Difference between revisions of "Team:ETH Zurich/Proof"

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Our concept is based on three major devices, that can be tested and optimised seperatly, and can then, once they fulfill all of our strict criterions, be brought together.
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Our concept is based on three major devices that can be tested and optimised separately. Once they fulfill all of our strict criterions, they would be brought together to demonstrate our work. Initially to prove our concept is working, we here present the three devices we decided suit our needs best.
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To prove our concept is working, we here present the three devices we decided to suit our needs best.
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This part takes nitric oxyde (NO) and N-Acyl homoserine lactone (AHL) as it's inputs and activates downstream gene expression. We were able to construct multiple versions
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This part takes nitric oxide (NO) and N-Acyl homoserine lactone (AHL) as it's inputs and activates downstream gene expression. We were able to construct multiple versions
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of this AND gate, tested each of them, and decided on how to optimize it to fit our purpose most: detect the physiological concentration ranges of NO and AHL during
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of this AND gate <a href=https://2016.igem.org/Team:ETH_Zurich/Part_Collection>(check out our Part Collection)</a>. After characterisation we decided on how to optimize it to fit our purpose the best: detect the physiological concentration ranges of NO and AHL during inflammation.
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diseased as well as healthy state.  
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<p> We have chosen the variant that has shown the best fold-activation, <a href= >Bba_</a>, characterised through GFP expression. We could thus show that our first device is functional and works as expected(Figure 1). </p>
  
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Plot AND gate: Activation of the AND gate leads to expression of a fluorescent reporter (sfGFP, Part:BBa_K....). <i>E. coli</i> cells were induced with ...
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<p><b>Figure 1:</b> Demonstration of AND gate behaviour in presence of NO and AHL. Measured x hours after induction. Error bars indicate S.D. of 3 technical replicates </a>.</p>
  
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<p>Our <a href=https://2016.igem.org/Team:ETH_Zurich/Switch_Module>model</a> shows that 4-fold activation would be sufficient for activating the next device; our switch.</p><b>MATTIA can you insert some stuff HERE </b>
  
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We can show that our AND gate has a X-fold activation compared to the negative control. This activation is assumed to be sufficient to activate its downstream device: The switch.
 
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Our switch consists of a recombinase, that once expressed, irreversibly switches the reporter construct. Different recombinases were constructed: TP901, Bxb1, phiC31.
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Our switch consists of a recombinase, that irreversibly switches the reporter construct. Different recombinases were constructed: TP901, Bxb1.
 
Here again, we focus on optimisation before assembly of all the parts. We tested the recombinases codon optimized, non-optimized, with different RBS' as well as different
 
Here again, we focus on optimisation before assembly of all the parts. We tested the recombinases codon optimized, non-optimized, with different RBS' as well as different
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degradation tags. Our favourite recombinase (codon optimized bxb1, Part: BBa_K...) exhibits the most desired kinetic data.
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degradation tags. Our favourite recombinase (codon optimized bxb1, <a href=>Part: BBa_K...)</a> exhibits the most desired kinetic data(Figure 2). According to our <a href=https://2016.igem.org/Team:ETH_Zurich/Switch_Module>model</a>, <b>MATTIA TALK ABOUT THE 6HOUR FLIPPING AND LOW LEAKINESS</b>
  
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<p><b>Figure 2:</b> Demonstration of functionality and ideal flipping kinetics of bxb1 expressed under Ptet promoter.  Measured x hours after induction. Error bars indicate S.D. of 3 technical replicates </a>.</p>
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<p>We conclude, that this switching rate is most desired for the whole construct.</p>
  
  
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We conclude, that this switching rate is most desired for the whole construct.
 
