Difference between revisions of "Team:Yale/Description"

Project Overview

Let's make today's most important microbes easier to engineer

Overview

Our iGEM research project involves developing a framework for the implementation of genetic manipulation techniques'specifically, multiplex automated genome engineering (MAGE) and CRISPR-Cas9 systems into non-model, environmentally significant microbes. MAGE was developed as a rapid, high efficiency tool for increasing the genetic diversity of a cell population at targeted loci within the genome, and has so far been ported into the model organism Escherichia coli and a few other members of the family Enterobacteriaceae (unpublished data). CRISPR (clustered regularly interspaced short palindromic repeats)/Cas systems, based on prokaryotic adaptive immunity mechanisms, have emerged as a powerful cleavage-based genome editing technique (Cong et al. 2013).

The organisms we used to develop our framework are Rhizobium tropici CIAT and Synechococcus sp. PCC 7002. Rhizobium tropici CIAT 899 (hereafter RT-CIAT) is a nitrogen-fixing bacterium capable of forming root nodules with certain legume-producing Leucaena and Phaseolus trees (Martinez-Romero et al). Synechococcus sp. PCC 7002 (hereafter PCC7002) is a fast-growing marine cyanobacterium capable of photosynthesis and free fatty acid (FFA) production (Ruffing 2014).

We envision numerous potential applications for MAGE, CRISPR-Cas9, and other genetic manipulation techniques in these organisms; for example, the nitrogen fixation mechanisms in Rhizobium tropici CIAT could be modified to enable plant growth in otherwise hostile environments, and the FFA biosynthesis pathway of Synechococcus sp. PCC 7002 could be optimized for the production of molecules that serve as precursors to lipid biofuels. Back

Multiplex Automated Genome Engineering (MAGE) in Escherichia coli

Mutate. Grow. Screen. Repeat.

Multiplex automated genome engineering (MAGE) is an iterative, oligonucleotide-mediated genetic manipulation developed by the George Church lab at Harvard Medical School in 2009 (Wang et al. 2009). The technique relies on the introduction of degenerate (randomized) ssDNA oligos into a bacterial cell. The oligos are designed with 30-45 bp homology arms that are complimentary to the target sequence in the cell's genome up- and downstream of the degenerate sequence. Phage homologous recombination proteins Gamma, Exo, and Beta (Lambda-Red cassette) are induced, and allow the oligo to anneal to the lagging target strand as the cell's chromosome separates into single strands for replication (Gallagher et al. 2014). Desirable genotypes are then selected based upon a phenotypic change in cell populations. MAGE can be used to optimize biosynthetic pathways which are then selected for based on a desirable phenotype.

MAGE can be used to create insertions and mismatches of up to 12 bp per cycle, and deletions of up to 1 kb per cycle (Gallagher et al. 2014). Each cycle, which involves introducing degenerate oligos into cells via electroporation, inducing the Lambda-Red cassette, and growing transformed cell populations, takes approximately 2.5 hours; thus, a highly diverse population of cells resulting from multiple MAGE cycles can be created in a matter of days. Since mutations created by MAGE rely upon DNA mismatches in the chromosome to go unnoticed, mutS-deficient E. coli strains are used when executing MAGE cycles. mutS is a highly-conserved gene in the DNA mismatch repair pathway whose associated protein is responsible for identifying and marking single-base mismatches after replication (Culligan et al. 2000). ∆mutS cell populations demonstrated increased mutagenesis efficiency per MAGE cycle; thus, creating cell populations deficient in the gene is an important aspect of porting MAGE technology into non-model organisms. Back

Target Organism: Synechococcus sp. PCC 7002

A fast-growing cyanobacterium for lipid biofuel production

Synechococcus sp. PCC 7002 is a marine cyanobacterium capable of rapid growth in a wide variety of environmental conditions (Song et al. 2015). The bacterium was first isolated from the waters off Magueyes Island, in southwestern Puerto Rico, in 1962 (Ludwig and Bryant 2012). The doubling time of PCC7002 in optimized, CO₂-enriched conditions is under 3 hours, making the organism an ideal model for photosynthetic prokaryotes. PCC7002 is also a prime candidate for genetic modification: The organism's genome is fully sequenced (NCBI Taxonomy ID: 32049), its metabolic pathways are well-characterized (Hamilton and Reed 2012), and a system for protein overexpression has been developed for PCC7002 (Xu et al. 2011). Since it is native to marine environments and grows readily in seawater, the use of PCC7002 as a large-scale producer of small molecules would not place a burden on increasingly limited freshwater sources (Ruffing 2014). PCC7002 is naturally competent and readily undergoes homologous recombination with linear DNA fragments (Widger et al. 1998).

Despite its favorable characteristics for basic research and environmental applications, PCC7002s capabilities as a chassis for genetic modification have not been fully realized (Ramey et al. 2015). The organism possesses no native CRISPR systems, making it an ideal candidate to receive a foreign CRISPR/Cas system (such as that of S. pyogenes) for genomic editing (Cong et al. 2013). Its tendency towards natural recombination suggests that a high-efficiency MAGE protocol could be developed for PCC7002 with relative ease. We sought to address the gap in genetic manipulation technologies available for PCC7002 by testing and implementing our framework on the organism. Back

Potential Outcomes

Genetic Manipulation Technologies in PCC7002 and RT-CIAT

We chose PCC7002 and RT-CIAT as organisms for developing our framework due to their high potential for making a positive impact in carbon-neutral and ecological industries. PCC7002 could be engineered to produce and tolerate higher concentrations of free fatty acids (FFAs), which could be harvested and developed into lipid biofuels. MAGE is an ideal technology for modifying PCC7002's FFA biosynthesis pathway due to its ability to produce large numbers of genetic variants at targeted loci. The nitrogen-fixation mechanisms of RT-CIAT could be optimized, reducing the need for artificial, nitrate-based fertilizers in plant crops. Back