chiba university 2010 igem team2010.igem.org/files/poster/chiba.pdf · japan, eppendorf japan,...
TRANSCRIPT
a
30
$me
$me
1st 2nd
1st
TEAM MEMBERS Ryuji Yamamoto Ling Li Manabu Onoda Ike Kohei Gunhye Lee Gyuwoon Jung Yuki Kawamura
Concept Circuit
Summary
Chiba University 2010 iGEM Team
Pulse generator
0 50 100 Time [min]
AHL + Fluo
resc
ence
[AU
]
GFP_LVA pulse generator circuit
AHL -
AHL +
AHL +
AHL - mut3.1
Plux cI434 Plux/cI434
BBa_K396006 on pSB3C5
gfp LVA
Pc luxR
BBa_K396011 on pSB1A3
GFP
GFP_LVA Pc luxR
BBa_K396011
Pc luxR
BBa_K396011
Plux/cI434 gfp
BBa_K396001
Plux/cI434 gfp LVA
(did not submit)
0 50 100 Time [min]
Fluo
resc
ence
[AU
]
Chiba Parts
7 new parts were submitted!
WORKFLOW
Acknowledgement
Slow pulse
Fast pulse
PT7/CI GFP PC
luxR
λ‐CI tetR Plux Ptet
T7RNAP CI434 Plux Plux/CI434
AHL
We thank to: Prof. Ron Weiss for providing pulse generating plasmids. Prof. D. Umeno and his group members for space and technical supports. M. Furubayashi (2006 iGEMer) for instruction (bench works) Many companies/ organizations for financial supports: Yakuken-sya, Knowledge service, Neo-Morgan Laboratory Inc., Greiner Japan, Eppendorf Japan, Cosmo-Bio, Chiba University, Alumni Society for Engineering Department,Chiba University / Chemistry division (Suzuna-no-Kai)/ Venture Business Laboratory.
T7/cI hybrid promoter was ac5vated by T7 RNAP, only in the absence of lambda cI repressor.
T7 ac5vity was compared between under condi5ons of CI non‐expression and CI expression.
T7/cI hybrid promoter
1 3
6
fluor
esce
nce
[AU
]
time [hours]
AHL ( cI)
IPTG ( T7RNAP)
T7/lac hybrid promoter [1] Dubendorf, J.W. 1991 JMB. 219 : 45
Characterization
How it works: ‐ 1st input: no output Slow pulse comes only aGer the fast pulse disappear (no overlap between these pulse) and last for a while (temporally memorized) ‐2nd input: Before slow pulse cleared out: output! AGer slow pulse cleared out: no output ($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.