UI-Indonesia Championship Poster

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Reprogramming Genius E. coli as a Pathogen Seeking Killer (V. cholerae) directed by Quorum Sensing Signals
BIOFILM
Causing
OUR MODEL: V. cholerae, a
pathogenic bacteria that can make
biofilm.
65%
Most of the pathogenic bacteria
could make biofilm that increases
their antibiotic resistance and its
pathogenicity.
CHOLERA
20,49% world population is at risk
5 million people infected annually
100-120 thousands death
Infection
In Hospital
cdc.gov
How Genius E. coli can solve the problem?
2. BIOFILM DEGRADING
1. HUNTING
SENSING
BIOFILM COMPOSITION
1-2%
CAI-1 ((S)-3-hydroxytridecan-4-one) is a quorum sensing signal molecule that is
specific for V. cholerae and used for communication. In high cell density, CqsS
histidine kinase transmembrane receptor will receive CAI-1 and triggers the
cascading autophosporilation and regulates virulence factor in V. cholerae
2-5 %
1-2%
2-3%
Incubated on starch agar
Flooded after 18 hours
Water
Polysaccharides
Extracellular DNA/RNA
Microbial Cells
Protein
(Shao Yi, 2014).
CqsS receptor
v
90% water
CAI-1
YbeL CqsS YaiN
Note:
CheZ
Moving towards V. cholerae
Our design construct for motility includes CqsS induced promoter to CAI-1
molecules. The CheZ gene can be expressed and it optimize the motility of E. coli to
run (smooth swimming) towards CAI-1 as attractant.
(Lengsfeld C, dkk., 2008)
J23100
MalS
J23100
HlyA
Prof.
Masafumi
Yohda
Tokyo University
of Agriculture and
Technology
“If you can make
your own bacteria,
what will it be?”
And collected a lot of
amazing idea!
“Synthetic biology application
projection in Asia, also
problems involving application
of Synbio in Japan.”
Aroem Naroeni,
DEA, Ph. D
Biosafety Officer,
IHVCB-UI
“About our project’s safety
assessment and the prospect of
synthetic biology development
in Indonesia.”
“How do movies
influence people?”
Watching movies
and programs on
the television as we
relax takes us away
into this different
realm. Through
movies, we're trying
to put a new point
of view of synthetic
biology, where
people will be able
to visualize the idea
and creates exciting
adventure in their
mind.
REFERENCES
Shao Yi (2014). Regulation of Quorum sensing in Vibrio harveyi and Vibrio cholerae. Dissertation of Ph.D.
Princeton University
Eisenbach, M. (2001). Bacterial Chemotaxis. Encyclopedia Of Life Sciences.
Molobela P, Cloete TE, Beukes M (2010). Protease and Amylase Enzyme for Biofilm Removal and
Degradation of Extracellular Polymeric Substances (EPS) Produced by Pseudomonas flourescens bacteria.
Ajmr. 4(4),pp.1515-1524
Lengsfeld C. Schonert S. Dippel R. Boos W (2009). Glucose- and Glucokinase-Controlled mal Gene
Expression in Escherichia coli. J. Bacteriol. February. vol. 191 no. 3 701-712
Sohrabi B (2012). Nuclease from Staphylococcus aureus. International Genetically Engineered Machine
(iGEM) London. Accessed: 28 August 2014. Available on: <http://parts.igem.org/Part:BBa_K729004>
Our team modify MalS and Nuc that will
be tagged with HlyA by erasing the part‘s
stop codons so the HlyA tag protein would
be expressed.
