Download Fig. 1. - iGEM 2010

Mosquito Intelligent Terminator
An Environment-friendly Pesticide for Killing Wrigglers
Students: Hui-Ching Tsai, Chia-Chun Kuo, Lam Sok Han, Yu-Han Liu, Chia-Le Meng, Pei-Hua Tsai, Hua-Yi Hsu, Hui-Ying Huang, Shao-I Wu, Chin-Han Huang, Chen-Hsi Tang, Han-Ting Tsai, Ting- Yao Wang
Instructors: Huai-En Lu, Min-Chih Kao, Shih-Ju Ho, Chih-Yuan Hsu, Davey Leu
Advisor: Hsaio-Ching Lee, Ching-Ping Tseng
Abstract
Low-temperature release system
The Mosquito Intelligent Terminator is designed and optimized to be an
ecological and environmental friendly mosquito pesticide. The
Terminator is an engineered E. coli secreting crystal proteins isolated
from Bacillus thuringiensis to kill mosquito larvae, or commonly known
as wrigglers. These crystal proteins are toxic to certain types of
mosquitoes only and are not pathogenic to mammals. We designed a
low-temperature release genetic circuit expressing high levels of crystal
proteins at room temperature only, thus production does not occur at
incubation temperature (37°C). In order to make an environmentally safe
insecticide, our design also incorporates a genetic circuit controlling the
population size of E. coli, thus a surplus will never exist as E. coli
population is self-maintained within the system (Fig. 1). Our design may
potentially serve as a promising pest control solution in the future.
A temperature-sensitive RBS (BBa_K115002) with high translation
activity at high temperature (>= 37°C) and low translation activity at
room temperature was used to design this circuit. It has two statuses:
Fig. 1. Terminators can be
sprayed into foul water which
is the wrigglers’ habitat. When
wriggler eats the Terminators,
it will die.
Design overview
The reproduction status when bacterium grows at >=37°C
Fig. 9. The population control system is not
activated during bacterium growth. This
design allows us to amplify the terminators
without any potential limiting factors.
The self-maintain status when grows at less than 37°C
Fig. 10. When external bacteria aggregate,
the concentration of AHL will rise pass
threshold level and resulting the formation of
AHL-LuxR complexes, and this complex
translates
downstream
ccdB
gene.
Accumulation of ccdB protein results in
subsequent suicide to restrict the population
size.
2. Downstream protein release status at less than 37°C.
Fig. 5. When the Terminator are
released in the environment at room
temperature (<37°C), the translational
activity of temperature-sensitive RBS
is repress by low temperature. Thus,
the
promoter
Ptet
promoter
transcribes the downstream protein
constitutively.
The self-destruction status by AHL administration
Fig. 11. After Terminators have served its
purpose, we can manually kill all
Terminators by spraying AHL to active Plux.
ccdB's are produced and results in E. coli
self-destruction.
Experimental results & modeling analysis
Our experimental results indicate that high temperature decreased the
translation rate of the target protein, and this temperature-dependent
genetic circuit can control the expression level of the target protein by
the host cell's incubation temperature.
Fig. 6. The green fluorescence
intensity of the low-temperature
release system was measured
using a flow cytometer at different
incubation temperature.
NCTU Formosa
Population control system
1. Bacterial reproduction status at >=37°C.
Fig. 4. The constitutive promoter
transcript tetR repressor inactivates
the promoter Ptet, subsequently
downstream protein is not transcribed
to restrict the host growth.
2010 iGEM team
Modeling analysis
Model equations of the self-maintain status can be as follows.
d[LuxI]
= αc − γLuxI [LuxI]
dt
d[LuxR]
= αc − γLuxR [LuxR]
dt
d[ccdB]
αD ([AHL][LuxR])n2
=
− γccdB [ccdB]
n3
dt
k1 + ([AHL][LuxR])
d[AHL]
= k 2 [LuxI][bac] − γAHL [AHL]
dt
d bac
αbac
=
dt
1 + ccdB
This system can be modeled by differential equations as
follows.
d[TetR]
dt
Fig. 2. The design of Mosquito Intelligent Terminator. (i) Cry weapon is located
on the strand B. (ii) Low-temperature release system is controlled by the strands
A and B. (iii) Population control system is controlled by the strands C and D.
Cry weapon system
Cry11Aa is highly toxic to certain dipteran larvae, such as Aedes, and
Anopheles larvae. To make cry11 gene to be a iGEM standard part, we
clone the cry11Aa gene from Bacillus thuringiensis, and process point
mutations on the cry11Aa gene to remove two of the enzyme sites
(EcoRI and SpeI) respectively (Fig. 3).
= αTemp − γTetR TetR − d(t) GFP
d GFP
αB
=
− γGFP GFP − d(t) GFP
n
dt
1 + TetR
n1
− γbac bac
Fig. 12. The simulation result of the population control system. When the
population size of E. coli rises to the threshold level, ccdB proteins are
induced by AHL to restrict the population size.
Advantages
The model equations present interesting mathematical properties that
can be used to explore how qualitative features of the genetic circuit
depend on reaction parameters (Fig. 7).
Fig. 7. Simulated results
(line) of the dynamic model
successfully approximated
the behavior of our lowtemp release system.
Fig. 13. This Mosquito Intelligent
Te r m i n a t o r i s n o t l i m i t e d t o
mosquito's only. we can create
different kinds of E. coli carrying
different cry genes to broaden the
pesticide range.
Fig. 3. DNA sequencing results of Cry11Aa (BBa_K332011).
Fig. 8. Using least squares
estimation from experimental data, the
relative translation activity of this RBS
(BBa_K115002) at 25°C, 30°C, 37 °C
and 40°C can be quantitative by our
model equations.
Contributions
Create a new Cry biobrick :BBa_K332011
Create and quantify a low temperature release device : BBa_K332032
Quantify the translation activity of temp-sensitive RBS: BBa_K115002
Design a new protocol for generation of RBS library with different
translational activity: BBF RFC=79