Difference between revisions of "Team:IIT Delhi/Design"

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             <h2 class="h2font">PROJECT OVERVIEW</h2>
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             <h2 class="h2font">Model for<br> Introduction</h2>
  
 
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<h2 id="pfont">Several circuits have been proposed, constructed, and implemented, leading to landmark discoveries in synthetic biology. These include systems such as the bi-stable toggle switch, and the repressilator, which brought about a paradigm shift in the field. Since then, several systems have been constructed to employ memory modules, create counters, adders, digital biosensors, and a whole wide range of other products.
  
<h2 id="pfont"><u id="pfont2">Digital Logic in Synthetic Biology</u><br><br>
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<br><br>
A large chunk of effort in synthetic biology has been aimed at attempting to view genes as parts of a circuit. Thus, a lot of focus has been directed toward creating biological analogues of digital logic gates, such as an AND or a NOT gate, which give a digital 1 or 0 response, depending on the truth table of the gate.<br><br>
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<img src = "https://static.igem.org/mediawiki/2017/4/45/T--IIT_Delhi--picture3.png" style='border:3px solid #000000'><br>
  
<img src = "https://static.igem.org/mediawiki/2017/b/b3/T--IIT_Delhi--notgate.png" width="720" height="500" style='border:3px solid #000000'>
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Figure – A brief timeline of major notable events in the creation and development of synthetic biology (Source: Del Vecchio, Domitilla et al, Journal of The Royal Society Interface 13.120 (2016): 20160380.)<br><br>
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However, there are several limitations that still need to be overcome, as the field continues to make strides in every area. These involve the fact that biological systems have a lot of noises that cannot be modeled accurately to date, and the fact that metabolic burden is a major issue. Along these lines, one of the central issues is the distinct lack of digital responses in synthetic biology. <br>
  
<h6>Figure – A typical example of a biological NOT Gate, considered to be ON when the concentration of inducer is below 10-6, and OFF when it is greater than 10-4. Such a response gives a corresponding gene expression level (au) of ~1 in the ON state, and ~0.4 in the OFF state (Source: “Generation of Pulse of a Bacterial Species in E.coli”, Kshitij Rai, Department of Biochemical Engineering and Biotechnology IIT Delhi, Master’s Thesis, 2017)</h6>
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Thus, as described in the project overview, we wished to use the high cooperativity TetR homologs in such a manner so as to generate a square wave oscillator circuit. Such a system could have a whole multitude of applications; some of which were mentioned briefly in the overview, and the same are also discussed below,
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<h2 id="pfont">
 
However, there is a serious issue in the scale up of these circuits. While that can be attributed to several reasons, one of the major reasons is this simplification under which the systems work. We can see that the output response from the gate is not close to the actual digital “1 or 0” kind that one would ideally want from a logic gate, and in the range that is neither in the ON nor the OFF regime, the response is really graded.  <br><br>
 
  
NThus, when a combination of gates is used in conjunction, one could not expect them to remain digital. Just imagine using a 10 input AND gate as shown below, that could possibly be used as an environmental biosensor. If the concentration of a few inputs is in the ON range, while the others are in the middle range, should the device show an ON or an OFF state? It may show neither, as we have seen above, leaving the researcher confused as to how the results must be perceived. <br><br>
 
<img src = "https://static.igem.org/mediawiki/2017/3/39/T--IIT_Delhi--shreya2.png" width="600" height="200"><br>
 
  
<h6>Figure: input AND gate which can act as a biosensor, sensing a particular environmental condition, and generating an output such as fluorescence through GFP.</h6>
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<header class="major">
</h2>
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            <h2 class="h2font">Applications</h2>
  
