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