Transcript Development of the Moss Physcomitrella patens

Development of the Moss
Physcomitrella patens for
Assessment in Space
Virginia Slater
Kirk Findlay, TJ White, Dr. Maria Ivanchenko and
Dr. Terri Lomax
Role of Plants on Earth
The Role of Plants in Space
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Life Support
Food
Medicines
Water Recycling
Waste Recycling
Exploration……
…..colonization…..
…..and habitation
The Unique Conditions of a Space
Environment
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Microgravity
Cosmic Radiation
Low Atmospheric Pressure
High CO2
Temperature
Microarray Technology
• Measures temporal and spatial gene
expression.
• Provides information of gene involvement
in a process or pathway.
Microarray Technology
Microarray Technology
Microarray Technology
Microarray Technology
NASA Free-Flyer
Plant Genetic
Assessment
and
Control Module
Resource
Inputs:
Resource
Adjustments:
Biological
Plants
Seed/spores
Microbes
Biological
Activate Genes
Deactivate Genes
Alter Genes
Physical
Light
Water
Gases
Nutrients
Plant-Centered
Life-Support
System
Life Support Outputs:
Food
Medicines
Vitamins
Gas Recycling
Water Recycling
Waste Recycling
Aesthetics/Avocation
Physical
Change light
Change water
Change gases
Change nutrients
Objective
• Design plants to be used to assess the
NASA Fundamental Space Biology FreeFlyer Satellite Program.
• Design and produce genetic constructs
that are coupled with fluorescent tags.
• Transform the constructs into the moss
Physcomitrella patens.
• Monitor protein expression to establish
baseline standards.
Why Moss?
• Physcomatrella patens is a model
organism for the study of plant
development.
Why Moss?
• Protonema
– Cell division and
growth.
– Signal transduction
• Gametophore
– Organogenesis
– Establishment of the
body plan.
– Plant development
Why Moss?
• Physcomatrella patens is a model
organism for the study of plant
development.
• Contains a super efficient gene targeting
system.
– 90% efficiency
Hypothesis
• Rubisco is located in the chloroplasts.
• We hypothesize that the Rubisco/GFP will
cause the chloroplasts to fluoresce
producing a signal that will be detectable
from ground control.
Methodology
• Design primers for a portion of the
Rubisco gene.
• Use designed primers in PCR to amplify
that portion of Rubisco.
• Restrict the moss PCR product (Rubisco).
• Restrict plasmid pMBL5.
• Ligate pMBL5 and the Rubisco fragment
together.
• Repeat with GFP (next slide).
Methodology
Not I
isco
Plasmid DNA
Bam HI
Kpn I
GFP
Homologous Recombination
Genomic DNA
Rub isco
isco
Plasmid DNA
GFP
Homologous Recombination
Stop
Codon
Rub isco
Recombinant
DNA
GFP
isco
Results
1 kb Ladder
pMBL5 and
GFP fragment
pMBL5 w/o insert
pMBL5 and
Rubisco
fragment
1st
Cut
Results
1 kb Ladder
pMBL5 and
GFP fragment
pMBL5 w/o insert
pMBL5 and
Rubisco
fragment
2nd
Cut
Additional Strategy
• Repeat the experiment using a structural
protein.
Digestion of Expansin
1 kb Ladder
pMBL5 BamH I/Kpn
GFP fragment - BamH I/Kpn I
?!?!?!?