<h2 id="pfont"><u id="pfont2">High Cooperativity Repressors – A Possible Solution</u><br>
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<br>The solution to the problem lies in the cooperativity of the parts that are used as actuators in these digital devices. These are mostly created using repressors or activators, which can repress or activate their respective promoters. Cooperativity is basically the phenomenon where the repressor (or activator) molecules do not act independently of each other, and two or more molecules of the same are needed to first bind to each other, before binding to the promoter that they are to repress.
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What this means is that if the cooperativity of the repressor is “n”, then unless n molecules of the repressor combine, they would not be able to repress the promoter and control gene expression. This kind of behavior becomes desirable, since when low quantities of the repressor are present, we would expect lesser repression in the case of a high cooperativity repressor. This would generate a response that would be closer to the digital output, which is desired. This is further explained in the modeling section, but just for a brief idea, here is what the picture looks like, for increasing cooperativity.
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<img src = "https://static.igem.org/mediawiki/2017/a/a8/T--IIT_Delhi--shreya3.png" width="720" height="400" style='border:3px solid #000000'> <br>
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<h6>Figure – Variation of output with repressor concentration. As can be seen, the response for n = 1 is the furthest away from a digital response, while as n increases, the output moves from 1 to 0 in a more digital fashion.</h6>
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<h2 id="pfont">
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<h2 id="pfont"><u id="pfont2">The Square Wave Generator</u><br>
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<br>With the goal of searching and employing high cooperativity repressors, we looked at a paper by Voigt et al (Nature Chem Biol. 2013), in which they reported 73 analogs of the TetR repressor, of which 16 of them were found to be perfectly orthogonal to each other. A few of these repressors had really high cooperativities, with the highest n value being 6.1 for the Orf2 repressor.
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We therefore decided to use repressors which were orthogonal and had the highest possible cooperativity, to demonstrate computationally, how these could be used in simple devices such as NOT gates and novel collapsible AND gates (where once the output switches from 1 to 0, it cannot be switched back. Think of it as a fuse box, which melts if the input voltage goes higher than a certain point). This entire module was called the Basic Logic Assessment and Signaling Toolbox, or the BLAST Toolbox.
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Having demonstrated these successfully, we moved ahead to our main aim, which was to engineer and demonstrate a square wave oscillator in E.coli. Square waves are commonly used in electrical circuits, and can have a wide array of applications in various areas in biology such as clock inputs for timing events, time dependent drug delivery, switching of metabolic pathways and shunt activation, and would also help understand variations of biological clocks such as the circadian clock, whose gene regulatory network still remains largely unknown. This has been discussed at length in the next section. <br><br>
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<img src = "https://static.igem.org/mediawiki/2017/5/59/T--IIT_Delhi--shreya4.png" width="700" height="350" style='border:3px solid #000000'><br>
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<h6>Figure – Comparison of a sine wave and a square wave. While both sinusoidal and square inputs are used in electrical engineering, the analogue of square waves in biological systems has not yet been reported. This largely limits the applications of oscillators to regions where a sine wave is required, since a proper, well characterized square wave oscillator has not been reported. </h6>
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          </header>
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<ul> Generating a clock input to time biological events - The potential of genetic clock lies in its role to triggering logic reaction for sequential biological circuits. A square wave generator could be used as a genetic clock, since square waves lie at the heart of clocks. Further, these clocks could be used in any cellular system to time particular events.
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<ul> Characterising gene regulatory network nodes through impulses - This could be used to study correlation between two genes, by coupling one of the genes to the oscillator, then observe the dynamics of the second gene. In this manner, the effect of one gene on the others could be studied.
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<ul> Periodic Drug delivery - Like diabetic patients, insulin needs to be provided externally through injections or other means. Our oscillator system could be used as a patch containing bacteria that are oscillating to produce square levels of insulin to the patient once. This then would deliver insulin automatically at intervals, guided by our system .Further when the requirement for insulin would be high, the amount of insulin being delivered to them could be changed through some source which could dive a change in frequency of the oscillations (future applications could be focused on engineering frequency modulation).
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<ul> Metabolic Switching – A bacterial species could be engineered to produce levels of the permease for a particular sugar in the form of a square wave. Thus, at varying intervals of time, the permease (say lac permease) would be expressed, which would cause the bacteria to start metabolizing lactose. When it goes off, the bacteria would not express the lac permease and consume glucose. In this manner, we could tune the frequency of the oscillations to ensure metabolic switching and activation of pathway shunts in the manner that we want.
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<ul> Temporal Bar codes – If we can control the frequency of the oscillations and modulate the time spent by the wave in the ON and OFF state (which is basically a function of the frequency itself), we could generate a combination of 0’s and 1’s, in order to generate a bar code. This bar code would be read in time, and therefore would be a temporal bar code. A typical example of this would be to encode the word “iGEM” by 11001001, where 1 represents an ON state for say, 20 minutes. Thus, an 11 response, which represents the letter i, would then be read if the fluorescence stays on for 40 minutes. In this fashion, our device could be used for encryption of data.
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<ul> For memory storage:-The system could be used as the bacterial analogue of memory storage. The base of the oscillations could be called the 0 bit, and the high point could be called the 1 bit, and these combinations of 0 and 1 bits could be used to store short term memory in biological cultures, performing the functions that the RAM (random access memory) does in computers.
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Revision as of 17:25, 1 November 2017

