Transcript The Mitochondria as a Minimal Chassis:

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
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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
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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
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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.”