Team:Wash U/Project

From 2009.igem.org

(Difference between revisions)

Revision as of 20:42, 6 July 2009

Abstract

Our project goal is to maximize the photosynthetic productivity for a culture of Rhodobacter sphaeroides under both high and low light intensities in a bioreactor by synthetically regulating the size of the light-harvesting antenna. We chose to undertake such a project in R. sphaeroides due to its well-characterized photosynthetic and genetic system.

The antenna system functions to expand the spectrum of light available for photosynthesis by absorbing different wavelengths than that of the reaction center. Essentially, it is like the dish around the main receiver of any antenna. Marine bacteria, such as R. sphaeroides, evolved very large antenna complexes to absorb light in a natural environment where there is great competition for photons. As a result, the photosynthetic machinery is saturated at a rather low light intensity in a synthetic non-competitive environment, such as a bioreactor. This causes up to 95% of incidental photons to be dissipated as heat or fluorescence by the bacteria at the exterior of a bioreactor through a process called Non-Photochemical Quenching (NPQ) (Mussgnug et al., 2007). In essence, these photons are being wasted as NPQ reduces light penetration into a bioreactor and starves cells on the interior for photons. One method that has been shown to improve photosynthetic efficiency is the reduction of light-harvesting antenna sizes (Polle et al., 2002, Mussgnug et al., 2007). Though, current approaches to this end rely on genetic knockouts and as such are difficult to precisely control from the perspective of metabolic engineering and synthetic biology. Our intention is to create a more dynamic system to vary antenna size that is dependent on incidental light intensity and that can be readily optimized using synthetic biology principles. This system should result in the bacteria at the exterior of the bioreactor expressing fewer light harvesting antenna proteins than the cells at the interior, reducing NPQ while maintaining a high absorbance of incidental photons.

We will focus on altering the quantity of the Light Harvesting Complex 2 (LH2) by regulating the pucB/A genes that code for the two subunits of the complex. LH2 absorbs photons maximally at the wavelength of 842 nm and funnels its energy to LH1 and the reaction center. The ratio of LH2 complexes to LH1 naturally ranges from 3.0 to 6.7 under varying light conditions (Scheuring et al., 2005). We propose that if this ratio of LH2 to LH1 is altered to range from 0-7 or more in response to incidental light intensity, then the photosynthetic efficiency and productivity for a culture of R. sphaeroides may be maximized.
Back To Top

