Biodetection of the hCG hormone - Development of a biodegradable pregnancy test kit Team Chalmers, Gothenburg
Pregnancy and the hCG hormone • A historical need for detecting pregnancy ” Could yeast be used as a • Human chorionic gonadotropin (hCG) pregnancy test ?” [1] Team Chalmers : Development of a biodegradable pregnancy test kit 2
Why? • Tests today: difficult to recycle • Biodegradable • Lower cost • A specific application of the GPCR-pheromone pathway-system [2] Team Chalmers : Development of a biodegradable pregnancy test kit 3
How? 1 2 3 Detection Signal transduction Output signal Team Chalmers : Development of a biodegradable pregnancy test kit 4
Detection - Expression of the LH/CG receptor in yeast • The hCG hormone binds to the LH/CG receptor • G protein-coupled receptor (GPCR) [3] Team Chalmers : Development of a biodegradable pregnancy test kit 5
How? 2 Detection Signal transduction Team Chalmers : Development of a biodegradable pregnancy test kit 6
Signal transduction - Coupling of the LH/CG receptor with the yeast pheromone pathway α -factor binding to Ste2 Activation of G-protein Yeast cell membrane • The pheromone pathway Ste18 Gpa1 activates transcription factor Ste12 Ste4 GTP GTP Ste20 GDP Ste5 • Ste12 binds to the FIG1 promoter Sst2 Ste11 Ste11 Phosphorylation of subsequent kinases in Ste7 MAPK cascade Fus3 Far1 Ste12 P P Transcription Cell cycle arrest activation [4] 7 Team Chalmers : Development of a biodegradable pregnancy test kit
How? 3 Detection Signal transduction Output signal Team Chalmers : Development of a biodegradable pregnancy test kit 8
Output signal - Production of indigo in yeast • Indigo: intensive colour • Tryptophan as precursor • Tryptophanase and monooxygenase [5] Team Chalmers : Development of a biodegradable pregnancy test kit 9
The biosensor put together 1 3 Detection Signal transduction Output signal 10 Team Chalmers : Development of a biodegradable pregnancy test kit
The IMFD-73 strain hCG hormone binding LH/CG receptor with to the LH/CG receptor high affinity for hCG Deleted endogenous Activation of the G protein GPCR (Ste2)) G γ Chimeric G α G β GTP Activation of the yeast GDP pheromone pathway Sst2 Deleted negative feedback regulator Sst2 Ste12 Ste12 P(TEF1) LHCGR gene Activated transcription factor acting on the FIG1 promoter P(TEF1) P(FIG1) Tryptophanase gene Monooxygenase gene h CWP2 gene P(FIG1) GFP gene Indigo production from precursor tryptophan Increased cell wall permeability Blue cells as a response to hCG hormone binding [6] 11 Team Chalmers : Development of a biodegradable pregnancy test kit
Modelling 1 3 Signal transduction Detection Output signal Team Chalmers : Development of a biodegradable pregnancy test kit 12
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Modelling results Concentration of Ste12 for different parameters and different initial concentrations of hCG. Team Chalmers : Development of a biodegradable pregnancy test kit 15
Modelling results Concentration of Ste12 with and without the Sst2 negative feedback and with different initial concentrations of hCG. Team Chalmers : Development of a biodegradable pregnancy test kit 16
Conclusions from modelling • The binding has to be efficient enough • Disassociation cannot be too high • The LH/CG receptor has to be able start the G-protein cycle with a rate that is at least 10% of the rate in the real yeast pheromone pathway • The Sst2 negative feedback has no significant effect Team Chalmers : Development of a biodegradable pregnancy test kit 17
Results Survival of yeast in urine Deletion of the CWP2 gene Production of indigo Functionality of the LH/CG receptor Achievements Team Chalmers : Development of a biodegradable pregnancy test kit 18
Survival of yeast in urine • Yeast cultivated in urine did not grow • But did survive for at least 6 hours 19 Team Chalmers : Development of a biodegradable pregnancy test kit
Deletion of the CWP2 gene • Confirmed CWP2 gene deletion • Weakened cell wall of the Δ cwp2 deletion strain Team Chalmers : Development of a biodegradable pregnancy test kit 20
Results and conclusions Production of indigo • Successful cloning of both enzyme genes 50 shades of brown! • Colour production by cells harbouring genes for indigo production Team Chalmers : Development of a biodegradable pregnancy test kit 21
Absorbance spectrum 6 5 Galactose, tryptophan 4 addedafter 24h added after 24h Absorbance Galctose, control 3 Glucose, tryptophan added 2 after 24h Glucose, control 1 0 200 300 400 500 600 700 wavelenght (nm) Team Chalmers : Development of a biodegradable pregnancy test kit 22
