Mitochondrial transformation in Chlamydomonas reinhardtii'

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Mitochondrial transformation in Chlamydomonas
reinhardtii.

• By Susan Frappier and Dr. Barbara B. Sears

Michigan State University, Department of Plant
Biology
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Can Chlamydomonas reinhardtii mitochondria be
transformed by electroporation?
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Overview

• Introduction to Chlamydomonas reinhardtii
• Introduction to electroporation
• Chloroplast electroporation
• Nuclear electroporation
• Conditions for mitochondrial electroporation

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Chlamydomonas reinhardtiiThe Green Yeast

• Unicellular, green algae
• haploid
• both asexual and sexual reproduction
• two mating types (mt and mt-)
• forms colonies which grow quickly on plates
• easily transformable

Picture courtesy of Mike Adams, Biology Dept ECSU
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Chlamy transformation--So how do you get DNA in?

• What is Transformation??
• Biolistic method
• Glass Bead method
• Electroporation method

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What is electroporation?

• A short controlled pulse of electricity to cell
  momentarily disrupting lipid bilayer.
• Small pores (40-120nm) reseal quickly.

Cell wall
Nucleus
DNA enters
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Basic conditions for electroporation in Chlamy

• Cell wall-less strain
• Transforming DNA
• Selectable markers
• Carrier DNA

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Transformation by electroporation in Chlamy

• Chloroplast transformation
• Nuclear transformation
• Mitochondrial transformation

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Chloroplast transformation

• Gene replacement by homologous recombination

x
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Transformation by electroporation in C.
reinhardtii

• Chloroplast transformation
• Nuclear transformation
• Mitochondrial transformation

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Nuclear transformation

• Random insertion of DNA into nuclear genome

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Conditions for electroporation in Chlamy nucleus

• Cell wall-less strain
• carrier DNA
• transforming DNA
• Selectable markers

• dW15-1
• sheared sonocated salmon sperm
• pMN24ee w/ Nit1 gene
• once transformed will grow on TAP Nitrate media

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Nuclear Transformation
Maximum resistance
Varied resistance
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Transformation by electroporation in C.
reinhardtii

• Chloroplast transformation
• Nuclear transformation
• Mitochondrial transformation

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Can Chlamy mitochondria be transformed by
electroporation ?
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Chlamy mitochondria

• Many per cell
• Small (15Kb)
• gene order highly conserved
• Linear DNA
• Mitochondrial inheritance is through the mt-
  strain

cob nad4 nad5 cox1 nad2 nad6 nad1 rtl
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Conditions for electroporation in Chlamy
mitochondria

• Cell wall-less strain
• Selectable markers
• transforming DNA
• carrier DNA

• Cc3400 w/mitochondrial mutation IS NOT CELL
  WALL-LESS
• Unless transformed, it dies under dark conditions
• p85 and COX1 gene from C. smithii
• Sheared, sonocated salmon sperm

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Chlamy Crosses

• Cc3400 (mt-)and a cell wall-less strain, cc400,
  were starved for nitrogen, placed in water, and
  mated.
• Progeny were plated and tested for cell walls

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Testing for cell walls

• Progeny were placed in liquid culture
• 1ml placed in water, 1ml placed in detergent
  (Triton)
• Two progeny found without cell walls

Chlamy with cell wall in detergent
Cellular debris from chlamy in detergent without
cell wall
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Dilution Platings (to confirm mt mutations)

• Cell wall-less progeny were grown in a liquid
  culture, concentrated to a density of 2x108, and
  diluted in a serial dilution to 10-6.
• The 10-5 and 10-6 dilutions were plated on media
  and 1 plate placed in dark, one in light.

• Wild Type Chlamy
• Cc3400xcc400 progeny

Light treatment
Dark treatment
Light treatment
Dark treatment
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Obtaining transforming DNA from C. smithii

• C smithii DNA isolated
• primers designed from known C. reinhardtii COX1
  gene sequence (1.3 kb) --added BAM HI cut site
• PCR (Polymerase Chain Reaction) done using C
  smithii DNA.

?DNA Pvu11
?DNA BstE11
cc1373 DNA
cc124 DNA
dd H2O
1.3 Kb cut site
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Summary

• We know good parameters for electroporation.
• We have cell wall-less strain of Chlamy with
  mitochondrial mutation.
• We have carrier DNA.
• We have transforming DNA.

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Future Experiments

• Mitochondrial electroporation.
• Insertion of PCR product into plasmid for
  electroporation into bacteria.
• If Mitochondrial electroporation can be
  standardized, may compare mutation frequencies of
  same transgene in different organelles.

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Acknowledgements

• Dr. Barbara B. Sears for giving me this wonderful
  research opportunity.
• The Sears Lab for all of their help and support.