Multiplex automated genome engineering (MAGE) allows for large-scale programming and evolution of cells. Mediated by λ-Red ssDNA-binding protein β, oligos are incorporated into the lagging strand of the replication fork during DNA replication, creating a new allele that will spread through the population as the bacteria divide. The efficiency of oligo incorporation depends on several factors, but the frequency of the allele can be increased by performing multiple rounds of MAGE on the same cell culture. MAGE facilitates rapid and continuous generation of a diverse set of genetic changes (mismatches, insertions, deletions). This multiplex approach embraces engineering in the context of evolution by expediting the design and evolution of organisms with new and improved properties.
Recent studies have isolated a novel, hyperactive antifreeze protein from the hemolymph of the cold tolerant Siberian cerambycid beetle, Rhagium inquisitor (RiAFP) (Kristiansen, 2011). The only reported details of this protein thus far are its primary sequence, and its thermal hysteresis activity. Thermal hysteresis measurements indicate that RiAFP is the most active AFP isolated thus far. The protein seems to have significantly fewer disulfide bonds and fewer repeated sequences compared to other active insect AFPs. Whereas the Tenebrio molitor antifreeze protein has a cysteine content of 19% and a total of eight disulfide bonds in its core, the Rhagium Inquisitor AFP has a cysteine content of <1% and has only one disulfide bond. These features, together with its small size (12kDa), lead our team to hypothesize that RiAFP would make a good candidate for recombinant expression. Our hope is to demonstrate that RiAFP is an attractive reagent for applications requiring freeze resistance or the control of ice growth and morphology. Our goal was to provide some of the first characterizations of this protein, since little is known about its structure, or activity (other than thermal hysteresis).
Fig 1
Each cell contains a different set of mutations, producing a heterogeneous population of rich diversity (denoted by distinct chromosomes in different cells). Degenerate oligo pools that target specific genomic positions enable the generation of a diverse set of sequences at each chromosomal location. (Wang et al, 2009)
Fig 2
This graphic, from Farren Isaacs, describes how to design oligos for MAGE. Our oligos were approximately 90 bases long with the first 5’ base phosphorothioated (increases recombination efficiency). Since we integrated the RiAFP into the genome in replichore 1 (sites 78110 and 1415740; determined at ecocyc.org), we designed oligos to target the appropriate strand. Mismatches, insertions, and deletions were centered on the oligo to increase recombination efficiency.
Fig 3
In order to perform MAGE, we needed to first integrate RiAFP into the genome of the EcNR2 strain. The RiAFP gene was linked to kanamycin by crossover PCR. dsDNA recombination efficiency data from Conjugative Assembly Genome Engineering (Isaacs, et al 2011). For further details, please see protocols section.
Fig 3
Gel confirming success of PCR reactions for eventual crossover PCR. 1: 100 bp ladder, 2: 1kb ladder, 3: 1kb ladder, 4: GR7 - i (PCR reaction for RiGFP, 78 integration site, and Kan crossover site), 5: replicate of above, 6: replicate of above, 7: 141Kan - i (PCR reaction for Kanamycin, 141 integration site), 8: replicate of above, 9: replicate of above, 10: GR14 - i (PCR reaction for RiGFP, 141 integration site, and Kan crossover site), 11: replicate of above, 12: replicate of above, 13: 78Kan - i (PCR reaction for Kanamycin, 78 integration site), 14: replicate of above, 15: replicate of above
Fig 4
Gel confirming success of PCR reactions for eventual crossover PCR. 1: 1kb ladder, 2: 1 kb ladder, 3: 100 bp ladder, 4: did not work - AR7 (supposed to be RiAFP gene only with 78 integration site and Kan crossover site), 5: did not work - replicate of above, 6: worked: AR14 ii - (RiAFP with 141 integration site and Kan crossover site)
Fig 5
Gels confirming success of crossover PCR for lambda red integration.1: ladder:, 2: GR7 + 78 kan (failed), 3: replicate, 4: replicate, 5: GR14 + 141 Kan (worked), 6: replicate, 7: replicate, 8: AR14 + 141 Kan (worked), 9: replicate, 10: replicate, 11: replicate
The lambda-red recombination of the AFP-Kan and AFP-eGFP-Kan in the EcNR2 strain worked; there were colonies on the appropriate Kanamycin plates and no colonies on the -ve control Kanamycin plates (no DNA added for electroporation). See below for sample colony plates (top row, negative control plates with no colonies; bottom row left plate with colonies of RiAFP-GFP-Kanamycin ; bottom row right plate with colonies of RiAFP-Kanamycin
Fig 6
We ordered 22 degenerate oligos to target the theorized ice-binding site of RiAFP. These oligos were designed to insert additional Tx repeats, delete Tx repeats, delete entire TxT segments, and replace regions with degenerate TxTxTxT repeats. Essentially the same protocol used for lambda red recombination was used to introduce the oligos to mid-log phase EcNR2 bacteria containing RiAFP or eGFP-RiAFP.
Thus far, we have generated a diverse population of mutants for the antifreeze protein sequence. Based on data from the original MAGE paper, we have generated four hundred and thirty four million predicted genomic variants thus far. An average change of 0.62 bp per cell across the population is detected after one MAGE cycles, multiplied by 7x108 cells across the cell population. This represents more potential “biobricks” than currently exist in the iGEM registry, generated in one experiment! We are currently applying the selective pressure of multiple freeze thaw cycles. We intend to run additional MAGE cycles on mutants that survive multiple freeze thaw cycles to hopefully generate and then characterize “superactive”, soluble antifreeze proteins.
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