Difference between revisions of "Team:USP-Brazil/Design"

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We use a genetic circuit that sense blue light. We use the blue part of the RGB circuit from Voigt and associates. This circuit uses a membrane protein that, in the presence of blue light (470nm) inactivates phIF gene, that works as a NOT gate. When phIF is not beeing transcript, it frees the expression of the promoter  PphIF (2) witch activates transcription of sigma fragment T3, that, with RNAP core, promotes transcription of PT3 promoter, that controls de expression of the output.
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We use a genetic circuit that sense blue light. We use the blue part of the <a href="<a href=""><b>RGB circuit from Voigt and associates</b></a>. This circuit uses a membrane protein that when stimulated by blue light (470nm) inactivates phIF , working as a NOT gate . When phIF is not being transcript, it frees the expression of PphIF promoter (2) which enables transcription of sigma fragment T3. This sigma fragment associates with a RNAP core promoting transcription of PT3 promoter, that controls de expression of the output of the system.
  
 
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We idealize a genetic circuit that switches between two states of gene expression, based on a single signal, the blue light.  The output of the circuit that senses blue light (T3 sigma fragment) is the input of our circuit  . In order to have a real memory stored in the cells and not only a specific response to distinct inputs, we use two approches:  
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We idealize a genetic circuit that switches between two states of gene expression, based on a single signal, the blue light.  The output of the circuit that senses blue light (T3 sigma fragment) is the input of our circuit  . In order to have a real memory stored in the cells and not only a specific response to distinct inputs, we use two approches:  
 
1) Utilize invertases for storing the states of memory in the cells. The hbiF and fimE enzymes were chosen, as the paper of Christopher and associates indicates that those enzymes are capable inverting DNA sequences back and forth in an accurate and efficient way;
 
1) Utilize invertases for storing the states of memory in the cells. The hbiF and fimE enzymes were chosen, as the paper of Christopher and associates indicates that those enzymes are capable inverting DNA sequences back and forth in an accurate and efficient way;
 
2) Utilize two types of repressor, each one repressing one another acting as a repressor loop.
 
2) Utilize two types of repressor, each one repressing one another acting as a repressor loop.
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Approach B
 
Here we design a genetic circuit using the repression handle to characterize the repression of our final circuit. The reporter mScarlet represents state 1 and mNeongreen represents state 2. The reporter mTagBFP2 is expressed constitutively and is use as a basal expression to correct cell density, as performed in the project of 2018 USP-Brazil’s team.  
 
Here we design a genetic circuit using the repression handle to characterize the repression of our final circuit. The reporter mScarlet represents state 1 and mNeongreen represents state 2. The reporter mTagBFP2 is expressed constitutively and is use as a basal expression to correct cell density, as performed in the project of 2018 USP-Brazil’s team.  
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The sequence has two PT3 promoters (responsive to the presence of blue light) that are never responsive at the same time, if the handle works. In the absence of arabinose and IPTG and in the presence of blue light (A), only mScarlet and mTagBFP2 (reporters), and LacI (repressor) are being expressed. LacI binds to the lacO (operator) blocking the expression of mNeongreen. When IPTG and arabinose are added (B), IPTG binds LacI protein and free the lac operator, so mNeogreen can be expressed in the presence of blue light. Arabinose activates the promoter pBAD, expressing TetR. This protein binds to tetO, blocking the expression of mScarlet.
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The sequence has two PT3 promoters (responsive to the presence of blue light) that are never responsive at the same time, if the handle works. In the absence of arabinose and IPTG and in the presence of blue light (A), only mScarlet and mTagBFP2 (reporters), and LacI (repressor) are being expressed. LacI binds to the lacO (operator) blocking the expression of mNeongreen. When IPTG and arabinose are added (B), IPTG binds LacI protein and free the lac operator, so
  
 
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Revision as of 01:12, 21 October 2019



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_The project

We use a genetic circuit that sense blue light. We use the blue part of the RGB circuit from Voigt and associates. This circuit uses a membrane protein that when stimulated by blue light (470nm) inactivates phIF , working as a NOT gate . When phIF is not being transcript, it frees the expression of PphIF promoter (2) which enables transcription of sigma fragment T3. This sigma fragment associates with a RNAP core promoting transcription of PT3 promoter, that controls de expression of the output of the system.

Genetic Circuit

We idealize a genetic circuit that switches between two states of gene expression, based on a single signal, the blue light. The output of the circuit that senses blue light (T3 sigma fragment) is the input of our circuit . In order to have a real memory stored in the cells and not only a specific response to distinct inputs, we use two approches: 1) Utilize invertases for storing the states of memory in the cells. The hbiF and fimE enzymes were chosen, as the paper of Christopher and associates indicates that those enzymes are capable inverting DNA sequences back and forth in an accurate and efficient way; 2) Utilize two types of repressor, each one repressing one another acting as a repressor loop. The project focus in two approaches to characterize the circuit.

Approach A

Here we aim to construct the circuit that switches, researching in the literature the best repressors, recombinases and promoters for our purpose. See below step by step how it works.

Approach B

Approach B Here we design a genetic circuit using the repression handle to characterize the repression of our final circuit. The reporter mScarlet represents state 1 and mNeongreen represents state 2. The reporter mTagBFP2 is expressed constitutively and is use as a basal expression to correct cell density, as performed in the project of 2018 USP-Brazil’s team. The sequence has two PT3 promoters (responsive to the presence of blue light) that are never responsive at the same time, if the handle works. In the absence of arabinose and IPTG and in the presence of blue light (A), only mScarlet and mTagBFP2 (reporters), and LacI (repressor) are being expressed. LacI binds to the lacO (operator) blocking the expression of mNeongreen. When IPTG and arabinose are added (B), IPTG binds LacI protein and free the lac operator, so