biodetection of hcg hormone -...
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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
• Human chorionic gonadotropin (hCG)
”Could yeast be used as a pregnancy test?”
[1]
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Why?
• Tests today: difficult to recycle
• Biodegradable
• Lower cost
• A specific application of the GPCR-pheromone pathway-system
[2]
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How?
1 2 3
Detection Signal transduction Output signal
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- Expression of the LH/CG receptor in yeast
• The hCG hormone binds to the LH/CG receptor
• G protein-coupled receptor (GPCR)
Detection
[3]
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How?
2
Detection Signal transduction
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- Coupling of the LH/CG receptor with the yeast pheromone pathway
• The pheromone pathway activates transcription factor Ste12 • Ste12 binds to the FIG1 promoter Phosphorylation of
subsequent kinases in MAPK cascade
α-factor binding to Ste2
Sst2
GTP GDP
P P
Transcription activation
Cell cycle arrest
Yeast cell membrane
Ste11
Ste20
Ste4 Ste18 Gpa1
GTP
Ste12 Far1
Ste5
Ste11
Ste7
Fus3
Activation of G-protein
Signal transduction
[4] 7 Team Chalmers : Development of a biodegradable pregnancy test kit
How?
3
Detection Signal transduction Output signal
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• Indigo: intensive colour
• Tryptophan as precursor
• Tryptophanase and monooxygenase
- Production of indigo in yeast
Output signal
[5]
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3
Detection Signal transduction Output signal
1
The biosensor put together
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The IMFD-73 strain
h
Chimeric Gα
Tryptophanase gene P(FIG1) Monooxygenase gene P(TEF1)
CWP2 gene
Increased cell wall permeability
Activated transcription factor acting on the FIG1 promoter
Indigo production from precursor tryptophan
Blue cells as a response to hCG hormone binding
LH/CG receptor with high affinity for hCG
Deleted endogenous GPCR (Ste2))
LHCGR gene P(TEF1)
Activation of the G protein
Sst2 Deleted negative feedback regulator Sst2
hCG hormone binding to the LH/CG receptor
GFP gene P(FIG1)
Ste12
Gβ Gγ
GTP GDP Activation of the yeast
pheromone pathway
Ste12
11
Team Chalmers : Development of a biodegradable pregnancy test kit
[6]
Modelling
Signal transduction
3
Output signal Detection
1
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13
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Modelling results
Concentration of Ste12 for different parameters and different initial concentrations of hCG.
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Modelling results
Concentration of Ste12 with and without the Sst2 negative feedback and with different initial concentrations of hCG.
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Conclusions from modelling
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• 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
Survival of yeast in urine
Deletion of the CWP2 gene
Production of indigo
Functionality of the LH/CG receptor
Achievements
Results
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• Yeast cultivated in urine did not grow
• But did survive for at least 6 hours
Survival of yeast in urine
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• Confirmed CWP2 gene deletion
• Weakened cell wall of the Δcwp2 deletion strain
Deletion of the CWP2 gene
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• Successful cloning of both enzyme genes
• Colour production by cells harbouring genes for indigo production
Results and conclusions Production of indigo
50 shades of brown!
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Absorbance spectrum
0
1
2
3
4
5
6
200 300 400 500 600 700
Ab
sorb
ance
wavelenght (nm)
Galactose, tryptophanaddedafter 24h
Galctose, control
Glucose, tryptophan addedafter 24h
Glucose, control
22
added after 24h
Team Chalmers : Development of a biodegradable pregnancy test kit
Production of indigo
Blue bubbles!
I'm Blue Da Ba Dee Da Ba Daa
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• Successful cloning of two different versions of the LH/CG gene
• Ability to detect hCG assessed by fluorescence screening
Functionality of the LH/CG receptor
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• Background noise present
• No successful detection
• Finalized biosensor system:
Functionality of the LH/CG receptor
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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
Achievements
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Future perspectives
Indigo production
The LH/CG receptor
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Indigo production: Future perspectives
[5]
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Indigo production: Future perspectives
• Presence of greenish cultures and blue bubbles – indications of indigo being produced?
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1. The LH/CG receptor only mediates weak signals when binding hCG
- Improved analysis by using flow cytometry
- Amplification of the signal
LH/CG receptor: Future perspectives
[6]
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Amplification of the signal - a method developed by Fukuda et al 2011
Gα GTP
Gβ Gγ
Activation of the pheromone signaling pathway
Expression of reporter gene Overexpression of Gβ and
Gα GDP
Gβ Gγ
Gβ
[7]
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1. The LH/CG receptor only mediates weak signals when binding hCG
2. The LH/CG receptor is not functional when expressed in yeast
- Improved analysis by using flow cytometry
- Amplification of the signal
LH/CG receptor: Future perspectives
[6] - Fluorescent tag on the LH/CG receptor
- Reengineer the receptor
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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.
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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.
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Thank you for listening!
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Do you want to know more about our project?
Visit our website at:
http://2012.igem.org/Team:Chalmers-Gothenburg
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