2014.igem.org/Team:UI-Indonesia
(%)
16.0
MalS-HlyA
14.0
Nuc-HlyA
12.0
Mix
10.0
8.0
6.0
4.0
2.0
0.0
3. KILLING
Peptide 1018
•Broad spectrum antibiofilm and antimicrobial substance
•Identified as innate
defense regulator
•Blocking (p)ppGp,
stress response 2nd
messenger,
preventing biofilm
formation and killing
the cells
HUNTING
1. Sensing New
Part: CqsS
(reversed
histidine kinase
quorum sensing
system from
Vibrio cholerae;
Bba_K1344010)
2. Sensing New
Parts: YbeL
(Bba_K1344011)
and YaiN
(Bba_K1344012)
3. Motility Device:
CheZ-GFP
BIOFILM
DEGRADING
1. Device:
J23100-(RBSMalS)-HlyA and
J23100-RBS
Nuc-HlyA
2. Device: (RBSMalS)-HlyA and
(Nuc-HlyA)
3. Improving HlyA
Parts from
UNICAMP
EMSE Brazil
4. Doing the
characterizatio
n successfully
Etri
Toxicity Assay
Peptide 1018 4-hour incubation Activation in LB
0.250
Y = -0.006x +0.1738
T5-lac
promoter
1018
SAFETY MODULE
The killing module is under
weak promoter so the enzyme
will be produced continuously
even there is no quorum
sensing signal until the
concentration of enzyme reach
the toxic concentration that
cause cells death.
KILLING
1. New Part:
Peptide 1018
2. Device: T5RBS-Peptide
1018
3. Improvement
Parts: Thermo
regulated
promoterPeptide 1018
4. Doing the
characterizatio
n with positive
results
TEAM MEMBER
Anggoro
Conclusion
As the result, our devices
work more effective in
degrading biofilm of Grampositive bacteria than Gramnegative bacteria. We
hypotize that the composition
of polysaccharides that
digested by alpha-amylase
and extracellular DNA/RNA in
Gram-positive bacteria are
greater than Gram-negative’s.
0.200
- No HlyA
secretion tag
Peptide 1018 have a
characteristic called
cell penetrating
peptides, meaning to
be able to freely
move across
membrane. So we do
not add HlyA
secretion tag in the
down stream region
of circuit.
Pseudomonas Staphylococcus Klebsiella Escherichia coli
aeruginosa (-) aureus (+) pneumonia (-)
(-)
Parts Characterization
0.150
IPTG Variation Curve
Linear (IPTG Variation
Curve)
0.100
0.050
0.000
0.000
0.500
1.000
1.500
2.000
2.500
IPTG [mM]
In this experiment, the
activation was conducted
with 18 hours culture and
diluted 1:20. The data
above shows no inhibitory
effect of IPTG as the
concentration increases.
But when we measured
the OD 595, all
treatments show lower
result than 0 mM IPTG.
(Nunez CF, Reffuveille F, Haney EF,
Hancock REW., 2014)
Based on the acquired data , it is safe to assume that Peptide 1018 works as we
expected. However, further research needs to be conducted to determine the optimum
concentration of IPTG.
Quorum sensing is a principal bacterial
communication. We can detect other
bacteria quorum sensing signal molecule by
making a system to detect quorum sensing
molecule in Gram-negative and Grampositive bacteria, i.e. Autoinducer-2 (AI-2);
not only Vibrio cholerae but also other
bacteria: Pseudomonas aerugoinosa, Bacillus
substillis, Klebsiella pneumonia, Staphylococcus
aureus, and E. coli . So we hope that our
degrading matrix enzymes can be applied to
those bacteria and other bacteria forming
biofilm using quorum sensing.
Fuente Nunez et al. for the discovery and
characterization of peptide 1018, a substance that
specifically kill biofilm-making bacteria. The discovery of
peptide 1018 has allowed us to design a synthetic
bacteria which kills biofilm-makin pathogen but harmless
to native, planktonic bacteria.
UNICAMP EMSE Brazil iGEM Team 2009 for the
development and characterization of Type I secretion
system in E. coli. We used the E. coli type I secretion
system to export biofilm degrading enzyme to
extracellular environment.
Budiman Bela for his insight on pathogen behaviour and
stress response that helps us to decide which quorum
sensing mechanism works best for our purposes.
