biodetection of hcg hormone -...

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]

2 Team Chalmers : Development of a biodegradable pregnancy test kit

Why?

• Tests today: difficult to recycle

• Biodegradable

• Lower cost

• A specific application of the GPCR-pheromone pathway-system

[2]


How?

1 2 3

Detection Signal transduction Output signal


- Expression of the LH/CG receptor in yeast

• The hCG hormone binds to the LH/CG receptor

• G protein-coupled receptor (GPCR)

Detection

[3]


How?

2

Detection Signal transduction


- 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



• Indigo: intensive colour

• Tryptophan as precursor

• Tryptophanase and monooxygenase

- Production of indigo in yeast

Output signal

[5]


3


1

The biosensor put together


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


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

Survival of yeast in urine

Deletion of the CWP2 gene

Production of indigo

Functionality of the LH/CG receptor

Achievements

Results


• Yeast cultivated in urine did not grow

• But did survive for at least 6 hours

Survival of yeast in urine


• Confirmed CWP2 gene deletion

• Weakened cell wall of the Δcwp2 deletion strain



• 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!


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


• Successful cloning of two different versions of the LH/CG gene

• Ability to detect hCG assessed by fluorescence screening



• Background noise present

• No successful detection

• Finalized biosensor system:



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


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


Future perspectives

Indigo production

The LH/CG receptor


Indigo production: Future perspectives

[5]


Indigo production: Future perspectives

• Presence of greenish cultures and blue bubbles – indications of indigo being produced?


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]


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]


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


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.


http://www.rcsb.org/pdb/explore/explore.do?structureId=1HRP

http://www.okokchina.com/product/Medicine-Health-Environment/Medical-Surgical-Instruments/Test-Tubes-Incubators/Pregnancy-Test/















http://www.abcam.com/index.html?pageconfig=resource&rid=11446

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.


Thank you for listening!


Do you want to know more about our project?

Visit our website at:

http://2012.igem.org/Team:Chalmers-Gothenburg





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