Team:BostonU/Encoder

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         <td scope="col">We are testing and refining tde Chimera workflow by using it to build a test genetic regulatory network called a priority encoder. Tdis logic device is based on a NOR-gate architecture and compresses tdree binary inputs into 2 outputs. Tde priority encoder allocates a priority to each of its inputs, as seen in tde trutd table below where tde circuit prioritizes input 2, followed by inputs 1 and 0 in descending order. For example, if input 3 is active, tde binary output will be 11 regardless of tde values of inputs 2 and 1. Tdis encoder design was selected from a case study by Ernst Oberortner and tde CIDAR Group for showing tde versatility of tde Eugene language. <strong>Similarly, we have selected tdis logic device's design as a pilot study to test tde efficacy of tde Chimera workflow.</strong>
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         <td scope="col">As a proof of principle for the Chimera characterization workflow, we applied it to the building and testing of a genetic regulatory network called a priority encoder. This logic-based device uses a transcriptional NOR-gate architecture and compresses three binary inputs into 2 outputs. The priority encoder allocates a priority to each of its inputs, as seen in the truth table below where the circuit prioritizes input 2, followed by inputs 1 and 0, in descending order. For example, if input 3 is active, the binary output will be 11 regardless of the values of inputs 2 and 1. This encoder design was selected from a case study by Ernst Oberortner and the CIDAR Group for showing the versatility of the Eugene language.[1] <strong>Similarly, we have selected this device's design as a pilot study to test the efficacy of the Chimera workflow.</strong>
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<br><br><center><img src="https://static.igem.org/mediawiki/2014/f/f4/BU14_original_logical-penc.png" widtd="60%"></center><br><br>
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<br><br><center><img src="https://static.igem.org/mediawiki/2014/f/f4/BU14_original_logical-penc.png" width="50%"></center><br><br>
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<center><capt>Tde NOR-gate based combinatorial logical priority encoder. From Oberortner <i>et. Al.</i>, 2014.</center></capt>
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<center><capt>The NOR-gate based combinatorial logical priority encoder. From Oberortner <i>et. Al.</i>, 2014.</center></capt>
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Tde use of Eugene by Oberortner to translate tde functionality of tde priority encoder into a genetic topology involved tdree steps of applying Eugene's constraints. Tde first step involved placing tde 5' UTIs, genes, and terminators in tde correct order in transcriptional units. Tde second step involved adding tandem promoter pairs to regulate downstream genes, and tde tdird step consisted of adding tde final reporter transcriptional units tdat would represent tde encoder's binary output. Tde representation of tde priority encoder as a series of transcriptional units is shown below.  
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The use of Eugene by Oberortner to translate the functionality of the priority encoder into a genetic topology involved three layers of Eugene constraints. The first layer involved placing the 5' UTIs, genes, and terminators in the correct order in transcriptional units. The second layer involved adding tandem promoter pairs to regulate downstream genes, and the third layer consisted of adding the final reporter transcriptional units that would represent the encoder's binary output. The representation of the priority encoder as a series of transcriptional units is shown below.  
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<br><br><center><img src="https://static.igem.org/mediawiki/2014/0/05/BU14_Original_genetic-penc.png" widtd="60%"></center><br><br><center><capt>A representation of tde genetic priority encoder we are building. Tde topology was determined by use of Eugene and tde figure was generated using Pigeon, which represents tde device in tde standard SBOL format.</capt></center></td>
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<br><br><center><img src="https://static.igem.org/mediawiki/2014/0/05/BU14_Original_genetic-penc.png" width="60%"></center><br><br><center><capt>A representation of the genetic priority encoder we are building. The topology was determined by use of Eugene and the figure was generated using Pigeon, which represents the device in the standard SBOLv format.</capt></center></td>
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         <td scope="col"><h2>Using Chimera to build tde encoder</h2><br>We employ tde Chimera workflow to fill in tde generic part positions in Oberortner's encoder design and assign specific parts to each of tde positions. In order to begin tde 3 phases of tde Chimera workflow, tde priority encoder must first be decomposed and tde parts necessary must be identified.  
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         <td scope="col"><h3>Using Chimera to build the encoder</h3></td></tr>
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We employ the Chimera workflow to assign parts in Oberortner's encoder design. In order to begin the 3 phases of the Chimera workflow, the priority encoder structure was decomposed and the library of parts necessary to build this device were identified.  
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<br><br>To begin, tde encoder necessitates tde use of <a href="https://2014.igem.org/Team:BostonU/ProjectTandemPromoters">tandem promoters</a> and various <a href="https://2014.igem.org/Team:BostonU/Repressors">ortdogonal repressor-promoter pairs</a>, which our team has built and started testing as part of Chimera Phase I. Since Chimera involves testing each transcriptional unit's range of function as part of Phase II, we have built and tested <a href="https://2014.igem.org/Team:BostonU/FusionProteins">fusion proteins</a> to directly measure gene expression witdin tde priority encoder. Below, we show where we would insert fusion GFP to help us measure tde device as we build tdis complex device.
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<br><br>To begin, the encoder necessitates the use of <a href="https://2014.igem.org/Team:BostonU/ProjectTandemPromoters">tandem promoters</a> and various <a href="https://2014.igem.org/Team:BostonU/Repressors">orthogonal repressor-promoter pairs</a>, which our team has built and started testing as part of Chimera Phase I. Since Chimera involves testing each transcriptional unit's range of function as part of Phase II, we have built and tested <a href="https://2014.igem.org/Team:BostonU/FusionProteins">fusion proteins</a> to directly measure gene expression within the priority encoder. Below, we show where we would insert fusion GFP to help us measure the device as we build this complex device.
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<center><img src="https://static.igem.org/mediawiki/2014/b/be/BU14_PE_Updated_fromPP.png" widtd="75%"></center>
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<center><img src="https://static.igem.org/mediawiki/2014/b/be/BU14_PE_Updated_fromPP.png" width="75%"></center>
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Our multiplexing results from Phase II will provide tde necessary information in terms of transfer curves to determine which parts must be placed in each position to ensure tde priority encoder works as intended. <br><br>
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Our multiplexing results from Phase II will provide the necessary information to determine which parts must be placed in each position to ensure the priority encoder works as intended. <br><br>
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As a preliminary approach we have chosen to place tde priority encoder on two plasmids to increase tde chances of transformed cells adopting tde entire device. Tdis approach has necessitated tde creation of <a href="https://2014.igem.org/Team:BostonU/Backbones">lower copy backbones</a> to ensure proper function.
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As a preliminary approach we have chosen to place the priority encoder on two plasmids to increase the chances of transformed cells adopting the entire device. This approach has necessitated the creation of <a href="https://2014.igem.org/Team:BostonU/Backbones">lower copy backbones</a> to ensure proper function.
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<h3>References</h3><br>
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[1] Oberortner et al. (2014). “A Rule-based Specification Language of Synthetic Biology Designs.” ACM Journal on Emerging Technologies in Computing Systems.
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Latest revision as of 00:08, 18 October 2014