Muhammad Hanifi for his insight on the structure and
composition of bacterial biofilm and thus, leading to the
right substance to degrade them.
ADVISORS AND INSTRUCTORS
Nunez CF, Reffuveille F, Haney EF, Hancock REW (2014). Broad Spectrum Antibiofilm Peptide that
Targets a Cellular Stress Response. PloS Pathog 10(5):e1004152.doi:10.1371/journal.ppat.1004152
World Health Organization (2014). Cholera Fact Sheet.. http://www.who.int/mediacentre/
factsheets/fs107/en/.
Average Reduction of Biofilm in Several Bacteria
Vibrio cholerae
Bacillus
(-)
substilis (+)
Siska
BIOFILM REMOVAL ASSAY
We intend to see the application of our device in
removing biofilm produced by other bacteria, beside V.
cholerae. Absorbance of the biofilm removal assay is
read by using ELISA Reader.
HlyA
RBS is not used on MalS device because the native
RBS is present in the MalS part we’re using
(BBa_K523001)
Parts Achievement
HUMAN PRACTICE
MOVIE
Parts Characterization
18.0
Human Practice
EXPERT TALKING
Nuc
Mals-HlyA is expected to secrete alphaamylase into supernatant
Plasmid only as negative control, no expression
WT is wild type E. coli top10, no plasmid
Attribution
v
(Sohrabi B, 2012)
HlyA is a signal peptide found in Cterminal sequence of alphahemolysin (HlyA). Gram negative
bacteria uses this protein tag via
the Type I Secretion System.
Fusion of the HlyA signal peptide
to the target protein causes the
excretion of protein to
extracellular medium .
Dark color (positive reaction)
indicates that the starch is
present in the agar.
Clear zone (negative reaction)
indicates the degradation of
polysaccharide.
OD 595
After sensing CAI-1, Genius E. coli will approach V. cholerae with an effective
motility. E. coli generally moves in tumbles because of the phosphorylated CheY
gene (CheY-P) and this case can be stopped by CheZ gene expression which will
dephosphorylate the CheY-P, so E. coli can run forward (Eisenbach M, 2001)
• Coded by Nuc gene
• Cut phosphodiester
bond
• Indigenous from
Staphylococcus aureus
+ Adding HlyA
secretion tag
Further Application
MOTILITY
• Coded by MalS
(periplasmic αamylase) gene
• Hydrolyse α-1,4glicosidic bond
• Indigenous from
E. coli
Nuclease
after
Our team would like to overexpress the CqsS receptor in Genius E. coli
transmembrane. But overexpressed transmembrane receptor is difficult to express
and highly toxic for the cells. Therefore we use two protein to help CqsS into
transmembrane region, YaiN and YbeL, both indigenous from E. coli. They are fused
into the N and C terminus to help expression.
Alpha-amylase
before
V. Cholerae
(biofilm)
We triggered some
high school students
with a question,
Incubated on Starch Agar
Flooded after 24 hours
(Molobela P, Cloete TE, Beukes M., 2010)
BIOFILM DEGRADING ENZYMES
SYNBIO SHOUT OUT
HYDROLISIS TEST
Genius E. coli aims to break
down polysaccharides and
extracellular DNA/RNA, two
components of biofilm, with
two degradation enzymes:
alpha-amylase and nuclease.
Mr. GE
CqsS induced
Promoter
Parts Characterization STARCH
Yuda
Diana
Vanessa
Robby
Anasthasia
drh. Yulianty,
M. Biomed
Nada Fithria,
S. Si, M.
Biomed
M. Hanifi
Wian
Taufik
Teguh
Danny
Dr. Drs.
Dr. Muhamad Dr. dr. Budiman drg. Endang
Sahlan, M. Bela, Sp.MK(K) W. Bachtiar M. Abinawanto,
M.Si
Eng.
Biomed, Ph.D

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