Biodetection of the hCG hormone - Development of a biodegradable - - PowerPoint PPT Presentation

▶
biodetection of the hcg hormone
SMART_READER_LITE
LIVE PREVIEW

Biodetection of the hCG hormone - Development of a biodegradable - - PowerPoint PPT Presentation

Sep 28, 2023 374 likes •789 views

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

slide-1
SLIDE 1

Biodetection of the hCG hormone

  • Development of a biodegradable pregnancy test kit

Team Chalmers, Gothenburg

slide-2
SLIDE 2

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

slide-3
SLIDE 3

Why?

  • Tests today: difficult to recycle
  • Biodegradable
  • Lower cost
  • A specific application of the GPCR-pheromone pathway-system

[2]

3 Team Chalmers : Development of a biodegradable pregnancy test kit

slide-4
SLIDE 4

How?

1 2 3

Detection Signal transduction Output signal

4 Team Chalmers : Development of a biodegradable pregnancy test kit

slide-5
SLIDE 5
  • Expression of the LH/CG receptor in yeast
  • The hCG hormone binds to the LH/CG receptor
  • G protein-coupled receptor (GPCR)

Detection

[3]

5 Team Chalmers : Development of a biodegradable pregnancy test kit

slide-6
SLIDE 6

How?

2

Detection Signal transduction

6 Team Chalmers : Development of a biodegradable pregnancy test kit

slide-7
SLIDE 7
  • 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

slide-8
SLIDE 8

How?

3

Detection Signal transduction Output signal

8 Team Chalmers : Development of a biodegradable pregnancy test kit

slide-9
SLIDE 9
  • Indigo: intensive colour
  • Tryptophan as precursor
  • Tryptophanase and monooxygenase
  • Production of indigo in yeast

Output signal

[5]

9 Team Chalmers : Development of a biodegradable pregnancy test kit

slide-10
SLIDE 10

3

Detection Signal transduction Output signal

1

The biosensor put together

10 Team Chalmers : Development of a biodegradable pregnancy test kit

slide-11
SLIDE 11

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]

slide-12
SLIDE 12

Modelling

Signal transduction

3

Output signal Detection

1

12 Team Chalmers : Development of a biodegradable pregnancy test kit

slide-13
SLIDE 13

13

slide-14
SLIDE 14

14

slide-15
SLIDE 15

Modelling results

Concentration of Ste12 for different parameters and different initial concentrations of hCG.

Team Chalmers : Development of a biodegradable pregnancy test kit 15

slide-16
SLIDE 16

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

slide-17
SLIDE 17

Conclusions from modelling

17 Team Chalmers : Development of a biodegradable pregnancy test kit

  • 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
slide-18
SLIDE 18

Survival of yeast in urine Deletion of the CWP2 gene Production of indigo Functionality of the LH/CG receptor Achievements

Results

18 Team Chalmers : Development of a biodegradable pregnancy test kit

slide-19
SLIDE 19
  • Yeast cultivated in urine did not grow
  • But did survive for at least 6 hours

Survival of yeast in urine

19 Team Chalmers : Development of a biodegradable pregnancy test kit

slide-20
SLIDE 20
  • Confirmed CWP2 gene deletion
  • Weakened cell wall of the Δcwp2 deletion strain

Deletion of the CWP2 gene

20 Team Chalmers : Development of a biodegradable pregnancy test kit

slide-21
SLIDE 21
  • 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!

21 Team Chalmers : Development of a biodegradable pregnancy test kit

slide-22
SLIDE 22

Absorbance spectrum

1 2 3 4 5 6 200 300 400 500 600 700

Absorbance wavelenght (nm)

Galactose, tryptophan addedafter 24h Galctose, control Glucose, tryptophan added after 24h Glucose, control

22

added after 24h

Team Chalmers : Development of a biodegradable pregnancy test kit

slide-23
SLIDE 23

Production of indigo

Blue bubbles!

I'm Blue Da Ba Dee Da Ba Daa

23 Team Chalmers : Development of a biodegradable pregnancy test kit

slide-24
SLIDE 24
  • 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

24 Team Chalmers : Development of a biodegradable pregnancy test kit

slide-25
SLIDE 25

25 Team Chalmers : Development of a biodegradable pregnancy test kit

slide-26
SLIDE 26

26 Team Chalmers : Development of a biodegradable pregnancy test kit

slide-27
SLIDE 27

27 Team Chalmers : Development of a biodegradable pregnancy test kit

slide-28
SLIDE 28
  • Background noise present
  • No successful detection
  • Finalized biosensor system:

Functionality of the LH/CG receptor

28 Team Chalmers : Development of a biodegradable pregnancy test kit

slide-29
SLIDE 29

 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

29 Team Chalmers : Development of a biodegradable pregnancy test kit

slide-30
SLIDE 30

Future perspectives

Indigo production The LH/CG receptor

30 Team Chalmers : Development of a biodegradable pregnancy test kit

slide-31
SLIDE 31

Indigo production: Future perspectives

[5]

31 Team Chalmers : Development of a biodegradable pregnancy test kit

slide-32
SLIDE 32

Indigo production: Future perspectives

  • Presence of greenish cultures and blue bubbles – indications of indigo

being produced?

32 Team Chalmers : Development of a biodegradable pregnancy test kit

slide-33
SLIDE 33
  • 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]

33 Team Chalmers : Development of a biodegradable pregnancy test kit

slide-34
SLIDE 34

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]

34 Team Chalmers : Development of a biodegradable pregnancy test kit

slide-35
SLIDE 35
  • 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

35 Team Chalmers : Development of a biodegradable pregnancy test kit

slide-36
SLIDE 36

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.

36 Team Chalmers : Development of a biodegradable pregnancy test kit

slide-37
SLIDE 37

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.

37 Team Chalmers : Development of a biodegradable pregnancy test kit

slide-38
SLIDE 38

38 Team Chalmers : Development of a biodegradable pregnancy test kit

slide-39
SLIDE 39

Thank you for listening!

39 Team Chalmers : Development of a biodegradable pregnancy test kit

slide-40
SLIDE 40

Do you want to know more about our project?

Visit our website at: http://2012.igem.org/Team:Chalmers-Gothenburg

40 Team Chalmers : Development of a biodegradable pregnancy test kit