iGEM IIT Delhi


Model for
Introduction

                                                                                                                                                                                                                 

Several circuits have been proposed, constructed, and implemented, leading to landmark discoveries in synthetic biology. These include systems such as the bi-stable toggle switch, and the repressilator, which brought about a paradigm shift in the field. Since then, several systems have been constructed to employ memory modules, create counters, adders, digital biosensors, and a whole wide range of other products.


Figure – A brief timeline of major notable events in the creation and development of synthetic biology (Source: Del Vecchio, Domitilla et al, Journal of The Royal Society Interface 13.120 (2016): 20160380.)

However, there are several limitations that still need to be overcome, as the field continues to make strides in every area. These involve the fact that biological systems have a lot of noises that cannot be modeled accurately to date, and the fact that metabolic burden is a major issue. Along these lines, one of the central issues is the distinct lack of digital responses in synthetic biology.
Thus, as described in the project overview, we wished to use the high cooperativity TetR homologs in such a manner so as to generate a square wave oscillator circuit. Such a system could have a whole multitude of applications; some of which were mentioned briefly in the overview, and the same are also discussed below,

Applications

                                                                                                                                                                                                                 

    Generating a clock input to time biological events - The potential of genetic clock lies in its role to triggering logic reaction for sequential biological circuits. A square wave generator could be used as a genetic clock, since square waves lie at the heart of clocks. Further, these clocks could be used in any cellular system to time particular events.
      Characterising gene regulatory network nodes through impulses - This could be used to study correlation between two genes, by coupling one of the genes to the oscillator, then observe the dynamics of the second gene. In this manner, the effect of one gene on the others could be studied.
        Periodic Drug delivery - Like diabetic patients, insulin needs to be provided externally through injections or other means. Our oscillator system could be used as a patch containing bacteria that are oscillating to produce square levels of insulin to the patient once. This then would deliver insulin automatically at intervals, guided by our system .Further when the requirement for insulin would be high, the amount of insulin being delivered to them could be changed through some source which could dive a change in frequency of the oscillations (future applications could be focused on engineering frequency modulation).
          Metabolic Switching – A bacterial species could be engineered to produce levels of the permease for a particular sugar in the form of a square wave. Thus, at varying intervals of time, the permease (say lac permease) would be expressed, which would cause the bacteria to start metabolizing lactose. When it goes off, the bacteria would not express the lac permease and consume glucose. In this manner, we could tune the frequency of the oscillations to ensure metabolic switching and activation of pathway shunts in the manner that we want.
            Temporal Bar codes – If we can control the frequency of the oscillations and modulate the time spent by the wave in the ON and OFF state (which is basically a function of the frequency itself), we could generate a combination of 0’s and 1’s, in order to generate a bar code. This bar code would be read in time, and therefore would be a temporal bar code. A typical example of this would be to encode the word “iGEM” by 11001001, where 1 represents an ON state for say, 20 minutes. Thus, an 11 response, which represents the letter i, would then be read if the fluorescence stays on for 40 minutes. In this fashion, our device could be used for encryption of data.
              For memory storage:-The system could be used as the bacterial analogue of memory storage. The base of the oscillations could be called the 0 bit, and the high point could be called the 1 bit, and these combinations of 0 and 1 bits could be used to store short term memory in biological cultures, performing the functions that the RAM (random access memory) does in computers.




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