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Cleanse-Attachment

Purpose

To facilitate the efficiency of our killing and anti-biofilm parts, we designed a cohesion module. The cohesion module will attach our modified E. coli to the surface of the S. mutans, so we can shorten the distance between our helper E.coli and our targeted enemy, S. mutans. Then, we can evaluate the efficiency of the other mechanism, the “Completion” part in our project.

For the Completion-killing part, when the helper E. coli received the signal sent by modified phage, the antibiotic secreted E. coli can kill the S. mutans faster if they are closer to each other. Additionally, E. coli can sense the increase of biofilm formation in a shorter period of time and thus activate the antibioflim module.In conclusion, the “Cohesion” part allows the E. coli to serve as a supervisor around S. mutans and reduces the time and distance of the attacks.

Background

In order to make E. coli a supervisor of the population of S. mutans, we have to find an anchor to attach E. coli to S. mutans. Thus, we take advantage of the CSP. CSP is a kind of quorum sensing pheromone, which enables Streptococcus mutans to alter their gene expression when the critical density of cell population is reached. Recent studies showed that density-dependent quorum sensing (QS) system is primarily comprised of the Competence Stimulating Peptide (CSP) and the ComD/ComE two-component signal transduction system. In addition to biofilm formation, the CSP-mediated QS system in S. mutans also affects its acidogenicity, aciduricity, genetic transformation and bacteriocin production. Most importantly,CSP is specificly secreted by S. mutans, which is the reason why we chose it as anchor targeting S. mutans.

Design

We use competence stimulating peptide (CSP) as a targeting material to attach E. coli to the surface of Streptococcus mutans. To do so, we utilize Surface display protein INPNC (designed by 2011 iGEM Edinburgh team) to display CSP on the membrane of E. coli, which act as the “anchor”. CSP binds to both the receptors on the surface of Streptococcus mutans and our engineered helper E. coli.

!fig1 not yet!

!circuit fig not yet!

Functional Measurement

Working circuit:

(圖1)

Testing circuit:

(圖2)

Method:
Use 5cc LB culture and 5 ul chloramphenicol antibiotic to incubate bacteria transformed by function test circuit( K523013 + CSP16 + E1010 and B0015)and control circuit (K523013 + CSP16 + and B0015 )12~16 hour. Then centrifuge 5cc bacteria culture at 13000 rps for 2 minute.

(圖3)

Result

(圖4)

Reference

1. Targeted Killing of Streptococcus mutans by a Pheromone-Guided “Smart” Antimicrobial Peptide(2006)Randal  Eckert1, Jian He2, Daniel K. Yarbrough2, Fengxia Qi2,Maxwell H. Anderson3 and Wenyuan Shi
2. Quorum sensing and biofilm formation by Streptococcus mutans. Senadheera.(2008) D1, Cvitkovitch DG