pMBL5 Not I/BamH I
Expansin fragment - Not I/BamH I
Expansin fragment - Not I/BamH I
PP Expansin Gene
AGATCTAACCACGAGAGTTTGGTGTGATCTCTGCAGTTTAAGCTAGTGAGCTGGTAGCAAGGGGCGATGGCGAG
GCATAATGCAACAAAGCCTGTGACACTCATTCTTGCTGCACTGATGGTTCTTTCAGCCACCGACAACGTCGAAG
GTCGTCATCACGTCAGAGATGGAAAAAACTGGCGCAAAGCTCATGCAACTTTCTACGGGGGTGCTGATGCTTCA
GGAACTATGGGTAACTTTTTTCAACCTCTTGTTCAACTTCGGAGGCGTCCCATGAATCCTTACAAGTAGTAATT
AAAACTTAAGTTTCTTGACAATGTGTATGCTTCCTATCTATTTGGAACTAAACCATTCCTCTGCTCTGATCAGA
AACTTGAATCAGCTGCAAAGAGAATAAGACGCCAATATACATGTATCAGAAAACTAACGAAGAGGACTACAAAT
TTTGGATCTCTTCCATGTAGCTCTTGTCCATAAGGCACCACCTTATGGAGAAATTTTTTTTCGAAAGTTTTGAA
TTCAGAGATCGGTTGACTAAATAGTAACCTTCGAATGTGCAGACGGTGCATGCGGTGCATGCGGTTACGGAAAC
CTCTACAGCACTGGCTATGGAGTCGATTCGACAGCTTTGAGTACAGCTCTTTTCAACAATGGGGCAAAATGCGG
AGCTTGTTTTGCGATCCAATGCTATCGTTCACAGTATTGCGTTCCAGGTTCACCTGTAATCACTGTCACAGCTA
CAAACTTCTGCCCTCCAACCACAAAGGTGATGGCACGCCAGGATGGTGTAATCCGCCAATGCGTCACTTCGACC
TTGCGCAGCCTAGCTTCACCAAAATCGCTAAGTATAGAGCCGGCATCGTCCCCGTTCTCTTCAGAAGGTGTGCA
TTGCGTTGAAGACTGATTTGTAAATTGTGACTTTAAGCCTTAATTACTGAGGATGGAGACAGCTGTGCAATCAC
TTCGCAAATTAAACCATGCATGTTTTTAAGAAAACAGAAACGGCAGAACAAACCGTCAGCCAATTGAAAGAGAT
GTTCTGAAACTTAGTAAAAGAGGTTGTCTTAGTCCTGTTTGGATTGGTAGTTTGATATTACGAAGTCCGTACTG
ACCAAAACTTTGTTATATGCCTTGCACAATGCAGGGTACCATGCGAGAAAAAAGGTGGCGTCAGGTTCACTATC
AATGGAAATAAGTATTTCAATCTCGTCCTAGTTCACAATGTTGGTGGAAAAGGCGATGTGCACGCAGTAGACAT
AATACAGAATGGATTCCCATGAAGCGAAACTGGGGAATGAACTGGCAACAGATGCT
GTTATGACCAAG GGATCC
TGGCCAGGCACTCTCCTTCCGAGTGACAACCAGTGATGGTAAGACCATAGTCTCTATGAACGCAACGCCATCTC
ACTGGAGCTTCGGCCAGACCTTCGAGGGAGGTCAGTTCGCTATGAATTGAATTCTGTAACCCCAAGAGCGGTGC
CACTCGATGAATGCTTTAGGCGAAGAGTTGATCCACAAGGGAACCTAGACGCAGTTGAGTCTCAATCTAGCTTC
ATGATATTTGTTGATACCTATACTGGATACCAATCGGCGTCTTGAATCCTCAACATCTACCCTCCACGCTTTAC
CCAGATATCCCGAGATCTGGCCAACGTGAACGGTTTTGAAATTTACCAATATCAGTAGCACATAAAACCCATGG
GGTACAATGATTTGTAGGTAGTGCGCAATCATGGGGAT
Future Plans
• Transform the moss cultures and select for
stable transformations.
• Repeat the experiment using a structural
protein.
• Monitor and analyze proteins levels to
establish baseline standards for ground
plants.
• Test moss cultures in space.
Acknowledgements
• Howard Hughes Medical
Insitute
• McNair Scholar’s Program
• Dr. Terri Lomax
• Dr. Maria Ivanchenko
• Kirk Findlay
• TJ White
• Dr. Kevin Ahern
• Dr. Indira Rajagopal
• The Lomax Lab
• HHMI and McNair Scholars
• Dept. of Botany and Plant
Pathology