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Killing Legionella pneumophila softly

Killing Legionella pneumophila

Killing Legionella pneumophila softly

Başkent Meds

Legionella pneumophila – G(-) bacteria – Infectious agent of atypic pneumonia; Legionellosis

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LEGIONNELLOSIS: CLINICS

• Symptomatic infections are primarily
• bserved in lungs
• Two forms of the disease:

PONTIAC FEVER LEGIONNAIRES’ DISEASE

IDENTIFICATION

• Clinical diagnosis of the disease
• Methods used for determining

contamination risk at water systems

Selective medium culturing ELISA QPCR

PROTECTION & PREVENTION

• Environmental Legionella monitorization
• Prevention by disinfection:

– Thermal eradication method (66°C) – Hyperchlorination – Ozonation – UV – Copper-silver ionization

Legionella pneumophila “talk” to each other

?

Ready for biofilm? No, we aren’t enough yet! Divide!

Quorum Sensing

Tiaden A, et al. Environ Microbiol, 12: 1243-59, 2010.

Legionella quorum sensing (lqs) autoinducer synthase response regulator sensor kinase

The system which we are trying to develop is E. coli cells that can recognize L. pneumophila specifically at species level by using its QS mechanism, and respond to that recognition by producing and secreting anti-Legionella peptide which is a peptide produced by some Staphylococcus strains.

Our system is composed of 3 expression “cassettes”. In the first cassette; a translational lqs response unit is generated with lqsS and lqsR system elements Source of lqs region is pNT-1 plasmid.

(Cellular Microbiol 9:2903-20, 2007).

The LqsR and LqsS genes have illegal restriction enzyme recognition sites inside their coding regions. So that composite part was amplified with primers having SpeI cutting sites, and cloned into the downstream of a constitutive promoter in the pSB1C3.

In the second cassette; anti-legionella unit Biobrick is transcribed under lqsR promoter which is regulated by response regulator lqsR itself. Promoter part was designed as a Biobrick. The regulatory part also contains an RBS.

LqsR promoter

Anti-legionella (Warnericin RK) peptide induces channel formation at a low concentration, and a detergent-like solubilization of the membrane at high concentration

Verdon et al. Peptides 30:817-23, 2009.

ORF for anti-legionella peptide specifically encodes for a 22 amino acid peptide. Anti- legionella unit is a composite Biobrick for suitable suffix and prefix regions. It contains a PelB leader sequence in the upstream of the anti-legionella peptide ORF to direct the peptide outside the cell. Source is synthetic oligonucleotides.

Anti-legionella

• ligonucleotide

PelB leader sequence

66 bp 66 bp E-X S-P

Verdon et al. Biophy J 97:1933-40, 2009.

The third cassette contains febA L8T pore protein which is responsible for diffusion

• f synthesized proteins through cell

membrane to outside of the cell. It is transcribed under a constitutive promoter and with RBS in the 5' region of ORF. Source of the parts is the iGEM distribution kit.

Our models

A B C D

Safety

Future plan

• Complete all system
• Kill switch
• Detection system
• How are we planing to use it
• Biofilm formation on

contaminated surfaces

• Our recombinant cells can be

entrapped into a system and placed in contaminated area

2012-2013 Baskent University Medical School Symposia

Human Practice

Presentation for Synthetic Biology Day

• rganized by METU iGEM Team

Presentation for organization of the ITU iGEM Team

Brief on High School iGEM to an International Baccalaureate School; Bilkent High School

Feride Özgür Berkhan Özlem Didem Mert Yunus Başak Sevecen Burak Cansu

This study was approved by Baskent University Institutional Review Board (Project No’s: DA12/05 and DA13/06) and supported by Baskent University Research Fund.

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