a

30

$me

$me

1st  2nd 

1st 

TEAM MEMBERS Ryuji Yamamoto Ling Li Manabu Onoda Ike Kohei Gunhye Lee Gyuwoon Jung Yuki Kawamura 

Concept Circuit

Summary

Chiba University 2010 iGEM Team

Pulse generator

0 50 100 Time [min]

AHL + Fluo

resc

ence

[AU

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GFP_LVA pulse generator circuit

AHL -

AHL +

AHL +

AHL - mut3.1

Plux cI434 Plux/cI434

BBa_K396006 on pSB3C5

gfp LVA

Pc luxR

BBa_K396011 on pSB1A3

GFP

GFP_LVA Pc luxR

BBa_K396011

Pc luxR

BBa_K396011

Plux/cI434 gfp

BBa_K396001

Plux/cI434 gfp LVA

(did not submit)

0 50 100 Time [min]

Fluo

resc

ence

[AU

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Chiba Parts

7 new parts were submitted!

WORKFLOW

Acknowledgement

Slow pulse

Fast pulse

PT7/CI GFP PC

luxR 

λ‐CI tetR Plux Ptet

T7RNAP CI434 Plux Plux/CI434

AHL

We thank to: Prof. Ron Weiss for providing pulse generating plasmids. Prof. D. Umeno and his group members for space and technical supports. M. Furubayashi (2006 iGEMer) for instruction (bench works) Many companies/ organizations for financial supports: Yakuken-sya, Knowledge service, Neo-Morgan Laboratory Inc., Greiner Japan, Eppendorf Japan, Cosmo-Bio, Chiba University, Alumni Society for Engineering Department,Chiba University / Chemistry division (Suzuna-no-Kai)/ Venture Business Laboratory. 

T7/cI hybrid promoter was ac5vated by T7 RNAP,   only in the absence of lambda cI repressor.

T7 ac5vity was compared between under  condi5ons of CI non‐expression and CI  expression.

T7/cI hybrid promoter

1 3

6

fluor

esce

nce

[AU

]

time [hours]

AHL ( cI)

IPTG ( T7RNAP)

T7/lac hybrid promoter [1] Dubendorf, J.W. 1991 JMB. 219 : 45

Characterization

How it works: ‐ 1st input: no output    Slow pulse comes only aGer the fast pulse disappear    (no overlap between these pulse)    and last for a while (temporally memorized) ‐2nd input:     Before slow pulse cleared out: output!    AGer slow pulse cleared out: no output                                                          ($meout → ini$alized)  

AND Fast Pulse

Slow Pulse Input Output

1st  2nd  2nd 

Genetic Double Click System -Eliminating the False-Input-

  Double click system is one of the most accepted, familiarized, and proven mechanisms to diminish the erroneous opera$ons. The system does not react to the single input, but does so when the same ac$ons (clicking) are given twice in a short period of $me.  Here, the first input is 'memorized' for a limited $me, and the second 'reconfirma$on' input is necessary to start the program. The system is also smart enough to dis$nguish double‐click from two separated single clicks; aGer a certain $me, the memory of the first input should be cleared out and the system must be back to the original state (ini$alized).    This year, Chiba Team designed the gene5c double click system, which should be of great use as the fail‐safe system for the reliable opera$on of gene$c circuits/ cellular func$ons. 

Fast pulse

BBa_K396007 Plux cI434 Plux/cI434

T7 RNAP LVA

T7 RNAP LVA Plux/cI434 Plux cI434 tetR

cI434 Plux Plux/cI434 T7 RNAP_LVA Assembly PCR + SOEing PCR + triple ligation!

Output device

BBa_K396000 Pc luxR PT7/cI

gfp LVA

gfp LVA PT7/cI Pc luxR Ptet λ-cI

Pc gfp LVA PT7/cI luxR

Slow pulse

We designed genetic double-click system. Construction is underway, but we had some progress. - All parts for the circuits are collected. - Most of the parts were confirmed their functions (Several need works). -  Some of the biobricks (new hybrid promoters) were deposited to the registry, and one existing biobrick (R0061) was characterized.  -  Core device (fast pulse) constructed and tested: didnot work. We are looking for the right parameter set in order to make it function as we designed.

Output

Design

TAATACGACTCACTATAGGGAGATCTGGCGGTGATAATGGTTGC

TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCC

T7 promoter Lambda cI operator (OR1)

T7 promoter lac operator

Newly designed parts: T7/cI hybrid promoter

T7/cI hybrid promoter sequence was designed based on the previously reported T7/lac hybrid promoter[1].

Input

No pulse observed...

Pulse observed in the previously reported pulse generator. (Basu et al. 2004 PNAS . 101: 6355)

Circuit Construction

Project Design

Design: - Input (AHL) generates two pulses Fast pulse (T7 RNAP) Inverted slow pulse (lambda CI) - Output under AND-gate of the two

BBa_K396000 on pSB1A3

Plux cI

(not biobrick) on pACYC

Plac T7RNAP

on BL21(DE3) genome

PT7/cI gfp LVA luxR Pc

IPTG

AHL

TO BE CONSTRUCTED…

Assembly PCR + SOEing PCR + triple ligation!

Alumni Society for Eng. Dept, Suzuna-no-kai, Venture Business Lab.