is it happy hour yet?
DESCRIPTION
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. Design and Testing of a Bacteriological Timer Device. Herman Armstrong - PowerPoint PPT PresentationTRANSCRIPT
Design and Testing of a Bacteriological Timer Device
Herman Armstrong
Morgan Schiermeier
Rachel Klapper
Jackie Schneider
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.
The Repressilator
Nature.com
<http://www.nature.com/nature/journal/v403/n6767/fig_tab/403335a0_F1.html>
Periodically induces the synthesis of green fluorescence protein (GFP).
Arabinose C Promoter
(araC)
Tetracycline Repressor +LVA
(TetR)
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.
Overall project
• Step 1 Build timer device – new repressilator
araC promoter
tetR
E coli cell
timer
GFP reporter
Overall project
• Step 2 Put timer device and reporter in bacteria
AraC TetR GFP Reporter
Negative Regulation
Arabinose
Sugar
No more sugar
TetR No GFP
(repressed)
No TetR GFP(fluorescence
activated)
INPUT
OUTPUT
• Step 3 Test timer conditions and visualize results
Overall project
Glowing E. coli from Elowitz and
Leibler
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
Constructing a Biological Breathalyzer
Cory Cheatham
Brian Pink
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.
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
• 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
Background
Background
Growth and Carbon utilization of P. pastoris
Methanol
Ethanol
Method
Construction
Isolation of AOXI promoter
• Amplified AOXI promoter using PCR• Flanked AOXI promoter with appropriate restriction sites using primers
EcoRI
AOXI promoter
XbaI SpeI PstI
EcoRI
AOXI promoter
XbaI SpeI PstI
EcoRI
AOXI promoter
XbaI SpeI PstI
AOXI promoter
PCR
Construction
Isolation of AOXI promoter
•Cloned PCR product into pCR2.1 vector
+
Transformation
EcoRI
AOXI promoter
XbaI SpeI PstI
EcoRI XbaI SpeI PstI
AOXI promoter
Construction
Isolation of AOXI promoter
• Transformed pCR2.1 vector with promoter into competent cells
Construction
Checking AOX1 promoter clones
• Isolated AOXI promoter from pCR2.1 plasmid using EcoRI
AOXI promoter
AOXI promoter
EcoRI EcoRI
+
Preparation of BBa_J61002 vector
• Obtained BBa_J61002 vector from registry• Transformed BBa_J61002 into E. coli cells to amplify
promoter RFP
E. coli
+ Transformation
Amplification
Construction
Preparation of BBa_J61002 vector
• Extracted amplified BBa_J61002 vectors
Extraction
Construction
Ligation of AOXI promoter and BBa_J61002 vector
• Digested AOXI promoter clone and BBa_J61002 vector with XbaI and SpeI
promoter RFPEcoRI XbaI SpeI PstIAOXI
+
XbaI SpeIAOXI
RFP
promoter
+
Construction
Ligation of AOXI promoter and BBa_J61002 vector
• Performed overnight ligation at 16 ºC• Transformed ligation into E. coli cells
AOXI promoter
XbaI SpeI
RFP
+ Ligation
AOXI RFP
Transformation
AOXI RFP
Construction
• Culture transformed yeast cells
• Test response of yeast cells to ethanol
• Design device
Method
Restriction enzyme cutting sites within AOX1 gene
Introducing methanol and ethanol simultaneously in breathalyzer
Obstacles