Reprogramming Genius E. coli as a Pathogen Seeking Killer (V. cholerae) directed by Quorum Sensing Signals

BIOFILM Most of the pathogenic bacteria could make biofilm that increases their antibiotic resistance and its pathogenicity.

Causing

65% Infection In Hospital

OUR MODEL: V. cholerae, a pathogenic bacteria that can make biofilm. CHOLERA

20,49% world population is at risk 5 million people infected annually

100-120 thousands death

How Genius E. coli can solve the problem?

3. KILLING

1. HUNTING 2. BIOFILM DEGRADING SENSING

MOTILITY

CqsS receptor

v

CqsS

V. Cholerae (biofilm)

CAI-1

YbeL YaiN

Mr. GE

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

(Shao Yi, 2014).

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.

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)

CheZ CqsS induced Promoter

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.

BIOFILM COMPOSITION WaterPolysaccharidesExtracellular DNA/RNAMicrobial CellsProtein

90% water

1-2% 2-3%

1-2% 2-5 %

Genius E. coli aims to break down polysaccharides and

extracellular DNA/RNA, two components of biofilm, with

two degradation enzymes: alpha-amylase and nuclease.

Alpha-amylase Nuclease + Adding HlyA secretion tag • Coded by Nuc gene

• Cut phosphodiester bond • Indigenous from Staphylococcus aureus (Sohrabi B, 2012)

• Coded by MalS (periplasmic α- amylase) gene • Hydrolyse α-1,4- glicosidic bond • Indigenous from E. coli (Lengsfeld C, dkk., 2008)

HlyA is a signal peptide found in C-terminal sequence of alpha-hemolysin (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 .

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.

Siska Anggoro Etri Yuda Diana Vanessa Robby Anasthasia

TEAM MEMBER

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> 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/.

Dr. Muhamad Sahlan, M.

Eng.

Dr. dr. Budiman Bela, Sp.MK(K)

drg. Endang W. Bachtiar M. Biomed, Ph.D

Dr. Drs. Abinawanto,

M.Si

ADVISORS AND INSTRUCTORS

SAFETY MODULE

Parts Characterization STARCH HYDROLISIS TEST

Nuc HlyA J23100 MalS HlyA J23100

RBS is not used on MalS device because the native RBS is present in the MalS part we’re using

(BBa_K523001)

Incubated on starch agar Flooded after 18 hours

Incubated on Starch Agar Flooded after 24 hours

Note: Dark color (positive reaction) indicates that the starch is present in the agar. Clear zone (negative reaction) indicates the degradation of polysaccharide.

Mals-HlyA is expected to secrete alpha-amylase into supernatant Plasmid only as negative control, no expression WT is wild type E. coli top10, no plasmid

Parts Characterization 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.

Conclusion As the result, our devices work more effective in degrading biofilm of Gram-positive bacteria than Gram-negative 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.

drh. Yulianty, M. Biomed

Nada Fithria, S. Si, M. Biomed

M. Hanifi Wian Taufik Teguh Danny

2014.igem.org/Team:UI-Indonesia

Peptide 1018 •Broad spectrum anti-

biofilm and anti-microbial substance • Identified as innate

defense regulator •Blocking (p)ppGp,

stress response 2nd messenger, preventing biofilm formation and killing the cells

BIOFILM DEGRADING ENZYMES

- 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.

1018 T5-lac promoter

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.

Parts Characterization Toxicity Assay 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)

IPTG [mM]

0.000 0.500 1.000 1.500 2.000 2.500 0.000

0.050

0.200

0.250

OD 5

95

Peptide 1018 4-hour incubation Activation in LB

Y = -0.006x +0.1738

IPTG Variation Curve Linear (IPTG Variation Curve) 0.100

0.150

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.

HUMAN PRACTICE Human Practice

SYNBIO SHOUT OUT EXPERT TALKING MOVIE

We triggered some high school students

with a question,

“If you can make your own bacteria,

what will it be?”

And collected a lot of amazing idea!

Prof. Masafumi

Yohda Tokyo University

of Agriculture and Technology

Aroem Naroeni, DEA, Ph. D Biosafety Officer, IHVCB-UI

“Synthetic biology application projection in Asia, also

problems involving application of Synbio in Japan.”

“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.

Parts

Ach

ievem

ent

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

1. Device:

J23100-(RBS-MalS)-HlyA and J23100-RBS Nuc-HlyA

2. Device: (RBS-MalS)-HlyA and (Nuc-HlyA)

3. Improving HlyA Parts from UNICAMP EMSE Brazil

4. Doing the characterization successfully

1. New Part:

Peptide 1018 2. Device: T5-

RBS-Peptide 1018

3. Improvement Parts: Thermo regulated promoter-Peptide 1018

4. Doing the characterization with positive results

HUNTING KILLING BIOFILM DEGRADING

Furth

er A

pplic

ation

Attri

butio

n

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.

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Vibrio cholerae(-)

Bacillussubstilis (+)

Pseudomonasaeruginosa (-)

Staphylococcusaureus (+)

Klebsiellapneumonia (-)

Escherichia coli(-)

Average Reduction of Biofilm in Several Bacteria (%)

MalS-HlyA

Nuc-HlyA

Mixv v

befo

re

afte

r

cdc.gov

Moving towards V. cholerae

(Molobela P, Cloete TE, Beukes M., 2010)

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 Gram-positive 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.