University of Waterloo iGEM

*Gene shuffling is used to create novel proteins

with improved functionality

*Antibodies, antibiotics, natural insecticides

(Cry proteins), interferon

*Usually extracellular shuffling, then

transformation back into cells

*Would be nice to do it all in the cells!

*Can introduce recombination sites within parent

proteins to allow rearrangements in vivo

*Eg Cre-lox system

* Problem: leftover DNA ‘scar’ may disrupt protein

expression or folding.

*Eg frame-shift

*Widespread in unrelated classes of phage,

bacteria, yeasts, as well as mitochondria and

chloroplasts

*Selfish, independently mobile elements (self-

excision)

*Self-catalysis dependent on complex secondary

structure

3’ OH GFPR 5’

3’ GFPL Intron GFPR 5’

GMP 3’OH

A mechanism of intron self-excision

Eg. GFP mRNA

3’ GFPL 5’

3’ GFPL GFPR 5’ 3’ Intron 5’

A mechanism of intron self-excision, continued

Translation of protein Degraded

3’ OH GFPR 5’

3’ GFPL 5’

3’ GFPL InL lox InR GFPR 5’

3’ GFPL GFPR 5’

3’ InL lox InR 5’

+

Combining self-excising introns with the lox

recombination site:

Parts

BBa_K576003 – Intron L BBa_K576004 – Intron R BBa_K576005 – GFP L BBa_K576006 – GFP R

Intron-L Intron-R GFP-L GFP-R pSB1c3 1Kb

Sub-Cloning Results

*Problems

*Small fragments

*Digest band locations

*An alternative design: multiple introns within a

gene

*Attempt to answer this with modeling and data

*We apply a principle of comparing the ratio of

fluorescence in the Intron Test Construct and the

Positive Control to elucidate system information

*Fluorescence is proportional to the quantity of GFP

I1 * I2 GFP1 GFP2

GFP1 GFP2

Intron Test Construct:

Positive Control:

Positive Control: GFP

Positive Control: GFP

Intron Construct: GF – intron – P

Intron Construct: GF – intron – P

• By measuring fluorescence levels and assuming that

growth rates and plasmid copy number in the cell

populations are the same, we determine the ratio of

the steady state protein levels:

• We assume αF+αS>> δM, which gives a measure of the

efficiency:

To raise awareness and broaden the

perspectives of science enthusiasts in

the field of synthetic biology.

*Engineering Science Quest

*Gr. 12 DIY Gel Electrophoresis

*Gr.11 Synthetic Biology Workshop

*EnviroPig

*DNA Manipulation 101

*Basic Training Lab Workshop for

Undergraduates

1. Community Bricks

- Grade 11 Synthetic Biology

Workshop Program

- DIY, At Home Gel Electrophoresis

- Basic Lab Training Workshops

- Communications Plan Template

and Example for Your Large-Scale

Outreach Programs

- Tips and Tricks to Have a

Successful Outreach Event

http://openwetware.org/wiki/IGE

M_Outreach:Activities

Educational Podcast on Synthetic Biology

and iGEM across Ontario

*BIOTALKS

*Open Panel Discussion involving industry

specialists from Pharma, Agriculture and Energy

*Entrepreneurship, innovation and

commercialization

A Multiple Regression Analysis

Determining the Factors which Affect

Perception of Synthetic Biology

What makes somebody more likely to have a favourable

or unfavourable opinion of synthetic biology?

Regression model of the form

yi = β0 + β1x1 + β2x2 +...+ βpxp

Where y = perception of synthetic biology

x = factor (e.g. age, field of study)

β = weighted impact on perception

*Multiple age ranges

*Mostly located in Ontario

*Distributed online

*Low response rate

*Model for 2013 project

*Built connections & gained experience

Synthetic biology typically consists of…

A. The creation of novel organisms from scratch

B. The recombination of existing genetic parts for

novel use

Advisors: Dr. Barb Moffatt

Dr. Trevor Charles

Dr. Marc Aucoin

Dr. Brian Ingalls