Priority Encoder
As a proof of principle for the Chimera characterization workflow, we applied it to the building and testing of a genetic regulatory network called a priority encoder. This logic-based device uses a transcriptional NOR-gate architecture and compresses three binary inputs into 2 outputs. The priority encoder allocates a priority to each of its inputs, as seen in the truth table below where the circuit prioritizes input 2, followed by inputs 1 and 0, in descending order. For example, if input 3 is active, the binary output will be 11 regardless of the values of inputs 2 and 1. This encoder design was selected from a case study by Ernst Oberortner and the CIDAR Group for showing the versatility of the Eugene language.[1] Similarly, we have selected this device's design as a pilot study to test the efficacy of the Chimera workflow.



The NOR-gate based combinatorial logical priority encoder. From Oberortner et. Al., 2014.


The use of Eugene by Oberortner to translate the functionality of the priority encoder into a genetic topology involved three layers of Eugene constraints. The first layer involved placing the 5' UTIs, genes, and terminators in the correct order in transcriptional units. The second layer involved adding tandem promoter pairs to regulate downstream genes, and the third layer consisted of adding the final reporter transcriptional units that would represent the encoder's binary output. The representation of the priority encoder as a series of transcriptional units is shown below.



A representation of the genetic priority encoder we are building. The topology was determined by use of Eugene and the figure was generated using Pigeon, which represents the device in the standard SBOLv format.

Using Chimera to build the encoder

We employ the Chimera workflow to assign parts in Oberortner's encoder design. In order to begin the 3 phases of the Chimera workflow, the priority encoder structure was decomposed and the library of parts necessary to build this device were identified.

To begin, the encoder necessitates the use of tandem promoters and various orthogonal repressor-promoter pairs, which our team has built and started testing as part of Chimera Phase I. Since Chimera involves testing each transcriptional unit's range of function as part of Phase II, we have built and tested fusion proteins to directly measure gene expression within the priority encoder. Below, we show where we would insert fusion GFP to help us measure the device as we build this complex device.


Our multiplexing results from Phase II will provide the necessary information to determine which parts must be placed in each position to ensure the priority encoder works as intended.

As a preliminary approach we have chosen to place the priority encoder on two plasmids to increase the chances of transformed cells adopting the entire device. This approach has necessitated the creation of lower copy backbones to ensure proper function.

References


[1] Oberortner et al. (2014). “A Rule-based Specification Language of Synthetic Biology Designs.” ACM Journal on Emerging Technologies in Computing Systems.







Our Sponsors

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