micro timer - 2015.igem.org
TRANSCRIPT
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MICRO TIMER
Sun Yat-Sen University, Guangzhou, China
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MICRO TIMER
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1.Circadian rhythm
2.Sensing the length of time
3.Behave accordingly Tyrosine family recombinase: Cre/Flpe Serine family recombinase:bxb1
recombinase
Recombination target site(RTS)
invertase
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? Can a single recombinase unit define a time length accurately? How can we construct a micro-timer based on these units?
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Answer: invertase dynamics
Core Problem
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activated phase
Timing length t
Product (inverted sequence)
Invertase
Mechanism How invertase counts time
time
Quantity
unactivated phase
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Major Concerns
How to construct an available
timing Module?
What factors determine the timing length?
• Real-time invertase dynamics testing system
Major concerns
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System construction
Real-time invertase dynamics testing system
Cmr Kmr
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Our timing module exactly works!
Results
• Relatively stable Cre generation
• A burst of Red signal
• A rough but obvious timing length
• Invertase can be a timer
Burst
8hrs
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• 5 invertases
• 16 pairs of pInv-rep and pInv-gen.
• Similar pattern
Our timing module exactly works!
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LoxP/FRT/ Rox/VloxP/ SloxM1/Vox
Ssra: a C-term tag leading to protein degradation
Ssra
Major Elements
With ssra or not
Cre/Flpe/Dre/ Vcre/Scre/Vika
With ssra or not 5 constitutive
promoters
2 Inducible promoters
What contributes to determination of timing length?
Promoter Invertase (and its RTS)
Variants
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Results - Promoter
Results Inversion efficiency is positively-correlated to promoter intensity. Stronger promoter has: • Higher mCherry
expression at plateau phase.
• Shorter timing length.
P_intensity: 2549
P_intensity: 1741
P_intensity: 844
P_intensity: 396
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Results – EGFP fusion site
Results
EGFP-Cre
Cre-EGFP
Cre-EGFP reporter
EGFP-Cre reporter
Fusing EGFP to invertase N-term deteriorates its activity. • Cre-EGFP: stable growth,
obvious mcherry burst.
• EGFP-Cre: Strong expression, deteriorated activity.
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Results – Ssra tag
Results Ssra might slightly reduce invertase expression, but not hinder inversion activity. • Test it for later use;
• Reduce the leakage expression.
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Results – Novel mechanisms
Results We introduced 4 new recombinases by de novo synthesis. • Dre-Rox • Scre-SloxM1 • Vika-Vox • Vcre-VloxP Rox, Scre, Vcre, and Flpe react at different rate.
Yat-sen Star Recombinases Tool Kit: • 7 recombinases, • 7 RTS, • 6 generators, • 6 reporters, Wide range of usage.
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1. We constructed invertase module that exactly works as a timer.
2. We introduced novel recombinases by de novo synthesis, and they works properly.
3. We understood how core elements determine timing length. We can create timers with flexible length of time.
Conclusion
Conclusion:
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We need a key!
To precisely measure timing length?
The key is modeling
To precise timing
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Purpose
1.Precisely measure the timing length 2. Timing prediction • Expand “1 element” to “n elements” • Expand “1 period module” to “n period module”
Tool Just design your own timer!