Production of indigo I'm Blue Da Ba Dee Da Ba Daa Blue bubbles! Team Chalmers : Development of a biodegradable pregnancy test kit 23
Functionality of the LH/CG receptor • Successful cloning of two different versions of the LH/CG gene • Ability to detect hCG assessed by fluorescence screening Team Chalmers : Development of a biodegradable pregnancy test kit 24
Team Chalmers : Development of a biodegradable pregnancy test kit 25
Team Chalmers : Development of a biodegradable pregnancy test kit 26
Team Chalmers : Development of a biodegradable pregnancy test kit 27
Functionality of the LH/CG receptor • Background noise present • No successful detection • Finalized biosensor system: Team Chalmers : Development of a biodegradable pregnancy test kit 28
Achievements Designing a system that could enable yeast to function as a pregnancy test Confirmation of survival of yeast in urine Modelling of the pheromone pathway Construction of needed plasmids Cloning of genes needed for indigo production Cloning of two different versions of the LH/CG gene Deletion of the CWP2 gene Creation of a strain with a weakened cell wall Production of colour in transformed cells Creation of a biosensor system BioBrick sent in to Registry Verified indigo production Verified detection of hCG Team Chalmers : Development of a biodegradable pregnancy test kit 29
Future perspectives Indigo production The LH/CG receptor Team Chalmers : Development of a biodegradable pregnancy test kit 30
Indigo production: Future perspectives [5] Team Chalmers : Development of a biodegradable pregnancy test kit 31
Indigo production: Future perspectives • Presence of greenish cultures and blue bubbles – indications of indigo being produced? Team Chalmers : Development of a biodegradable pregnancy test kit 32
LH/CG receptor: Future perspectives 1. The LH/CG receptor only mediates weak signals when binding hCG - Improved analysis by using flow cytometry - Amplification of the signal [6] Team Chalmers : Development of a biodegradable pregnancy test kit 33
Amplification of the signal - a method developed by Fukuda et al 2011 Gβ Gγ Gγ G α G α Gβ Gβ GTP GDP Activation of the pheromone signaling pathway Expression of reporter gene Overexpression of G β and [7] Team Chalmers : Development of a biodegradable pregnancy test kit 34
LH/CG receptor: Future perspectives 1. The LH/CG receptor only mediates weak signals when binding hCG - Improved analysis by using flow cytometry - Amplification of the signal 2. The LH/CG receptor is not functional when expressed in yeast [6] - Fluorescent tag on the LH/CG receptor - Reengineer the receptor Team Chalmers : Development of a biodegradable pregnancy test kit 35
References [1] http://www.rcsb.org/pdb/explore/explore.do?structureId=1HRP [2] http://www.okokchina.com/product/Medicine-Health-Environment/Medical-Surgical-Instruments/Test- Tubes-Incubators/Pregnancy-Test/ [3] Figure adapted from: Alberts B, Johnson A, Lewis J, Ra M, Roberts K, Walter P. Molecular Biology of the Cell. 5th ed. New York: Garland Science; 2008. [4] Figure adapted from: Ishii J, Fukada N, Tanaka T, Ogino C, Kondo A. Protein-protein interactions and selection: yeast-based approaches that exploit guanine nucleotide-binding protein signaling. Federation of European Biochemical Societies Journal. 2010;277(9):1982-95. [5] Figure adapted from: Han GH, Shin HJ, Kim SW. Optimization of bio-indigo production by recombinant E. coli harboring fmo gene. Enzyme and Microbial Technology. 2008;42(7):617-623. [6] http://www.abcam.com/index.html?pageconfig=resource&rid=11446 [7] Figure adapted from: Fukuda N, Ishii J, Kaishima M, Kondo A. Amplification of agonist stimulation of human G-protein-coupled receptor signaling in yeast. Analytical Biochemistry. 2011;417(2):182-187. Team Chalmers : Development of a biodegradable pregnancy test kit 36
Acknowledgements We would like to thank • Kondo Group, Kobe University, for the IMFD-73 strain • Kim Group, Chosun University, for the fmo gene • Our supervisors Dr. Verena Siewers, Assoc. Prof. Joakim Norbeck, Prof. Torbjörn Lundh and Assoc. Prof. Marcus Wilhelmsson for expertise and help. Team Chalmers : Development of a biodegradable pregnancy test kit 37
Team Chalmers : Development of a biodegradable pregnancy test kit 38
Thank you for listening! Team Chalmers : Development of a biodegradable pregnancy test kit 39
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