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Identification and Localization of Carbon Concentrating Mechanism Components in Chlamydomonas reinhardtii Zoe Friedberg 2014-2015 Introduction: Photosynthesis ● Most crops, including wheat, rice and soybean use C3 photosynthesis ● Limitations http://www.citruscollege.edu/lc/archive/biology/PublishingImages/c06_14.jpg Introduction: Chlamydomonas reinhardtii ● Photosynthetic apparatus similar to land plants http://protist.i.hosei.ac.jp ● Optional photosynthesis ● Partially sequenced genome flagellum eyespot basal body nucleus mitochondrion pyrenoid chloroplast Image provided courtesy of Dr. Luke Mackinder Introduction: Carbon Concentrating Mechanism (CCM) ● CCM increases the concentration of carbon dioxide available to the initial carboxylase of the Calvin cycle, RuBisCO ● Benefits include: o Increased tolerance to low concentrations of atmospheric CO2 o Reduced photorespiration o Greater tolerance to water stress ● Transformation of CCM into C3 crops to increase crop yield Review of Literature Pollock, Steve V., et al. "Rubisco activase is required for optimal photosynthesis in the green alga Chlamydomonas reinhardtii in a low-CO2 atmosphere." Plant physiology 133.4 (2003): 18541861. ● Generated a mutant that lacked the gene for RuBisCO Activase (Rca) ● Mutant grew at 60% of photosynthetic capacity ● CCM partially compensated for the absence of an active Rca Review of Literature Maurino, Veronica G., and Christoph Peterhansel. "Photorespiration: current status and approaches for metabolic engineering." Current opinion in plant biology 13.3 (2010): 248255. ● Identified mutants with reduced photorespiration and high photosynthetic yield ● Synthetic detours naturally occurring in cyanobacteria installed in Arabidopsis thaliana, to bypass photorespiration ● Enrichment of CO2 in the chloroplast and increased plant growth Review of Literature McGrath, Justin M., and Stephen P. Long. "Can the cyanobacterial carbon-concentrating mechanism increase photosynthesis in crop species? A theoretical analysis." Plant physiology 164.4 (2014): 2247-2261. ● Models predict the benefits of engineering a full cyanobacterial CCM into a C3 leaf ● Increased: ▪ Leaf photosynthesis by 25% ▪ Soybean yield by 15% ▪ Water use efficiency by 20% Key Questions ● What proteins are essential to the functioning of the CCM in Chlamydomonas reinhardtii? ● Which proteins should be transformed into C3 plants to improve photosynthetic efficiency? Methodology: Pipeline Protocol Gene Identification/ Amplification Gibson Assembly Purification Sequencing Transformation Localization Methodology: Identification and Amplification PCR Products Identification ● Multiple genome saturated mutant screen Amplification ● Touchdown PCR ● Products were run through Gel Electrophoresis 5A8 5A9 5A10 5A11 5A5 5A6 5A7 5A5 5A7 5A6 Amplicon Sizes: 5A5: 443 5A6: 705 5A7: 729 5A8: 772 5A9: 792 5A9 5A8 5A11 5A10 5A10: 840 5A11: 1018 5A12: 1022 2G11: 1115 2G11 5A12 (positive control) Methodology: PCR Alterations ● Annealing temperature o TD PCR o 66oC, 68oC, 70oC, 72oC ● Extension time o 30 seconds per 1000 base pairs ● DMSO concentrations o 3%, 4.5%, 6% ● Addition of Betaine Methodology: Purification Primer 5A5-5A11 Concentrations Purified products were run against samples of known concentration to determine DNA concentration 5A5 5A7 5A6 5A9 5A8 5A11 5A10 2G11 Methodology: Gibson Assembly ● Gel purified genes were cloned in frame with the yellow fluorescent protein, CrVenus ● Ampicillin resistance ● Transformed E.coli by heat shock Image provided courtesy of Dr. Luke Mackinder Methodology: Plasmid Sequencing ● Cells were plated on LB agar containing carbenicillin ● Plasmids were extracted then cut with the restriction enzyme Eco-RV to confirm successful cloning Methodology: Electroporation and plating ● Incubated in a 16oC water bath for 5 minutes ● DNA was added ● Shock was administered at 800V and 25µF ● TAP paromomycin plate www.btxonline.com Methodology: Localization ● Transformation plates were screened for strong VENUS expressing colonies using the Typhoon Imaging System ● Confocal imaging