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Our reporter consists of an AHL inducible promoter flanked by recombinase specific att-sites. While not flipped, the reporter will express red fluorescent protein (mNectarine, Part:BBa_K..) once induced.
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Our final reporter consists of an AHL-inducible promoter flanked by recombinase specific att-sites. While not flipped, the reporter will express red fluorescent protein (mNectarine, Part:BBa_K..). After flipping it would express <a href=>GFP</a>. For proof of concept we tested our reporter with a constitutive promoter <a href=>Bba_J23118</a> rather than an AHL-inducible promoter. Different reporter variants were tested in conjunction with our recombinases. We found that (p121) is most suitable for our purposes, and works with our selected switch module (insert link). </p>
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If flipping occurs, the promoter will face the other direction and is able to express green fluorescent protein (sfGFP, Part:BBa_K..). Different reporter variants were tested
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by changing degredation tags. We found .... most suitable for our purpose.  
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All three BioBrick devices were tested individually and show the desired function. The system was modeled, and shown to be functional. We conclude, that the  
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All three BioBrick devices were tested individually and display the desired function. The system was modelled <b>MATTIA, AGAIN PLEASE:)</B>, and shown to be functional. Thus we argue that the concept of <b>associative learning</b> with our genetic circuit has been proven. The system will be put together, and its function will be demonstrated under simulated real-world conditions (link to our demonstrate page).  
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concept works. The system will be put together, and its function will be demonstrated under real-world conditions.  
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Revision as of 23:19, 19 October 2016

PAVLOV'S COLI

PROOF OF CONCEPT

Our concept is based on three major devices that can be tested and optimised separately. Once they fulfill all of our strict criterions, they would be brought together to demonstrate our work. Initially to prove our concept is working, we here present the three devices we decided suit our needs best.

AND GATE

This part takes nitric oxide (NO) and N-Acyl homoserine lactone (AHL) as it's inputs and activates downstream gene expression. We were able to construct multiple versions of this AND gate (check out our Part Collection). After characterisation we decided on how to optimize it to fit our purpose the best: detect the physiological concentration ranges of NO and AHL during inflammation.

We have chosen the variant that has shown the best fold-activation, Bba_, characterised through GFP expression. We could thus show that our first device is functional and works as expected(Figure 1).

Figure 1: Demonstration of AND gate behaviour in presence of NO and AHL. Measured x hours after induction. Error bars indicate S.D. of 3 technical replicates .

Our model shows that 4-fold activation would be sufficient for activating the next device; our switch.

MATTIA can you insert some stuff HERE

SWITCH

Our switch consists of a recombinase, that irreversibly switches the reporter construct. Different recombinases were constructed: TP901, Bxb1. Here again, we focus on optimisation before assembly of all the parts. We tested the recombinases codon optimized, non-optimized, with different RBS' as well as different degradation tags. Our favourite recombinase (codon optimized bxb1, Part: BBa_K...) exhibits the most desired kinetic data(Figure 2). According to our model, MATTIA TALK ABOUT THE 6HOUR FLIPPING AND LOW LEAKINESS

Figure 2: Demonstration of functionality and ideal flipping kinetics of bxb1 expressed under Ptet promoter. Measured x hours after induction. Error bars indicate S.D. of 3 technical replicates .

We conclude, that this switching rate is most desired for the whole construct.

REPORTER

Our final reporter consists of an AHL-inducible promoter flanked by recombinase specific att-sites. While not flipped, the reporter will express red fluorescent protein (mNectarine, Part:BBa_K..). After flipping it would express GFP. For proof of concept we tested our reporter with a constitutive promoter Bba_J23118 rather than an AHL-inducible promoter. Different reporter variants were tested in conjunction with our recombinases. We found that (p121) is most suitable for our purposes, and works with our selected switch module (insert link).

CONCLUSION

All three BioBrick devices were tested individually and display the desired function. The system was modelled MATTIA, AGAIN PLEASE:), and shown to be functional. Thus we argue that the concept of associative learning with our genetic circuit has been proven. The system will be put together, and its function will be demonstrated under simulated real-world conditions (link to our demonstrate page).

REFERENCES

  • [1]

Thanks to the sponsors that supported our project: