Is it Happy Hour Yet?

1. University of Missouri - Rolla Is it Happy Hour Yet? Herman Armstrong Brian Pink Rachel Klapper Cory Cheatham Morgan Schiermeier Jackie Schneider Biological Timer and Breathalyzer Development of a

2. Design and Testing of a Bacteriological Timer Device Herman Armstrong Morgan Schiermeier Rachel Klapper Jackie Schneider

3. The Device We have decided to create a biological timer. This idea was spurred by observing some of the previously created projects, which included biological clocks. Building on this idea, we want to very precisely monitor the time between when an organism begins to feed upon until it finishes feeding on a food source – in this case, a sugar.

4. The Repressilator Periodically induces the synthesis of green fluorescence protein (GFP). Nature.com <http://www.nature.com/nature/journal/v403/n6767/fig_tab/403335a0_F1.html>

5. Arabinose C Promoter (araC) Tetracycline Repressor +LVA (TetR)

6. Assembly Using enzymes, araC can be cut out of its plasmid, and the plasmid containing tetR can be opened. araC can be inserted into the tetR plasmid through ligation.

7. Overall project • Step 1 Build timer device – new repressilator araC promoter tetR

8. Overall project • Step 2 Put timer device and reporter in bacteria E coli cell timer GFP reporter

9. AraC TetR GFP Reporter Negative Regulation INPUT No GFP (repressed) Arabinose Sugar TetR OUTPUT GFP (fluorescence activated) No more sugar NoTetR

10. Overall project • Step 3 Test timer conditions and visualize results Glowing E. coli from Elowitz and Leibler

11. Conclusions • Our goal was to build a new timer device to add to the parts registry • Obstacles • Gel extraction of fragments – spin column • Ligation efficiency • Next step • Alternative gel extraction methods • Sacrifice to cloning gods

13. Constructing a Biological Breathalyzer Cory Cheatham Brian Pink

14. The Device The Bio-Breathalyzer is a device designed to determine the blood alcohol concentration of an individual. It is constructed using DNA from Pichia pastoris, a strain of yeast with a diauxic metabolic pathway for ethanol and methanol. The alcohol sensor will utilize this metabolic activity, along with a fluorescent protein indicator fused with the alcohol oxidase gene (AOXI) promoter. When the ethanol is consumed, the E.coli transformed with this tagged gene will fluoresce. The amount of alcohol present will be based on the time it takes the bacteria to fluoresce.

15. Background • Complex pathway for metabolizing methanol in spp. of the Pichia taxa • Alcohol oxidase (AOX) serves as the major enzyme • AOX encoded in AOX1 and AOX2 genes • AOX converts methanol to formaldehyde

16. Background • Pathway for ethanol metabolism also exists • Ethanol is preferred • If both ethanol and methanol are present, ethanol will be consumed first • AOX gene will not be expressed until ethanol has been consumed

17. Growth and Carbon utilization of P. pastoris Methanol Ethanol Background

18. Method

19. AOXI promoter AOXI promoter AOXI promoter AOXI promoter EcoRI EcoRI EcoRI XbaI XbaI XbaI PstI PstI PstI SpeI SpeI SpeI Construction • Isolation of AOXI promoter • Amplified AOXI promoter using PCR • Flanked AOXI promoter with appropriate restriction sites using • primers PCR

20. AOXI promoter AOXI promoter EcoRI XbaI PstI SpeI EcoRI XbaI PstI SpeI Construction • Isolation of AOXI promoter • Cloned PCR product into pCR2.1 vector Transformation +

21. Construction • Isolation of AOXI promoter • Transformed pCR2.1 vector with promoter into competent cells

22. AOXI promoter AOXI promoter EcoRI EcoRI + Construction • Checking AOX1 promoter clones • Isolated AOXI promoter from pCR2.1 plasmid using EcoRI

23. promoter RFP Construction • Preparation of BBa_J61002 vector • Obtained BBa_J61002 vector from registry • Transformed BBa_J61002 into E. coli cells to amplify Transformation + Amplification E. coli

24. Construction • Preparation of BBa_J61002 vector • Extracted amplified BBa_J61002 vectors Extraction

25. EcoRI XbaI PstI AOXI AOXI SpeI promoter promoter RFP RFP Construction • Ligation of AOXI promoter and BBa_J61002 vector • Digested AOXI promoter clone and BBa_J61002 vector with XbaI and SpeI + + XbaI SpeI

26. AOXI promoter AOXI AOXI RFP RFP RFP XbaI SpeI Construction • Ligation of AOXI promoter and BBa_J61002 vector • Performed overnight ligation at 16 ºC • Transformed ligation into E. coli cells Transformation Ligation +

27. Method • Culture transformed yeast cells • Test response of yeast cells to ethanol • Design device

28. Obstacles Restriction enzyme cutting sites within AOX1 gene Introducing methanol and ethanol simultaneously in breathalyzer