MODELING
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Time interval can be observed between invertase generation and a burst of product accumulation
RFU
T1 T2
Invertase-EGFP
mCherry by reporter Timing length = T2-T1 Avoid random fluctuations
MODELING
T3
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} = = > leakage
+
MODELING
Main production part + leakage Green light cre EGFP
=
LOGISTIC EQUATION:
=
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Experiment data Fitting curve derived from model above
MODELING
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Equations: MODELING
Degradation
Michaelis-Menten equation
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RFU
MODELING
T1 T2 T
R(t)
G(t)
T1: time of ½ max growth rate of G(t) T2: time of ½ max growth rate of R(t)
The definition of precise timing length
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MODELING
• Cre • Dre • Flpe • Scre • Vcka • Vcre • … • …
• Loxp • Dox • FRT • SloxM1 • Vox • Vloxp • … • …
• Loxp • Dox • FRT • SloxM1 • Vox • Vloxp • … • …
• Yes • No
• Yes • No
• J23100 • J23101 • J23102 • J23103 • J23014 • … • …
The prediction of timing with any element
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MODELING
RFU • Change mCherry to
mCherry-Invertase
• Let R(t) = G2(t), we got R2(t), the second timer module
• Multiple module timer
The prediction of multiple-module timer
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Circuit 1 Circuit1
Fusion: From single to multiple
PROKARYOTIC TIMER
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Circuit1
Circuit2
PROKARYOTIC TIMER
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2015.04 2015.09
result PROKARYOTIC TIMER
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result PROKARYOTIC TIMER
RFU
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Eukaryotic Timer
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Eukaryotic Timer
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STATE 0
attP attP attP
attB attB attB
1
Eukaryotic Timer
Inte
gra
se3 attB attP attL attR
Int Int+Xis
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STATE 1 2
Eukaryotic Timer
Inte
gra
se3
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Inte
gra
se3
STATE 2 3
Eukaryotic Timer
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STATE 3 0
Eukaryotic Timer
Inte
gra
se3
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Eukaryotic Timer
Modified pAUR135 Bxb1 for the first step
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• Fundamental system
• Assemble different unit with different target genes
Significance
lifetime limitation
periodical administration
safer ferment engineering
suicide
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Significance
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Significance
Promoter1
Promoter2
Recombinase1
Recombinase2
RTS1
RTS2
Degradation tag1
Degradation tag 2
INPUT TIME LENGTH
OUTPUT COMBINATION
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l Summer School l 2015 iGEM Conference l Newsletter
l SKLBC Meet Up
l Team Selection l Biocamp l High School Science Camp l Freshmen Work
Human Practice
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Safety
• Use well-developed and relatively safe strains.
• Mainly use biobricks from iGEM kit plate
• Strictly follow the lab treatment disciplines of
disposable biological equipment.
• Strictly observed lab regulations.
• Provide a idea to deal with implanting strains
safety concerns.
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THANKS FOR YOUR ATTENTION
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Appendix
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Oscillator vs Our 3mer damping
damping
Flpping Threshold (Constant)
Recombinase Fast Accumulates
Recombinase Fast Degrades
S3mulator
Repressor
Stage A Stage B
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“2A” Assembly
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Excision Won’t Cause Chaos
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Excision Won’t Cause Chaos
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EcoRV
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Project Safety How will your project work? The purpose of our project is to build an internal clock in microbes, in order to let them perform complex self-regulating activities. We mainly used E.coli and yeast as experimental subjects. In E.coli, we produced proteins we need by plasmid transformation, while in yeasts, we integrate genes needed into the genome by gene recombination. There were different fluorescent proteins to report each gene expression. By testing the starting point and variation of their concentrations, we can infer how this system works from the fluorescent protein accumulation pattern. When the fluorescent proteins are expressed accordingly and regularly, the system was built successfully. With further development, this system may be applied to other fields.
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What risks might your project pose, if it were fully developed into a real product that real people could use? What future work might you do to reduce those risks? Our project aims at providing existing micro organism products a more self regulating control and a safer method, which can be used to deal with those safety problems lots of iGEM teams failed to answer. For example, for a currently impossible project about implanting redesigned microbes into human bodies, our system would provide it a very attractive alternative solution to its safety concerns. Certainly, our project itself has its own risks, if the self-regulating suicide genes lost control, precious strains might die. Also, this system cannot avoid the problem of genetic shift.
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With those concerns, we added a reset system in the yeast cycle system, in order to prevent the system from accidentally turning on by human error or other factors. This system could save the strains, though we have not done experiments of this area. As for genetic drift, we consider that there has not been any efficient technology to solve this problem, while our system can reduce the possibility of genetic drift to a certain extent by artificially controlling bacteria‘s life. Of course, risks need good managements, so these kind of products should be properly preserved to avoid the cost of inappropriate turning on. Laboratory Safety
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What risks does your project pose at the laboratory stage? What actions are you taking to reduce those risks? We have experiments on E.coli and yeasts at the same time, though the E.coli strain DH5a and Top10 are well-developed and relatively safe strains, treating them unscrupulously might still cause contamination to the environment, or even harm to humans. Yeasts cause less damage to human, but as fungi, they might contaminate prokaryotic culture and nearby cell culture rooms. For safety concern, we strictly acquired team members to wear properly during the experiments, which means they had to wear trousers, long sleeve tops, shoes that cover the whole feet, the lab gown and latex gloves. When using harmful reagents, team members must wear masks and operate in the fume hood.
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In order to prevent E.coli and yeasts from polluting the environment, we strictly follow the lab treatment disciplines of disposable biological equipments. All used petri dishes and culture will be sent to the fixed collecting locations and go through uniform disposal. Besides, the experiment region and resting area are strictly separated. Also, we have a spare rocking bed used on yeast culturing only and placed in another room, in order to prevent yeasts contamination.