1. Development of the Moss Physcomitrella patens for Assessment in Space Virginia Slater Kirk Findlay, TJ White, Dr. Maria Ivanchenko and Dr. Terri Lomax

2. Role of Plants on Earth

3. The Role of Plants in Space Life Support Food Medicines Water Recycling Waste Recycling

4. Exploration……

5. …..colonization…..

6. …..and habitation

7. The Unique Conditions of a Space Environment Microgravity Cosmic Radiation Low Atmospheric Pressure High CO 2 Temperature

8. Microarray Technology Measures temporal and spatial gene expression. Provides information of gene involvement in a process or pathway.

9. Microarray Technology

13. NASA Free-Flyer

14. 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-Centered Life-Support System Life Support Outputs: FoodMedicines Vitamins Gas Recycling Water Recycling Waste Recycling Aesthetics/Avocation Resource Inputs: Biological Plants Seed/spores Microbes Physical Light Water Gases Nutrients

15. 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.

16. Why Moss? Physcomatrella patens is a model organism for the study of plant development.

17. Why Moss? Protonema –Cell division and growth. –Signal transduction Gametophore –Organogenesis –Establishment of the body plan. –Plant development

18. Why Moss? Physcomatrella patens is a model organism for the study of plant development. Contains a super efficient gene targeting system. –90% efficiency

19. 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.

20. 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).

21. Methodology Plasmid DNA isco GFP Not I Kpn IBam HI

22. Homologous Recombination Genomic DNA Plasmid DNA Rubisco GFP

23. Homologous Recombination Rubisco GFP Recombinant DNA isco Stop Codon

24. Results 1 kb Ladder pMBL5 and GFP fragment pMBL5 w/o insert pMBL5 and Rubisco fragment 1 st Cut

25. Results 1 kb Ladder pMBL5 and GFP fragment pMBL5 w/o insert pMBL5 and Rubisco fragment 2 nd Cut

26. Additional Strategy Repeat the experiment using a structural protein.

27. 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 ?!?!?!?

28. AGATCTAACCACGAGAGTTTGGTGTGATCTCTGCAGTTTAAGCTAGTGAGCTGGTAGCAAGGGGCGATGGCGAG GCATAATGCAACAAAGCCTGTGACACTCATTCTTGCTGCACTGATGGTTCTTTCAGCCACCGACAACGTCGAAG GTCGTCATCACGTCAGAGATGGAAAAAACTGGCGCAAAGCTCATGCAACTTTCTACGGGGGTGCTGATGCTTCA GGAACTATGGGTAACTTTTTTCAACCTCTTGTTCAACTTCGGAGGCGTCCCATGAATCCTTACAAGTAGTAATT AAAACTTAAGTTTCTTGACAATGTGTATGCTTCCTATCTATTTGGAACTAAACCATTCCTCTGCTCTGATCAGA AACTTGAATCAGCTGCAAAGAGAATAAGACGCCAATATACATGTATCAGAAAACTAACGAAGAGGACTACAAAT TTTGGATCTCTTCCATGTAGCTCTTGTCCATAAGGCACCACCTTATGGAGAAATTTTTTTTCGAAAGTTTTGAA TTCAGAGATCGGTTGACTAAATAGTAACCTTCGAATGTGCAGACGGTGCATGCGGTGCATGCGGTTACGGAAAC CTCTACAGCACTGGCTATGGAGTCGATTCGACAGCTTTGAGTACAGCTCTTTTCAACAATGGGGCAAAATGCGG AGCTTGTTTTGCGATCCAATGCTATCGTTCACAGTATTGCGTTCCAGGTTCACCTGTAATCACTGTCACAGCTA CAAACTTCTGCCCTCCAACCACAAAGGTGATGGCACGCCAGGATGGTGTAATCCGCCAATGCGTCACTTCGACC TTGCGCAGCCTAGCTTCACCAAAATCGCTAAGTATAGAGCCGGCATCGTCCCCGTTCTCTTCAGAAGGTGTGCA TTGCGTTGAAGACTGATTTGTAAATTGTGACTTTAAGCCTTAATTACTGAGGATGGAGACAGCTGTGCAATCAC TTCGCAAATTAAACCATGCATGTTTTTAAGAAAACAGAAACGGCAGAACAAACCGTCAGCCAATTGAAAGAGAT GTTCTGAAACTTAGTAAAAGAGGTTGTCTTAGTCCTGTTTGGATTGGTAGTTTGATATTACGAAGTCCGTACTG ACCAAAACTTTGTTATATGCCTTGCACAATGCAGGGTACCATGCGAGAAAAAAGGTGGCGTCAGGTTCACTATC AATGGAAATAAGTATTTCAATCTCGTCCTAGTTCACAATGTTGGTGGAAAAGGCGATGTGCACGCAGTAGACAT AATACAGAATGGATTCCCATGAAGCGAAACTGGGGAATGAACTGGCAACAGATGCT GGATCC PP Expansin Gene GTTATGACCAAG TGGCCAGGCACTCTCCTTCCGAGTGACAACCAGTGATGGTAAGACCATAGTCTCTATGAACGCAACGCCATCTC ACTGGAGCTTCGGCCAGACCTTCGAGGGAGGTCAGTTCGCTATGAATTGAATTCTGTAACCCCAAGAGCGGTGC CACTCGATGAATGCTTTAGGCGAAGAGTTGATCCACAAGGGAACCTAGACGCAGTTGAGTCTCAATCTAGCTTC ATGATATTTGTTGATACCTATACTGGATACCAATCGGCGTCTTGAATCCTCAACATCTACCCTCCACGCTTTAC CCAGATATCCCGAGATCTGGCCAACGTGAACGGTTTTGAAATTTACCAATATCAGTAGCACATAAAACCCATGG GGTACAATGATTTGTAGGTAGTGCGCAATCATGGGGAT

29. 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.

30. 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