Overview

Our iGEM research project involves porting Multiplex Automated Genome Engineering (MAGE) technology into two prokaryotic organisms—Sinorhizobium meliloti 1021 and Synechococcus sp. PCC 7002—for the production of industrially-relevant small molecules. MAGE was developed as a rapid, high efficiency tool for increasing the genetic diversity of a cell population at targeted loci within the genome. The technique has so far been ported into the model organism Escherichia coli and a few other members of the family Enterbacteriaceae. Sinorhizobium meliloti 1021 is a nitrogen-fixing bacterium capable of forming root nodules with legume plants. Synechococcus sp. PCC 7002 is a fast-growing marine cyanobacterium capable of photosynthesis. We envision numerous potential applications for MAGE in these organisms; for example, the nitrogen fixation mechanisms in Sinorhizobium meliloti 1021 could be modified to enable plant growth in otherwise hostile environments, and the lipid biosynthesis pathway of Synechococcus sp. PCC 7002 could be optimized for the production of molecules that serve as precursors to lipid biofuels.

Project Rationale

Despite their potential as producers of industrially-relevant products, Synechococcus sp. PCC 7002 and Sinorhizobium meliloti 1021 lag far behind other model organisms (E. coli, S. cerivisiae) in terms of genome engineering technologies. Numerous genetic engineering techniques for prokaryotes have been developed in the last decade, including TALENs, CRISPR-Cas9 systems, and SCALE (citation). The implementation of one such technique, MAGE, would signify an important step towards building a comprehensive genetic manipulation system for our chosen organisms. These systems would be useful to a wide range of industries; we anticipate the technology to be most applicable in carbon-neutral and ecological industries.

Target Organism: Synechococcus sp. PCC 7002

Synechococcus sp. PCC 7002 (referred to as 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 PCC 7002 in optimized, CO2-enriched conditions is under 3 hours, making the organism an ideal model for photosynthetic prokaryotes, as well as 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 PCC 7002 (Xu et al. 2011).

Although PCC 7002 is naturally competent and readily undergoes homologous recombination with linear DNA fragments (Widger et al. 1998), its capabilities as a chassis for genetic modification has not been fully realized.

Target Organism: Sinorhizobium meliloti 1021

Potential Outcomes of MAGE Technology in Cyanobacteria and Rhizobia

References

Hamilton JJ and Reed JA. "Identification of Functional Differences in Metabolic Networks Using Comparative Genomics and Constraint-Based Models." PLOS One 2012 7(4) 1-19.

Ludwig M and Bryant DA. "Synechococcus sp. Strain PCC 7002 Transcriptome: Acclimation to Temperature, Salinity, Oxidative Stress, and Mixotrophic Growth Conditions." Front. in Microbiology 2012 3(324) 1-14.

Song HS, McClure RS, Beliaev AS et al. "Integrated in silico Analyses of Regulatory and Metabolic Networks of Synechococcus sp. PCC 7002 Reveal Relationships Between Gene Centrality and Essentiality." Life 2015 5(2) 1127-1140.

Widger WI, Chen X, and Samartzidou H. "Synechococcus PCC 7002." In Bacterial Genomes: Physical Structure and Analysis. De Bruijn FJ, Lupski JR, GM Weinstock, Eds. Springer US: 1998, 763-770.