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

• Life Support• Food• Medicines• Water Recycling• Waste Recycling

Exploration……

…..colonization…..

…..and habitation

The Unique Conditions of a Space Environment

• 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 Adjustments:Biological Activate Genes Deactivate Genes Alter Genes

Physical Change light Change water Change gases Change nutrients

Plant-CenteredLife-Support

System

Life Support Outputs:Food Medicines VitaminsGas RecyclingWater RecyclingWaste RecyclingAesthetics/Avocation

Resource Inputs:Biological Plants Seed/spores Microbes

Physical Light Water Gases Nutrients

Objective

• Design plants to be used to assess the NASA Fundamental Space Biology Free-Flyer 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

Plasmid DNA

isco GFP

Not I Kpn IBam HI

Homologous Recombination

Genomic DNA

Plasmid DNA

Rub isco

isco GFP

Homologous Recombination

Rub isco GFP

Recombinant DNA

isco

Stop Codon

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

?!?!?!?

AGATCTAACCACGAGAGTTTGGTGTGATCTCTGCAGTTTAAGCTAGTGAGCTGGTAGCAAGGGGCGATGGCGAGGCATAATGCAACAAAGCCTGTGACACTCATTCTTGCTGCACTGATGGTTCTTTCAGCCACCGACAACGTCGAAGGTCGTCATCACGTCAGAGATGGAAAAAACTGGCGCAAAGCTCATGCAACTTTCTACGGGGGTGCTGATGCTTCAGGAACTATGGGTAACTTTTTTCAACCTCTTGTTCAACTTCGGAGGCGTCCCATGAATCCTTACAAGTAGTAATTAAAACTTAAGTTTCTTGACAATGTGTATGCTTCCTATCTATTTGGAACTAAACCATTCCTCTGCTCTGATCAGAAACTTGAATCAGCTGCAAAGAGAATAAGACGCCAATATACATGTATCAGAAAACTAACGAAGAGGACTACAAATTTTGGATCTCTTCCATGTAGCTCTTGTCCATAAGGCACCACCTTATGGAGAAATTTTTTTTCGAAAGTTTTGAATTCAGAGATCGGTTGACTAAATAGTAACCTTCGAATGTGCAGACGGTGCATGCGGTGCATGCGGTTACGGAAACCTCTACAGCACTGGCTATGGAGTCGATTCGACAGCTTTGAGTACAGCTCTTTTCAACAATGGGGCAAAATGCGGAGCTTGTTTTGCGATCCAATGCTATCGTTCACAGTATTGCGTTCCAGGTTCACCTGTAATCACTGTCACAGCTACAAACTTCTGCCCTCCAACCACAAAGGTGATGGCACGCCAGGATGGTGTAATCCGCCAATGCGTCACTTCGACCTTGCGCAGCCTAGCTTCACCAAAATCGCTAAGTATAGAGCCGGCATCGTCCCCGTTCTCTTCAGAAGGTGTGCATTGCGTTGAAGACTGATTTGTAAATTGTGACTTTAAGCCTTAATTACTGAGGATGGAGACAGCTGTGCAATCACTTCGCAAATTAAACCATGCATGTTTTTAAGAAAACAGAAACGGCAGAACAAACCGTCAGCCAATTGAAAGAGATGTTCTGAAACTTAGTAAAAGAGGTTGTCTTAGTCCTGTTTGGATTGGTAGTTTGATATTACGAAGTCCGTACTGACCAAAACTTTGTTATATGCCTTGCACAATGCAGGGTACCATGCGAGAAAAAAGGTGGCGTCAGGTTCACTATCAATGGAAATAAGTATTTCAATCTCGTCCTAGTTCACAATGTTGGTGGAAAAGGCGATGTGCACGCAGTAGACAT

AATACAGAATGGATTCCCATGAAGCGAAACTGGGGAATGAACTGGCAACAGATGCT GGATCC

PP Expansin Gene

GTTATGACCAAG

TGGCCAGGCACTCTCCTTCCGAGTGACAACCAGTGATGGTAAGACCATAGTCTCTATGAACGCAACGCCATCTCACTGGAGCTTCGGCCAGACCTTCGAGGGAGGTCAGTTCGCTATGAATTGAATTCTGTAACCCCAAGAGCGGTGCCACTCGATGAATGCTTTAGGCGAAGAGTTGATCCACAAGGGAACCTAGACGCAGTTGAGTCTCAATCTAGCTTCATGATATTTGTTGATACCTATACTGGATACCAATCGGCGTCTTGAATCCTCAACATCTACCCTCCACGCTTTACCCAGATATCCCGAGATCTGGCCAACGTGAACGGTTTTGAAATTTACCAATATCAGTAGCACATAAAACCCATGGGGTACAATGATTTGTAGGTAGTGCGCAATCATGGGGAT

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