Presentation on theme: "The Mitochondria as a Minimal Chassis: Expanding the Toolkit for Mitochondrial Genomic Engineering Bustamante Lab (UC Berkeley): Brad Zamft, Anton Vila-Sanjurjo,"— Presentation transcript:
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,
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 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. (δ-ALA)
Transformation of ρ + strain with HEM1 m 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. DFS160 MH G418 + dALAG418 30° 2d
Thank You Carlos Bustamante Natalie Kuldell Anton Vila-Sanjurjo Tom Fox Peter Thorsness Jasper Rine Erin Osborne Adam Deutchbauer John Dueber Chris Anderson Judith Jaehnning Pawel Golik The Synthetic Biology Division of the Bustamante Lab Full TimePart Time Mariana Leguia Alyssa Rosenbloom 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
Incorporation of RPO41m into the Mitochondrial Genome: Problems, Problems, Problems
The Original Plan BMZ4-7: Synthetic - BMZ3-1: Final Mater (cox2-62)
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.
Cells Grow with 5FOA Counterselection 5FOA resistance must come from mutations in URA3 rather than absence of plasmid. Diploids Synthetic ρ - Final Mater rpo41Δ WT
Colonies Still Have Deleted Version of Cox2 Region Diploids cox2-62 WT
Colonies also have Full Version of Cox2 Diploids WT Synthetic ρ - Final Mater
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
Wang Y., Shadel G. S. PNAS 1999;96: Use a Temperature Sensitive Shuffle Vector
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 TimePart Time Mariana Leguia Alyssa Rosenbloom 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 ρ 0 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.”