Team:BostonU/Multiplexing

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<center><img src="https://static.igem.org/mediawiki/2014/c/ca/BU14_Phase_II_updated.png" width="100%"></center><br>
<h3>Why multiplexing?</h3>
<h3>Why multiplexing?</h3>
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As the key method of Phase II of the Chimera workflow, multiplexing is being employed by our group to determine the full range of function of our transcriptional units (TUs). We define full range of function as having a set of TUs with one varied constituent part, whose transfer curves display a difference across at least two decades of <a href="https://2014.igem.org/Team:BostonU/Software#flow">MEFLs</a> with varying peak fluorescence as measured by flow cytometry. This wide range is desirable and essential to the robustness of the workflow because it provides the constitutive parts of a device with diverse expression levels. Having this diversity makes having a desired combination of parts more likely, which can more easily result in a desired device behavior. We have selected multiplexing as our tool to achieve this diversity because of its compatibility with our <a href="https://2014.igem.org/Team:BostonU/MoClo">MoClo assembly method</a>, which facilitates the quick replacement of any set of parts in a transcriptional unit.<br><br></td></tr>
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As the key method of Phase II of the Chimera workflow, multiplexing is being employed by our group to determine the full range of function of our transcriptional units (TUs). We define full range of function as having a set of TUs with one varied constituent part, whose transfer curves display a difference across at least two decades of <a href="https://2014.igem.org/Team:BostonU/Software#flow">MEFLs</a> with varying peak fluorescence as measured by flow cytometry. This wide range is desirable and essential to the robustness of the workflow because it provides the constitutive parts of a device with diverse expression levels. Having this diversity makes having a desired combination of parts more likely, which can more easily result in a desired device behavior. We have selected multiplexing as our tool to achieve this diversity because of its compatibility with our <a href="https://2014.igem.org/Team:BostonU/MoClo">MoClo assembly method</a>, which facilitates the quick replacement of any set of parts in a transcriptional unit. Multiplexing with MoClo allows us to achieve our desired range of function with a small number of one-pot reactions, as opposed to building and testing each variant individually.<br><br></td></tr>
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<h3>Building multiplexed transcriptional units</h3>Our first multiplexing reactions vary the ribosomal binding sites (RBSs) of transcriptional units. We began by creating a <a href="https://2014.igem.org/Team:BostonU/Software">Eugene</a> file (<a href="https://static.igem.org/mediawiki/2014/5/5b/Multiplex_RBS_Alan_BU14.txt" target="blank">found here</a>) to plan our first reaction. By specifying constraints on the number and order of parts, in addition to the number of RBSs to be multiplexed, Eugene outputted five possible layouts from a possible design space of 65,536 possibilities. A MoClo reaction was prepared to multiplex the RBSs  
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<h3>Designing multiplexed transcriptional units</h3>
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Our first multiplexing reactions vary the ribosomal binding sites (RBSs) of transcriptional units. We began by creating a <a href="https://2014.igem.org/Team:BostonU/Software">Eugene</a> file (<a href="https://static.igem.org/mediawiki/2014/5/5b/Multiplex_RBS_Alan_BU14.txt" target="blank">found here</a>) to plan our first reaction. By specifying constraints on the number and order of parts, in addition to the number of RBSs to be multiplexed, Eugene outputted five possible layouts from a possible design space of 65,536 possibilities. </td></td>
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<img src="https://static.igem.org/mediawiki/2014/0/0b/Multiplex1_BU14.png" width="300"><br>
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<br><center><capt>Pigeon CAD images of the two initial multiplexing MoClo reactions that were carried out.</td></tr>
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<h3>Building multiplexed transcriptional units</h3>
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A MoClo reaction was prepared to multiplex the RBSs  
<a href="http://parts.igem.org/Part:BBa_K1114100">B0030</a>,  
<a href="http://parts.igem.org/Part:BBa_K1114100">B0030</a>,  
<a href="http://parts.igem.org/Part:BBa_K1114101">B0031</a>,  
<a href="http://parts.igem.org/Part:BBa_K1114101">B0031</a>,  
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<a href="http://parts.igem.org/Part:BBa_K1114103">B0033</a>,  
<a href="http://parts.igem.org/Part:BBa_K1114103">B0033</a>,  
and <a href="http://parts.igem.org/Part:BBa_K1114104">B0034</a>
and <a href="http://parts.igem.org/Part:BBa_K1114104">B0034</a>
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  in a TU with repressible promoter <a href="http://parts.igem.org/Part:BBa_K1114023">I13453</a>, GFP <a href="http://parts.igem.org/Part:BBa_K1114209">E0040</a>, terminator <a href="http://parts.igem.org/Part:BBa_K1114300">B0015</a>, and origin of replication pMB1 in a <a href="http://parts.igem.org/Part:BBa_K1114418">MoClo Level 1 backbone</a>. This set of parts was used as a starting point for directly observing the effect of changing a ribosomal binding site with a repressible promoter, which is a layout similar to what will be present in the priority encoder. A second MoClo reaction was prepared to multiplex the remaining RBSs from our lab:  
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  in a TU with repressible pBAD promoter <a href="http://parts.igem.org/Part:BBa_K1114023">I13453</a>, GFP <a href="http://parts.igem.org/Part:BBa_K1114209">E0040</a>, terminator <a href="http://parts.igem.org/Part:BBa_K1114300">B0015</a>, and origin of replication pMB1 in a <a href="http://parts.igem.org/Part:BBa_K1114418">MoClo Level 1 backbone</a>. This set of parts was used as a starting point for directly observing the effect of changing a ribosomal binding site with a repressible promoter, which is a layout similar to what will be present in the priority encoder. A second MoClo reaction was prepared to multiplex the remaining RBSs from our lab:  
<a href="http://parts.igem.org/Part:BBa_K1114106">BCD1</a>,  
<a href="http://parts.igem.org/Part:BBa_K1114106">BCD1</a>,  
<a href="http://parts.igem.org/Part:BBa_K1114107">BCD2</a>,  
<a href="http://parts.igem.org/Part:BBa_K1114107">BCD2</a>,  
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and <a href="http://parts.igem.org/Part:BBa_K1114110">BCD13</a>
and <a href="http://parts.igem.org/Part:BBa_K1114110">BCD13</a>
  in the same TU layout. </td></td>
  in the same TU layout. </td></td>
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Because MoClo requires an equal amount of DNA for each part, two RBS master mixes were prepared with 8fmol of each individual RBS, totaling 40fmol of RBS mix. Following MoClo, blue-white screening is carried out to pick colonies that incorporated the new plasmid. We have decided to begin by picking 10 colonies per plate to increase the possibility of picking at least one colony per RBS. Each colony that we screen must be sequence verified to link its flow cytometry results to the ribosomal binding site used.<br><br>
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<h3>Testing of multiplex reactions</h3>Testing of our multiplex reactions will follow our <a href="https://static.igem.org/mediawiki/2014/7/7c/Flow_Cytometer_WorkflowYABU.xls">flow cytometry workflow</a> and our <a href="https://2014.igem.org/Team:BostonU/Software#flow">testing scheme</a> with the appropriate single and double expression fluorescence controls for use with the TASBE Tools. For testing of the initial RBS multiplexing experiment, we will measure fluorescence induced with varying concentrations of arabinose and note the changes in expression observed across TUs due to changed RBSs.<br><br></td>
<td scope="col">
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<img src="https://static.igem.org/mediawiki/2014/0/0b/Multiplex1_BU14.png" width="300"><br><br><center><capt>Pigeon CAD images of the two initial multiplexing MoClo reactions that were carried out.</td></tr>
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<center><img src="https://static.igem.org/mediawiki/2014/3/3a/BU14_Multiplexing_results.png" width="200px"><br>
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<capt>When screening for function, we expect to see a range of transfer curves based on the strength of the RBS used in the device. Each colored line is meant to represent the same construct with a different RBS.</capt></center><br><br></td></tr>
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<tr>
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<td scope="col" colspan="2">
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Because MoClo requires an equal amount of DNA for each part, two RBS master mixes were prepared with 8fmol of each individual RBS, totaling 40fmol of RBS mix. Following MoClo, blue-white screening is carried out to pick colonies that incorporated the new plasmid. We have decided to begin by picking 10 colonies per plate to increase the possibility of picking at least one colony per RBS.<br><br>
+
Following characterization of our RBS multiplexing, we will carry out multiplexing reactions for our remaining individual parts: tandem promoters, fusion proteins, and vector backbones. These reactions will be similarly planned with the Eugene software, which will provide a preliminary visualization of the transcriptional unit variety possible by changing individual parts. Our multiplexed TUs will be assembled into a library of variants, and the transfer curves will be used to predictively assemble the priority encoder as part of Phase III.
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<h3>Testing of multiplex reactions</h3>
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Latest revision as of 01:00, 18 October 2014



