XylE – Rapid, Sensitive Reporter – Perfect for low copy number Promoter Characterisation.
Introduction
We’ve designed a parasite detection system with a rapid
response. A combination of modelling and human practices
specifications have influenced our design so that our
detection kit is as effective as possible!
Schistosomiasis: a neglected tropical disease According to the WHO, this is amongst the most devastating of parasitic
diseases. It impairs development, causes loss of academic potential and
retains countries in a state of poverty.
We are the first iGEM team to tackle an NTD and hopefully not the last!
Specifications
• Detects a waterborne parasite
• Produces an output in minutes
• Easy to use, store and transport
• Inexpensive (less than 50 cents per kit)
• Safe
• Results interpretable by eye without
specialist equipment
Our Chassis Bacillus Subtilis
• No outer membrane, so can attach cell wall anchoring system
• Non-pathogenic
• Capable of sporulation
Our Solution Detection Before Infection
A parasite detection kit with a rapid response
Modelling
Modelling has informed our project design
throughout the whole process. Here are some of the
most interesting simulations.
Modelling showed that a two-step amplification of the
reporter gave the optimal output.
This shows the relationship between a visible output and
the concentration of the parasite protease.
Enzymatic reaction modelled:
Linker 1
Linker 2
Linker 3
Linker 4
Linker 5
Linker 6 Linker AIP CWB
Final Product Design
We wanted to contextualise the detection
kit, so we decided to get some
prototypes made, as this might help
inform our design and improve its
efficiency. Below you can see the
potential products!
The linker domain can be
customised to be sensitive to
different proteases and other
proteins can be attached to the
cell wall.
Optimal absorbance spectrum of Yellow Product
Comparing activity of XylE under J23101 and pVeg
promoters in relative promoter units (RPU).
Having decided that we wanted to detect a specific
protease that Schistosoma releases, we engineered a
novel surface protein construct.
An autoinducing peptide (AIP) is anchored to the cell wall
which can be cleaved off by the parasite protease.
GFP-XylE fusion: x10 fold inactivation
compared to XylE
GFP-XylE fusion: change of activity on cleavage
Results
School Workshops We ran a series of synthetic biology school workshops
and created a toolkit so that other teams can do the
same!
We used the ComCDE quorum sensing system
from S. pneumoniae. When the AIP binds ComD,
a two component signalling cascade is activated.
ComE can now induce TEV protease expression.
0
1
2
3
4
5
6
7
8
9
10
0 2 4 6 8 10
Absorb
ance a
t 360nm
Time/minutes
Activity of GFP-XylE against XylE only
XylE only
GFP-XylE
Enzyme kinetics characterised for the
existing part XylE (BBa_J33204)
AIP His TEV cleavable CWB
domain Linker
3’ amyE dif 5’
amyE
CmR Lac I GAAA TTTC
XylE
1) XylE: used to characterise
pVeg and J23101 promoters
Our Favourite BioBricks
2) Modular surface protein construct
3) LacI transformation vector: targets the amyE locus in the
B. subtilis genome, integrating any DNA inserted in the
PmeI site
Piotr Faba Harriet Gliddon Nicolas Kylilis Benjamin Miller Anita Nguyen Wolfgang Pernice
Madeline Rounds Florian Sessler Kirill Shkura Kyasha Sri Ranjan
Schistosoma Today infection is diagnosed by
stool/urine sample, but no
effective system is in place to
detect the infective stage.
We designed a system called
Parasight to detect this infective
stage, allowing the improvement of preventative measures .
Detection Module
Human Practices Throughout our iGEM project we ensured that Human Practices
truly informed our design specifications.
At a Human Practices Panel Discussion, we addressed all the ethical, social and environmental concerns that our project raised.
Fast Response Module
Signalling Module
4.
P
2. The AIP activates receptor ComD
3. Response Regulator ComE is phosphorylated
4. ComE causes expression of target gene TEV protease
5. TEV cleaves a pre-made XylE-GFP fusion protein
6. The cleaved XylE tetramerises and gives a visible colour output
1. Protease released by Schistosoma cleaves AIP of the Detection Module
Detection
Fast Response
Signalling cascade results in transcription of TEV
protease. TEV protease results in GFP-XylE
monomer cleavage and XylE activation.
Fast response