Kuldell Lab (MIT): Natalie Kuldell 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 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 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) 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.

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

The Synthetic Biology Division of the Bustamante Lab Thank You The Synthetic Biology Division of the Bustamante Lab Full Time Part 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 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

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.

Cells Grow with 5FOA Counterselection WT Diploids rpo41Δ Synthetic ρ- Final Mater 5FOA resistance must come from mutations in URA3 rather than absence of plasmid.

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 Genome

Use 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;96:8046-8051

The Synthetic Biology Division of the Bustamante Lab Thank You The Synthetic Biology Division of the Bustamante Lab Full Time Part 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 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

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.”