igem groningen presents 7. aakanksha lactoaid 3....
TRANSCRIPT
The problem In severe burn injuries the skin is damaged
and the first defense against pathogens is
lost. Because of this, bacteria can easily
access deeper parts of the skin, which
provide a suitable habitat for bacterial
growth. The most common pathogens that
cause problems are Staphylococcus aureus
and Pseudomonas aeruginosa. If an
infection is suspected, wound swabs must be
taken to diagnose the pathogens. However,
antibiotics are often used preventively to fight
infections. Also, the treatment of burn
wounds is labor intensive, since the
bandages must be refreshed daily.
Therefore, we designed a smart bandage
which does not require daily refreshment or
treatment with antibiotics, but detects
pathogens and secretes Infection Preventing
Molecules (IPMs) upon detection.
Our Chassis
We introduced Lactococcus lactis in the
competition as a chassis because:
• It is known to be non-pathogenic
• It is a food grade bacterium (e.g.: cheese)
• It produces anti-microbial molecules
• There is no spore formation
This makes it a perfect bacterium for medical
purposes.
Detection Firstly, the pathogens are
detected by their quorum
sensing molecules, namely
AHLs (P. aeruginosa) and
AIPs (S. aureus). After detection the
secretion system is activated. Thereby, the
risk of antibiotic resistance is decreased.
1 Mark, B.L. et al. (2001). J. Biol. Chem. 276: 10330–10337
2 García-Contreras, R. et al. (2013) Appl. Environm. Microbiol. 79: 6840–6846
3 Papaioannou, E., et al. (2013) Microbiol. Mol. Biol. Rev. 77: 73–111
4 Kim, M.H. et al. (2005. PNAS 102
5 Dong, Y.H. et al. (2000. PNAS 97: 3526-3531
6 Wiedemann, I. (2001). J. Biol. Chem. 276: 1772-1779
LactoAid detects quorum
sensing molecules from
S. aureus and P. aeruginosa
and secretes the IPMs DspB,
AiiA and Nisin to prevent
infections in burn wounds.
Secretion of IPMs In response to detection, L. lactis will
produce three IPMs:
DspB degrades Biofilms 1
AiiA disrupts P. aeruginosa
quorum sensing 2-5
Nisin kills S. aureus 6
7 Boonmee, Mallika, et al. Biochemical Engineering Journal 14.2 (2003): 127-135
8 Shimizu, Hiroshi, et al. Applied and environmental microbiology 65.7 (1999):3134-
3141
9 Tong, Jane, and John L. Anderson. Biophysical journal 70.3 (1996): 1505-1513
10 Fischer, Hannes, et al. Protein Science 13.10 (2004): 2825-2828
Toolbox Since we introduced a new chassis for the
competition, a toolbox was designed which
makes the use of Lactococcus lactis easier
in the future. This toolbox consists of: a
vector, a set of constitutive promoters,
several genes from the nisin operon, and a
suitable fluorescent marker.
From Bacterium to Bandage To incorporate the bacterium in a safe way,
we enclosed it in a bandage consisting of
three layers:
• The top layer is transparent, hydrophobic
and permeable to gases.
• The middle layer of polyacrylamide
contains L. lactis.
• The bottom layer allows diffusion of IPMs
and quorum sensing molecules, while
isolating the wound from the bandage.
Results
Left: L. lactis expressing GFP showing
growth in the polyacrylamide gel. Right:
Polyacrylamide gel with nisin-secreting L.
lactis in a plate with a nisin-sensitive strain.
The zone of inhibition confirms that nisin
diffuses through the gel and kills bacteria.
Modelling
The dynamic model was discretized into
lattices each containing differential equations
for growth of bacteria and both diffusion and
production of molecules. 9-12
Six designs were
modelled to find
the best one.
Best design: Nisin diffuses out within 24
minutes, AiiA and DspB within 30 minutes.
Regulation and Public awareness
According to the Dutch Commission for
Genetic Modification LactoAid is a medical
product, which requires elaborate testing but
can be introduced in the market.
We increased awareness of our product and
synthetic biology on a number of events,
such as Discovery Festival and a TEDx event
and by educating high school students.
iGEM Groningen presents
LactoAid The Smart Bandage
AiiA DspB
DspB
A
B
D C
B
A
A
B
C
D
Team Members
1. Rick Elbert
2. Lisa Hielkema
3. Chandhuru
Jagadeesan
4. Anna Lauxen
5. Thomas Meijer
6. Sandra Mous
7. Aakanksha
Saraf
8. Otto Schepers
9. Oscar Smits
10. Andries de Vries
11. Lianne Wieske
12. Freek de Wijs
Supervisors
13. Ruud Detert
Oude Weme
14. Bayu Jayawardhana
15. Oscar Kuipers
16. Renske
van Raaphorst
17. Jan-Willem
Veening 1
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