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The Mitochondria as a Minimal Chassis: Expanding the Toolkit for Mitochondrial Genomic Engineering Bustamante Lab (UC Berkeley): Brad Zamft, Anton Vila-Sanjurjo, Carlos Bustamante Kuldell Lab (MIT): Natalie Kuldell 9/20/08 Benefits of a Minimal Chassis • First picture of the necessary and sufficient elements to define a living system. • Insight into the principles underlying the organization of the “living state.” • Thorough quantitative modeling of cellular physiology easier. • First step towards the construction in the laboratory of whole synthetic organisms. • Microbial engineering: A minimalistic cell could be more prone to accept new metabolic pathways than a more complex organism. Approaches • Top Down – Starts out with a biologically derived envelope and genome. – The minimized genome is evolutionarily derived, as opposed to rationally designed. – Does not teach us how to design a genome. – Size will depend on the choice of organism. • Bottom Up – Both genome and envelope are rationally designed. – The synthetic envelope must: • contain all essential cellular components. • allow all cellular functions to proceed in a coordinated manner. • all the components of the system must be present at once. The Middle-Ground: Mitochondria • Rationally designed genome into a biologically derived envelope. • Reduces the problem of creating a rationally designed organism to that of synthesizing its genome. S. cerevisiae mitochondria have already been transformed in vivo • • • • GFPm Arg8m BARSTm RIP1m Cohen, J.S., and Fox, T.D. (2001). Expression of green fluorescent protein from a recoded gene inserted into Saccharomyces cerevisiae mitochondrial DNA. Mitochondrion 1, 181-189. Successfully Transformed Mitochondrial Plasmid Incorporation into Genome • Requires mitochondrial deletion mutants. • Homology can cause difficulties. – Not scaleable. • Decreases versatility. Use of Plasmids • Have shown that plasmids can stably accompany mitochondrial genome if under selection. • Need to develop selectable marker. Collaboration with Natalie Kuldell HEM1: 5-aminolevulinate synthase (δ-ALA) •1647 bp, 549 aa. •First step in heme synthesis pathway. •Encoded in nucleus, translated in cytoplasm, imported in to mitochondria. •Mutants cannot grow on media lacking δ-ALA. Transformation of ρ+ strain with HEM1m δ-ala- δ-ALA+ Eventually Use as Autonomous Plasmid δ-ala- δ-ALA+ Progress on HEM1 Project • Bombardment plasmid synthesized by DNA2.0. • HEM1 knockout strain made by Natalie Kuldell. – δ-ALA auxotrophy confirmed • Transformations forthcoming. G418 + dALA G418 30° 2d 6 5 MH339 4 DFS160 3 1 2 Thank You • Carlos Bustamante • Natalie Kuldell • Anton Vila-Sanjurjo • • • • • • • • • QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. QuickTi me™ and a TIFF ( Uncompressed) decompressor are needed to see thi s pi ctur e. Quick Time™a nd a TIFF ( Unco mpre ssed ) dec ompr esso r ar e nee ded to see this pictur e. QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. Full Time •Mariana Leguia •Alyssa Rosenbloom Tom Fox Peter Thorsness Jasper Rine Erin Osborne Adam Deutchbauer John Dueber Chris Anderson Judith Jaehnning Pawel Golik Quic kTime™ and a TIFF (Unc ompres sed) dec ompres sor are needed to see this pic ture. The Synthetic Biology Division of the Bustamante Lab Part Time •Lourdes Dominguez •Bernadette Hapsari •Marta Kopaczynska •Fabian Stroehle •Celina Vila-Sanjurjo •Errol Watson •Natalie Benadum •Andrew Chen •DJ Cummings •Anaar Eastoak-Siletz •Tren Gu •Matthew Koh •Natalie Kolawa •Sandy Lao •Joe Marlin •Brett Schofield •Samuel Schumacher •Helen Yu •Richard