Antonio Bahilo Gmez Miriam Brines Julin Rubn Fernndez Santos Ana - - PowerPoint PPT Presentation

Antonio Bahilo Gómez Miriam Brines Julián Rubén Fernández Santos Ana Monserrat Martínez Elia Pérez León Tatiana Rodríguez Giraldo David Sanjaime Rodríguez

Introduction

• Team: 7 students from Valencia
• Worktime

Diseases transmited by insects like malaria or sleeping sickness

PROBLEM SOLUTION

Creating a yeast capable

• f repelling these insects

introduction aim materials and methods results conclusion

Aim

Create a biological platform within common yeast to develop a device capable of producing several monoterpenoids acting like aromas and repellents

S-linalool geraniol 1,8-cineole

• AROMA 1

linalool synthase from Clarkia breweri (Dudareva et al., 1996) – M. Orejas

• AROMA 2

geraniol synthase from Ocimum basilicum

• REPELLENT

1,8-cineole synthase from Arabidopsis thaliana (Demissie et al., 2012)

introduction aim materials and methods results conclusion

introduction aim materials and methods results conclusion

Inducible promoter Cup1: linalool synthase

Inducible promoter H2O2: geraniol synthase

introduction aim materials and methods results conclusion

S-linalool synthase

• From Clarkia breweri
• GPP  S-linalool
• 2760 bp
• mRNA linear
• Parts registry: BBa_M11053.

http://parts.igem.org/ Part:BBa_M11053:Design? title=Part:BBa_M11053:Design

• Restriction enzymes

incompatibilities: EcoRI, XbaI, BglII, XhoI.

introduction aim materials and methods results conclusion

Materials and methods

• Raw material: LIS in ER85
• Amplification using PCR (TOPO

vector to increase the concentration)

• Digestion of LIS by BamHI and

SalI

• Ligation into pYEX-4T
• Transformation into S.

cerevisiae ERG20 K197G

• Genome-scale metabolic

models to estimate the behavior of the organism.

• Organism metabolism

modeled by a network of metabolites and enzymes that must integrate all biochemical reactions present in the

• rganism.
• Flux Balance Analysis (FBA)

WET LAB DRY LAB

introduction aim materials and methods results conclusion

Results

• Starting from the initial objectives, good results

were not achieved in geraniol synthase and 1,8- cineole synthase. They could not be cloned in their respective plasmids neither expressed in yeast. In the first case, the cDNA could not be obtained; in the second one, the gene could not be amplified from the A. thaliana cDNA.

• LIS was transformed into S. cerevisiae. No

experiments were performed due to time limitation

WET LAB

introduction aim materials and methods results conclusion

• GPP was excreted into the medium.
• Simulation in steady-state

conditions  accumulation

• Decreasing yeast growth excess of GPP
• Point of optimal growth  production value
• f GPP is zero (all is used to grow)
• The points near to the optimum have higher

accuracy, since they are closer to the point validated experimentally

• Production of GPP by ERG20

higher than in the classical strain

• If the yeast was modified to

diminish its growth  more GPP for our reaction

DRY LAB

introduction aim materials and methods results conclusion

Conclusions

• S. cerevisiae ERG20 K197G is a good biologic

device

• Extra experiments could have been performed:
• Optimal concentration of copper for the expression
• Time needed after the addition of copper to
• bserve expression of the gene
• Measuring how the expression varies depending on

the concentration of copper

introduction aim materials and methods results conclusion

Human practices and others

• Survey about the impact of our project on society

Did you know that Saccharomyces is frequently used in research in the field of biology and health sciences? General opinions about transgenic

• rganisms.

Opinion about modified

• rganisms.

• Lipdub to promote science

• Lab practices with teenagers

References

• Butt, T. R., Sternberg, E. J., Gorman, J. A., Clark, P., Hamer, D., Rosenberg, M., & Crooke, S. T. (1984). Copper

metallothionein of yeast, structure of the gene, and regulation of expression. Proceedings of the National Academy

• f Sciences, 81(11), 3332-3336.
• Chen, F., Ro, D. K., Petri, J., Gershenzon, J., Bohlmann, J., Pichersky, E., & Tholl, D. (2004). Characterization of a

root-specific Arabidopsisterpene synthase responsible for the formation of the volatile monoterpene 1, 8-cineole. Plant Physiology.

• Dudareva, N., Cseke, L., Blanc, V. M., and Pichersky, E.(1996) Evolution of floral scent in Clarkia: Novel patterns
• f S-linalool synthase gene expression in the C. breweri flower Plant Cell, 8, 1137-1148.
• Förster J., Famili I., Fu P., Palsson B. Ø., and Nielsen J., 2003, “Genome-scale reconstruction of the

Saccharomyces cerevisiae metabolic network.,” Genome Res., 13(2), pp. 244–53.

• Fischer, M. J., Meyer, S., Claudel, P., Bergdoll, M., & Karst, F. (2011). Metabolic engineering of monoterpene

synthesis in yeast. Biotechnology and bioengineering, 108(8), 1883-1892.

• Mascorro-Gallardo, J. O., Covarrubias, A. A., & Gaxiola, R. (1996). Construction of a CUP1promoter-based vector

to modulate gene expression in Saccharomyces cerevisiae. Gene, 172(1), 169-170.

• Pichersky E, E Lewinsohn, R Croteau. Purification and characterization of S-linalool synthase, an enzyme involved

in the production of floral scent in Clarkia breweri. Arch Biochem Biophys. 316:803-807. (1995).

• Thiele I., and Palsson B. Ø., 2010, “A protocol for generating a high-quality genome-scale metabolic

reconstruction.,” Nat. Protoc., 5(1), pp. 93–121.

• Orth J. D., Thiele I., and Palsson B. Ø., 2010, “What is flux balance analysis?,” Nat. Biotechnol., 28(3), pp. 245–8.