1. Transformation of CCM into C3 crops could increase crop yield
Identification and Localization of Carbon Concentrating Components in Chlamydomonas reinhardtii
RESULTS
PIPELINE EFFICIENCY
INTRODUCTION
Most crops, including wheat, rice and soybean use C3 photosynthesis
C4 and CAM photosynthesis overcome the tendency of RuBisCO to wastefully fix oxygen rather than carbon dioxide (photorespiration) while C3 does not
CHLAMYDOMONAS REINHARDTII
Photosynthetic apparatus similar to land plants
Unicellular
Heterotrophic and autotrophic
photosynthetic apparatus
Partially sequenced genome
CARBON CONCENTRATING MECHANISM
CCM increases the concentration of carbon dioxide available to the initial carboxylase of the Calvin cycle, RuBisCO
Benefits include an increased tolerance to low
concentrations of inorganic carbon, reduced
photorespiration and a greater tolerance to
water stress
Transformation of CCM into C3 crops could increase crop yield
REVIEW OF LITERATURE
Maurino, Veronica G., et al., 2010
Synthetic detours naturally occurring in cyanobacteria were installed in Arabidopsis thaliana, to bypass photorespiration
McGrath, Justin M., et al., 2014
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?
Which proteins should be transformed into C3 plants to improve photosynthetic efficiency?
How can we optimize the High Throughput Tagging Pipeline?
467 DNA fragments were successfully amplified
83 fully amplified genes were transformed into E.coli
52 genes were localized efficiently
PCR
67%
Cloning
95%
METHODOLOGY
Identification and Amplification
Multiple genome saturated mutant screen
Touchdown PCR
Products were run through Gel Electrophoresis
Purification
Purified products were run against samples of known concentration to determine DNA concentration
Gibson Assembly
Gel purified genes were cloned in frame with yellow fluorescent protein, CrVenus
Ampicillin resistance
Transformed E.coli by heat shock
Plasmid Sequencing
Plasmids were extracted then cut with the restriction enzyme Eco-RV to confirm successful cloning
Electroporation and Plating
Cassettes were incubated in a 16oC water bath for 5 minutes
DNA was added, and a shock was administered at 800V and 25 uF
Localization
Transformation plates were screened for strong Venus expressing colonies using the Typhoon Imaging System
Confocal imaging determined subcellular localization
Transformation
95%
LOCALIZATION
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
PCR PRODUCTS
Localization
65%
ELECTROPORATION
EFFICIENCY
Image provided courtesy of
Dr. Luke Mackinder
PCR ALTERATIONS
6% DMSO
4.5% DMSO
CONCLUSIONS
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
REFERENCES
Eckardt, Nancy A. "Gene Regulatory Networks of the Carbon-Concentrating Mechanism in Chlamydomonas reinhardtii." The Plant Cell Online 24.5 (2012):
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):
Hom, Erik FY, and Andrew W. Murray. "Niche engineering demonstrates a latent capacity for fungal-algal mutualism." Science  (2014): 94-98
Kebeish, Rashad, et al. "Chloroplastic photorespiratory bypass increases photosynthesis and biomass production in Arabidopsis thaliana." Nature biotechnology 25.5 (2007):
Maurino, Veronica G., and Christoph Peterhansel. "Photorespiration: current status and approaches for metabolic engineering." Current opinion in plant biology 13.3 (2010):
Wang, Yingham, and Deqiang Duanmu. "Carbon dioxide concentrating mechanism in   Chlamydomonas reinhardtii: inorganic carbon transport and CO2 recapture."    pringer Science and Business Media (2010): n. pag. Print. 
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
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,
increasing crop yield