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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