Wang •Courtney Lane Questions? Incorporation of RPO41m into the Mitochondrial Genome: Problems, Problems, Problems The Original Plan (cox2-62) BMZ4-7: Synthetic r- BMZ3-1: Final Mater Results of Final Mating • Media is SEG+5FOA – SEG: Ethanol and Glycerol • Nonfermentable. • Cells must respire to grow. • Cells must have some source of full COX2 and RPO41. – 5FOA • Selects against strains that have URA3 protein. • Cells must have lost their URA3 gene. WT rpo41Δ Final Mater Synthetic ρ- Diploids Cells Grow with 5FOA Counterselection 5FOA resistance must come from mutations in URA3 rather than absence of plasmid. Diploids cox2-62 WT Colonies Still Have Deleted Version of Cox2 Region Diploids WT Final Mater Synthetic ρ- Colonies also have Full Version of Cox2 Colonies also have Cox2 as a Separate Plasmid Intramolecular Recombination of Original Plasmid PCRs and Sequencing Confirm Intramolecular Recombination Plasmid Genome Strategies Tried • Screen ~80 colonies by PCR to see if they still have plasmid version of RPO41. • Grow final mater first in 5FOA, then mate. • Grow final mater first in nonselectable media, then mate, then select on 5FOA. • Grow final mater first in nonselectable media, then select on 5FOA, then verify that it does not have shuffle plasmid and that it has some intact genome, then use this strain exclusively to mate. Future Directions • Use flipped plasmid. • Use different plasmid. • Use temperature-sensitive shuffle vector. Using a Flipped Plasmid Synthetic ρ- of Flipped Plasmid Isolated Using a Different Plasmid Plasmid Genome Use a Temperature Sensitive Shuffle Vector Wang Y., Shadel G. S. PNAS 1999;96:8046-8051 Thank You • SynBERC • Carlos Bustamante • Anton Vila-Sanjurjo • • • • • • • • • • Tom Fox Peter Thorsness Jasper Rine Erin Osborne Adam Deutchbauer John Dueber Chris Anderson Judith Jaehnning Pawel Golik Natalie Kuldell The Synthetic Biology Division of the Bustamante Lab Full Time •Mariana Leguia •Alyssa Rosenbloom Part Time •Lourdes Dominguez •Bernadette Hapsari •Marta Kopaczynska •Fabian Stroehle •Celina Vila-Sanjurjo •Errol Watson •Natalie Benadum •Andrew Chen •DJ Cummings •Anaar Eastoak-Siletz •Tren Gu •Matthew Koh •Natalie Kolawa •Sandy Lao •Joe Marlin •Brett Schofield •Samuel Schumacher •Helen Yu •Richard Wang •Courtney Lane Proving I Have Synthetic ρ- Use of a Mostly ρ- Final Mating Strain • Mated BMZ4-7 (synthetic ρ-) with BMZ3-1-1 (BMZ3-1 with the shuffle plasmid removed). • No discernable growth on YPEG. • After about 5 days of growth sitting on my bench, I noticed small colonies. • Streaked, grew in 50mL YPEG for 3 weeks! • Glyceroled, genomic prep. • PCR’d, found only plasmid in one strain, nothing in others. • Did controls, found that even ρ0 cells grow a little on YPEG. Take nothing for granted! Use of S. douglasii Sequences “To allow homologous recombination between the new construct and rho+ mtDNA, the last S. douglasii cox1 exon and part of its terminator region were cloned upstream of the cox1::RIP1m gene. This large additional region homologous to the 3′ part of the cox1 gene (886 bp) should promote integration of RIP1m between the cox1 and atp8 genes in rho+ mtDNA (Fig. 1C). S. douglasii rather than bona fide S. cerevisiae cox1 sequences were used, because repeated sequences in S. cerevisiae mtDNA are known to be highly unstable. Because the portion of S. douglasii sequence displays several polymorphic changes compared with the S. cerevisiae and S. capensis relevant sequences (Fig. 3F), we reasoned that this would lower the excision of the RIP1m gene by recombination and should allow us to discriminate wild-type and recombinant molecules.”