Analysis

full report
Back To Top

Reults

results
Back To Top

Conclusion

conclusion
Back To Top

References

"2007-2008 Catalog & Technical Reference." New England BioLabs Inc. (2007).
Sambrook, Joseph and David W. Russell. Molecular Cloning: A Laboratory Manual. Vol. 1-3. New York: Cold Springs Harbor Laboratory Press, 2001.
Smith, Harry and M. Geoffrey Holmes. Techniquies in Photomorphogenesis. London: Academic Press, Inc., 1984.
"Life Sciences Catalog 2009-2010." National Diagnostics. (2009).
Ausubel, Fred et al. Short Protocols in Molecular Biology: Third Edition. Canada: John Wiley and Sons, Inc., 1999.
"Entrez Nucleotide Search." National Center for Biotechnology Information. 2009. 6 June 2009 <http://www.ncbi.nlm.nih.gov/nuccore/49175990?from=3533887&to=3534606&report=gbwithparts>.
"NEBCutter V2.0." New England BioLabs. 2009. 6 June 2009 <http://tools.neb.com/NEBcutter2/index.php>.
"OligoAnalyzer 3.1." Integrated DNA Technologies. 2009. 6 June 2009. <http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/>
Lagarias, J. Clark. PCB from Spirulina. Personal Communication. June 2009.
Shimizu-Sato, Sae et al. "A Light-Switchable Gene Promoter System." Nature Publishing Group. Vol. 20 (2002): 1,041-1044.
Yakovlev, A. G. et al. "Light Control Over the Size of an Antenna Unit Building Block as an Efficient Strategy for Light Harvesting in Photosynthesis." Federation of European Biochemical Societies. (2002): 129-132.
Jones, M. R. et al. "Mutants of Rhodobacter sphaeroides Lacking One or More Pigment-Protein Complexes and Complementation with Reaction-Centre, LH1, and LH2 Genes." Molecular Microbiology. Vol. 6.9 (1992): 1,173-1,184.
Jager, Andreas et al. "The AppA and PpsR Proteins from Rhodobacter sphaeroides Can Establish a Redox-Dependent Signal Chain but Fail to Transmit Blue-Light Signals in Other Bacteria." Journal of Bacteriology. Vol. 189.6 (2007): 2,274-2,282.
Terry, Matthew J. "Biosynthesis and Analysis of Bilins." Heme, Chlorophyll, and Bilins: Methods and Protocols. (2002) 273-291. # Dragnea, Vladimira et al. "Time-Resolved Spectroscopic Studies of the AppA Blue-Light Receptor BLUF Domain from ''Rhodobacter sphaeroides'' Biochemistry. Vol. 44 (2005): 15,978-15,985.
Levskaya, Anselmetal et al. "Engineering Escherichia coli to See Light." Nature. Vol. 438 (2005): 441-442.
Laible, Philip D. "Foreign Gene Expression in Photosynthetic Bacteria." The Purple Phototropic Bacteria. (2009): 839-860.
Fowler, Gregory J. S. and C. Neil Hunter. "The Synthesis and Assembly of Functional High and Low Light LH2 Antenna Complexes from Rhodopseudomonas palustris in Rhodobacter sphaeroides." The Journal of Biological Chemistry. Vol. 271.23 (1996): 13,356-13,361.
Wang, Wanneng et al. "Heterologous Synthesis and Assembly of Functional LHII Antenna Complexes from Rhodovulum sulfidophilum in Rhodobacter sphaeroides Mutant." Molecular Biology Reports. Vol. 12.3 (2008).
Braatsch, Stephan et al. "A single Flavoprotein AppA, Integrates Both Redox and Light Signals in Rhodobacter sphaeroides." Molecular Microbiology. Vol. 45.3 (2002): 827-836.
Mizuno, Takeshi and Shoji Mizushima. "Characterization by Deletion and Localized Mutagenesis in Vitro of the Promoter Region of the Escherichia coli OmpC Gene and Importance of the Upstream DNA Domain in Positive Regulation by the OmpR Protein." Journal of Bacteriology. Vol. 168.1 (1986): 86-95.
Brown, Stanley B. et al. "Bile Pigment Synthesis in Plants: Incorporation of Haem into Phycocyanobilin and Phycocyanobiliproteins in Cyanidium Caldarium." Biochemistry. Vol. 194 (1981): 137-147.

Back To Top

@@ Line 50: / Line 50: @@
 # Laible, Philip D. "Foreign Gene Expression in Photosynthetic Bacteria."  <html><u>The Purple Phototropic Bacteria.</u></html>  (2009): 839-860.
 # Fowler, Gregory J. S. and C. Neil Hunter.  "The Synthesis and Assembly of Functional High and Low Light LH2 Antenna Complexes from ''Rhodopseudomonas palustris'' in ''Rhodobacter sphaeroides.''"  <html><u>The Journal of Biological Chemistry.</u></html>  Vol. 271.23 (1996): 13,356-13,361.
+# Wang, Wanneng et al.  "Heterologous Synthesis and Assembly of Functional LHII Antenna Complexes from ''Rhodovulum sulfidophilum'' in ''Rhodobacter sphaeroides'' Mutant."  <html><u>Molecular Biology Reports.</u></html>  Vol. 12.3 (2008).
+# Braatsch, Stephan et al.  "A single Flavoprotein AppA, Integrates Both Redox and Light Signals in ''Rhodobacter sphaeroides.''"  <html><u>Molecular Microbiology.</u></html>  Vol. 45.3 (2002): 827-836.
+# Mizuno, Takeshi and Shoji Mizushima.  "Characterization by Deletion and Localized Mutagenesis in Vitro of the Promoter Region of the ''Escherichia coli'' OmpC Gene and Importance of the Upstream DNA Domain in Positive Regulation by the OmpR Protein."  <html><u>Journal of Bacteriology.</u></html>  Vol. 168.1 (1986): 86-95.
+# Brown, Stanley B. et al.  "Bile Pigment Synthesis in Plants: Incorporation of Haem into Phycocyanobilin and Phycocyanobiliproteins in Cyanidium Caldarium."  <html><u>Biochemistry.</u></html>  Vol. 194 (1981): 137-147.
+#