibody were cloned by Golden Gate assembly. The yeast strain Saccharomyces Cerevisiae was used, and both proteins were displayed at the yeast surface. The expression analysis of the antibody and the antigen was done by Fluorescent-Activated Cell Sorting (FACS) and fluorescent microscopy. In parallel, several GPCRs were also cloned and expressed. These GPCRs are sensitive to pheromones, which trigger a signaling pathway that is coupled to the expression of a reporter gene were also cloned.
Analysis of protein interactions between the sender and receiver cells showed that the expression of the reporter gene seems to be independent of the production of the pheromone. This leaky background expression was attempted to be amilorated by fine-tuning the expression levels and by changing different receptor-pheromone pairs. A library of antigen-antibodies was created by error-prone PCR, which is a method to introduce random mutations at the DNA level which will be translated at the protein level. the frequency and density of the mutation introduced were analyzed by sequencing.To minimize this background, different approaches were explored: (a) The destination vector was digested with a third restriction enzyme and was purified by agarose gel. (b) New yeast strains were purchased from a commercial source. (c) Different media and reagents were used in a side-by-side comparison. Unfortunately, none of these approaches lead to finding a solution to this problem. Therefore, we could not deliver the WP4, and subsequently, the WP5.
Based on the difficulty to implant a co-directed evolution platform and a selection scheme, we decided to harness the DNA-repair reporter to create a novel genetic system to evaluate the recombination pattern of two given DNA sequences. After a literature survey, we decided to harness the power of synthetic biology to build a genetic circuit to discern and label the different DNA-reparation mechanisms in yeast. Due to its genetic background, the yeast strain Saccharomyces cerevisiae has become the model organism to study genetic recombination. Importantly, the knowledge generated has been successfully extrapolated to other organisms or cell lines such as mammalian cells. This genetic circuit is based on the conditional expression of fluorescent proteins driven by the different mechanisms of DNA reparation. The cloning of this reporter system was successfully done.
The reporter gene was tested in different genetic strains bearing a knock-out gene belonging to the epistasis family of the gene RAD52. As expected, the knock-pots of the RAD50, RAD52 and XRS2 genes, showed a different pattern, as the DNA-reparation mediated by HR is compromised. The turn-on of this reporter gene relies on a DNA-double strain break so I designed and cloned a CRISPR-Cas9 system to induce this cleavage in living cells. Satisfactorily, I observed that the cleavage was induced and the reporter gene got activated.
At this point, I expanded the nature of the reporter gene to enable the detection of another mechanism of DNA reparation which is called single-strand alignment (SSA). In addition, I also evaluated the differences in the performance of the reporter if it is encoded in an episomal vector or integrated into the genome.
I would like to add that I got involved in another project within the lab that involved the synthesis of the chromosome XI of Saccharomyces cerevisiae. This effort belongs to the Sc2.0 Project, led by Dr. Jef Boeke at the Johns Hopkins University (now at the New York University), and is the first attempt to synthesize a eukaryotic cell genome. The goal of the Sc2.0 Project is to synthesize the entire yeast genome, which consists of 16 linear chromosomes, about 6,000 genes and a total of 12 Mb nonredundant of DNA. My role will consist of evaluating the growth rates of yeast bearing a synthetic version of the Chromosome XI in different conditions.
Analysis of protein interactions between the sender and receiver cells showed that the expression of the reporter gene seems to be independent of the production of the pheromone. This leaky background expression was attempted to be amilorated by fine-tuning the expression levels and by changing different receptor-pheromone pairs. A library of antigen-antibodies was created by error-prone PCR, which is a method to introduce random mutations at the DNA level which will be translated at the protein level. the frequency and density of the mutation introduced were analyzed by sequencing.To minimize this background, different approaches were explored: (a) The destination vector was digested with a third restriction enzyme and was purified by agarose gel. (b) New yeast strains were purchased from a commercial source. (c) Different media and reagents were used in a side-by-side comparison. Unfortunately, none of these approaches lead to finding a solution to this problem. Therefore, we could not deliver the WP4, and subsequently, the WP5.
Based on the difficulty to implant a co-directed evolution platform and a selection scheme, we decided to harness the DNA-repair reporter to create a novel genetic system to evaluate the recombination pattern of two given DNA sequences. After a literature survey, we decided to harness the power of synthetic biology to build a genetic circuit to discern and label the different DNA-reparation mechanisms in yeast. Due to its genetic background, the yeast strain Saccharomyces cerevisiae has become the model organism to study genetic recombination. Importantly, the knowledge generated has been successfully extrapolated to other organisms or cell lines such as mammalian cells. This genetic circuit is based on the conditional expression of fluorescent proteins driven by the different mechanisms of DNA reparation. The cloning of this reporter system was successfully done.
The reporter gene was tested in different genetic strains bearing a knock-out gene belonging to the epistasis family of the gene RAD52. As expected, the knock-pots of the RAD50, RAD52 and XRS2 genes, showed a different pattern, as the DNA-reparation mediated by HR is compromised. The turn-on of this reporter gene relies on a DNA-double strain break so I designed and cloned a CRISPR-Cas9 system to induce this cleavage in living cells. Satisfactorily, I observed that the cleavage was induced and the reporter gene got activated.
At this point, I expanded the nature of the reporter gene to enable the detection of another mechanism of DNA reparation which is called single-strand alignment (SSA). In addition, I also evaluated the differences in the performance of the reporter if it is encoded in an episomal vector or integrated into the genome.
I would like to add that I got involved in another project within the lab that involved the synthesis of the chromosome XI of Saccharomyces cerevisiae. This effort belongs to the Sc2.0 Project, led by Dr. Jef Boeke at the Johns Hopkins University (now at the New York University), and is the first attempt to synthesize a eukaryotic cell genome. The goal of the Sc2.0 Project is to synthesize the entire yeast genome, which consists of 16 linear chromosomes, about 6,000 genes and a total of 12 Mb nonredundant of DNA. My role will consist of evaluating the growth rates of yeast bearing a synthetic version of the Chromosome XI in different conditions.
At present, the area of synthetic genomes and genome editing techniques have propelled the development of genetic systems that enable the understanding and evaluation of different DNA repair mechanisms. While there are some examples in mammalian cells, to the best of our knowledge, there is no reporter system that works in yeast, specifically in the yeast strain Saccharomyces cerevisiae, which is a model organism for genetics and microbiology with massive industrial applications. Moreover, its simple design facilitates its use in other microorganisms whose DNA repair mechanisms and preferences are less clear. In the future, this reporter could be harnessed to establish high throughput methods for the screening of drug candidates that can enhance one pathway over the other. Finally, I would like to add that I am still working on the development of the platform for the directed co-evolution of two proteins.