Download Isolation of AOXI promoter

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
Periodically induces the synthesis of green fluorescence protein (GFP).
Nature.com
<http://www.nature.com/nature/journal/v403/n6767/fig_tab/403335a0_F1.html>
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
Overall project
• Step 2 Put timer device and reporter in bacteria
E coli cell
timer
GFP reporter
Negative Regulation
AraC
TetR
GFP Reporter
INPUT
Arabinose
TetR
Sugar
No
GFP
(repressed)
OUTPUT
No more sugar
No TetR
GFP
(fluorescence
activated)
Overall project
• Step 3 Test timer conditions and visualize
results
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
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
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 XbaI
AOXI
promoter
PCR
EcoRI XbaI
EcoRI XbaI
AOXI
promoter
AOXI
promoter
AOXI
promoter
SpeI PstI
SpeI PstI
SpeI PstI
Construction
Isolation of AOXI promoter
•Cloned PCR product into pCR2.1 vector
Transformation
+
EcoRI XbaI
EcoRI XbaI
AOXI
promoter
SpeI PstI
AOXI
promoter
SpeI
PstI
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
EcoRI
EcoRI
+
AOXI
promoter
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
promoter
RFP
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
AOXI
EcoRI XbaI
SpeI PstI
promoter
+
RFP
+
AOXI
XbaI
RFP
SpeI
promoter
Construction
Ligation of AOXI promoter and BBa_J61002 vector
• Performed overnight ligation at 16 ºC
• Transformed ligation into E. coli cells
XbaI
AOXI
promoter
SpeI
AOXI RFP
Transformation
Ligation
+
AOXI RFP
RFP
Method
• Culture transformed yeast cells
• Test response of yeast cells to ethanol
• Design device
Obstacles
Restriction enzyme cutting sites within AOX1
gene
Introducing methanol and ethanol
simultaneously in breathalyzer