Multiplex Automated Genome Engineering

MAGE is a genetic engineering method that relies on recombineering to produce frequent and large scale genetic changes to cells. Simply, it is a cyclical and scalable recombineering system that allows multiple genetic changes in a high throughput manner. A given cycle in MAGE requires cell growth, the introduction of recombineering substrate, and provision of providing synthesized DNA con­structs a continuous and interlinked process. Target cells are continuously grown and drawn out of a cell growth chamber into an exchange chamber where the substrates required for recombineering are added under the right conditions. The induced cells are then mixed with the recombinant and synthetic DNA constructs in another chamber and then moved to an electroporator to induce the uptake of the synthetic DNA constructs. Those modified cells are then reintroduced into the growth chamber and grown. This whole process occurs continuously; and move­ment to each stage is facilitated by microfluidics, or any suitable pumping assembly (Isaacs et al. 2011).

Just like recombineering, MAGE can be used to create a mismatch, deletion or insertional genetic changes. The efficiency of each genetic change is dependent on the size of the homology flanking each introduced DNA construct, the size of the desired change, and the number of cycles. This efficiency is also correlated with the Gibbs free energy from the hybridization that occurs between the DNA strands. Knowledge of the efficiency of each genetic change and the parameters that affect it can be used to tune the MAGE process to produce colonies or strains with desired genetic traits (Isaacs et al. 2011). The MAGE system was initially demonstrated by modifying a 1-Deoxy-D-xylulose 5-phosphate (DXP) biosynthesis pathway in Escherichia Coli. The DXP pathway was modified to increase production of isoprenoid lycopene. New genetic modifications were introduced in greater than 30 % of the cell population every 2-2.5 h under optimum conditions. After just five cycles of MAGE, the average genetic change across the entire cell population was

3.1 bp, and increased to 5.6 bp afters 15 cycles. Ultimately, 24 genes were opti­mized simultaneously and about 15 billion genetic variants were produced at an average rate of 430 million bp changes per MAGE cycle in a total of 35 cycles. This translated to an up to 390 % increase in isoprenoid lycopene production, clearly demonstrating MAGE’s potential (Isaacs et al. 2011). Development of a system similar to MAGE in algae is dependent on the design of a suitable genetic engi­neering method; recombineering strategies are not established in algae.

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