determined subcellular localization Results: Pipeline Efficiency PCR 67% Cloning 95% Transformation 95% ● 467 DNA Fragments were successfully amplified ● 83 fully amplified genes were transformed into E.coli ● 52 genes were localized efficiently Localization 60% Results: PCR Condition Comparison A B D C Key: NA: No amplicon SA: Single Amplicon MA: Multiple Amplicon FA: Faint Amplicon Conditions: All under 70C annealing temperature A. 2 min ext. time, 4.5% DMSO B. 2min ext. time, 6% DMSO C. 1 min 20 sec ext. time, 6% DMSO D. 1min 20 sec ext. time, 4.5% DMSO Results: 6% DMSO 2min 1 min 20 sec ● 2 min vs 1 min 20 sec (6%DMSO) ○ 11 primers had more successful results with 1 min 20 sec extension time Key: NA: No amplicon SA: Single Amplicon MA: Multiple Amplicon FA: Faint Amplicon Results: 4.5% DMSO 2min 1 min 20 sec ● 2 min vs 1 min 20 sec (4.5% DMSO) ○ 1 primer had a more successful result with 2 min extension time ○ 13 primers had more successful results with 1 min 20 sec extension time Key: NA: No amplicon SA: Single Amplicon MA: Multiple Amplicon FA: Faint Amplicon Results: Electroporation Efficiency Results: Localization ● Organelles were marked to aid in determining subcellular localizations and identifications ● Cre04.g229300_F, a suggested RuBisCO Activase coding protein ● The concentrated green pocket indicated that the gene was localized in the pyrenoid ● In total, 83 genes were localized, and 60% were identifiable Conclusion ● High Throughput Tagging Pipeline can be successfully utilized to amplify and localize putative photosynthetic genes of the CCM in the alga Chlamydomonas reinhardtii ● Electroporation efficiency is negatively affected by increased time transformation cassettes were left in a 16oC water bath ● Failed PCR primers can be recovered by increasing DMSO levels and decreasing extension times Discussion ● With the high throughput tagging pipeline genes are continuing to be identified and localized at a rapid rate ● Their function and interaction with the genome are still unknown Discussion ● 82 genes will be imaged and analyzed at a later date ● Complementation vectors will be constructed with different resistance markers ● Increased availability of information to other labs will assist in all realms of C.reinhardtii research Discussion ● It is estimated that there will be about 11 billion people living on the earth by 2100 ● New innovations need to be made to increase food production ● Transforming a viable CCM into crop plants augments photosynthetic productivity Thank you! Luke Mackinder, Martin Jonikas, the Jonikas Lab, Mrs. Kleinman, Ms. Foisy and Ms. O’Hagan. Chris Chen Matt Rodman Zoe Friedberg Rachel Vasquez Bibliography Eckardt, Nancy A. "Gene Regulatory Networks of the Carbon-Concentrating Mechanism in Chlamydomonas reinhardtii." The Plant Cell Online 24.5 (2012): 1713-1713. Fang, Wei, et al. "Transcriptome-wide changes in Chlamydomonas reinhardtii gene expression regulated by carbon dioxide and the CO2-concentrating mechanism regulator CIA5/CCM1." The Plant Cell Online 24.5 (2012): 1876-1893. Hom, Erik FY, and Andrew W. Murray. "Niche engineering demonstrates a latent capacity for fungal-algal mutualism." Science 345.6192 (2014): 94-98 Kebeish, Rashad, et al. "Chloroplastic photorespiratory bypass increases photosynthesis and biomass production in Arabidopsis thaliana." Nature biotechnology 25.5 (2007): 593599. Maurino, Veronica G., and Christoph Peterhansel. "Photorespiration: current status and approaches for metabolic engineering." Current opinion in plant biology 13.3 (2010): 248255. Wang, Yingham, and Deqiang Duanmu. "Carbon dioxide concentrating mechanism in Chlamydomonas reinhardtii: inorganic carbon transport and CO2 recapture." Springer Science and Business Media (2010): n. pag. Print. Wang, Yingjun, Deqiang Duanmu, and Martin H. Spalding. "Carbon dioxide cocentrating mechanism in Chlamydomonas reinhardtii: inorganic carbon transport and CO2 recapture." Springer Science and Business Media (2011): 115-22. Print. Identification and Localization of Carbon Concentrating Mechanism Components in Chlamydomonas reinhardtii Zoe Friedberg 2014-2015