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