Presentation on theme: "Kuldell Lab (MIT): Natalie Kuldell"— Presentation transcript:
1Kuldell Lab (MIT): Natalie Kuldell The Mitochondria as a Minimal Chassis: Expanding the Toolkit for Mitochondrial Genomic EngineeringBustamante Lab (UC Berkeley): Brad Zamft, Anton Vila-Sanjurjo, Carlos BustamanteKuldell Lab (MIT): Natalie Kuldell9/20/08
2Benefits 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.
3Approaches Top Down Bottom Up 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 UpBoth 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.
4The 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.
5S. cerevisiae mitochondria have already been transformed in vivo GFPmArg8mBARSTmRIP1mCohen, J.S., and Fox, T.D. (2001). Expression of green fluorescent protein from a recoded gene inserted into Saccharomyces cerevisiae mitochondrial DNA. Mitochondrion 1,
7Incorporation into Genome Requires mitochondrial deletion mutants.Homology can cause difficulties.Not scaleable.Decreases versatility.
8Use of PlasmidsHave shown that plasmids can stably accompany mitochondrial genome if under selection.Need to develop selectable marker.
9Collaboration with Natalie Kuldell HEM1: 5-aminolevulinate synthase (δ-ALA)Catalyzes first step in heme synthesis pathway. Also involved in the regulation of transcription of genes involved in Fe & Cu transport. Human analog: ALAS2. Mutations cause X-linked sideroblastic anemia.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.
10Transformation of ρ+ strain with HEM1m δ-ala-δ-ALA+
11Eventually Use as Autonomous Plasmid δ-ala-δ-ALA+
12Progress on HEM1 Project Bombardment plasmid synthesized by DNA2.0.HEM1 knockout strain made by Natalie Kuldell.δ-ALA auxotrophy confirmedTransformations forthcoming.G418 + dALAG418 30° 2d65MH3394DFS160312
15Incorporation of RPO41m into the Mitochondrial Genome: Problems, Problems, Problems
16The Original Plan(cox2-62)BMZ4-7: Synthetic r-BMZ3-1: Final Mater
17Results of Final Mating Media is SEG+5FOASEG: Ethanol and GlycerolNonfermentable.Cells must respire to grow.Cells must have some source of full COX2 and RPO41.5FOASelects against strains that have URA3 protein.Cells must have lost their URA3 gene.
18Cells Grow with 5FOA Counterselection WTDiploidsrpo41ΔSynthetic ρ-Final Mater5FOA resistance must come from mutations in URA3 rather than absence of plasmid.
19Colonies Still Have Deleted Version of Cox2 Region Diploidscox2-62WT
20Colonies also have Full Version of Cox2 DiploidsWTSynthetic ρ-Final Mater
22Intramolecular Recombination of Original Plasmid
23PCRs and Sequencing Confirm Intramolecular Recombination PlasmidGenome
24Strategies TriedScreen ~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.
25Future Directions Use flipped plasmid. Use different plasmid. Use temperature-sensitive shuffle vector.
29Use a Temperature Sensitive Shuffle Vector Characterization of amino-terminal deletions in mtRNA polymerase. (A) A schematic representation of yeast mtRNA polymerase and deletion mutations used in this study is shown with important features labeled as follows: mitochondrial targeting sequence, black box; region containing bacteriophage homology, open box; amino-terminal extension, hatched box. The number of amino acids deleted (Δ) in each protein is given in parentheses. (B) Phenotypic analysis of RPO41 deletion mutants by plasmid shuffle. Mitochondrial function was assessed in each strain by comparing growth on YPD and YPG at 30°C and 37°C. The RPO41 genotype of each strain after plasmid shuffle is given as follows: Δ, rpo41; wt, RPO41; Δ2-Δ5, rpo41Δ2-Δ5. (C) Western analysis of rpo41Δ3-encoded mtRNA polymerase from GS129. After growth at 37°C for ≈20 generations, protein from whole cells (wc) and from a purified mitochondrial fraction (mito) were analyzed. Signals for the wild-type (wt) and rpo41Δ3-encoded (Δ3) protein are indicated with arrows. Protein molecular mass standards are indicated on the left.Wang Y., Shadel G. S. PNAS 1999;96:
32Use 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!
33Use 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.”