Multiplexing


Why multiplexing?

As the key method of Phase II of the Chimera workflow, multiplexing is being employed by our group to determine the full range of function of our transcriptional units (TUs). We define full range of function as having a set of TUs with one varied constituent part, whose transfer curves display a difference across at least two decades of MEFLs with varying peak fluorescence as measured by flow cytometry. This wide range is desirable and essential to the robustness of the workflow because it provides the constitutive parts of a device with diverse expression levels. Having this diversity makes having a desired combination of parts more likely, which can more easily result in a desired device behavior. We have selected multiplexing as our tool to achieve this diversity because of its compatibility with our MoClo assembly method, which facilitates the quick replacement of any set of parts in a transcriptional unit. Multiplexing with MoClo allows us to achieve our desired range of function with a small number of one-pot reactions, as opposed to building and testing each variant individually.

Designing multiplexed transcriptional units

Our first multiplexing reactions vary the ribosomal binding sites (RBSs) of transcriptional units. We began by creating a Eugene file (found here) to plan our first reaction. By specifying constraints on the number and order of parts, in addition to the number of RBSs to be multiplexed, Eugene outputted five possible layouts from a possible design space of 65,536 possibilities.

Pigeon CAD images of the two initial multiplexing MoClo reactions that were carried out.

Building multiplexed transcriptional units

A MoClo reaction was prepared to multiplex the RBSs B0030, B0031, B0032, B0033, and B0034 in a TU with repressible pBAD promoter I13453, GFP E0040, terminator B0015, and origin of replication pMB1 in a MoClo Level 1 backbone. This set of parts was used as a starting point for directly observing the effect of changing a ribosomal binding site with a repressible promoter, which is a layout similar to what will be present in the priority encoder. A second MoClo reaction was prepared to multiplex the remaining RBSs from our lab: BCD1, BCD2, BCD8, BCD12, and BCD13 in the same TU layout.
Because MoClo requires an equal amount of DNA for each part, two RBS master mixes were prepared with 8fmol of each individual RBS, totaling 40fmol of RBS mix. Following MoClo, blue-white screening is carried out to pick colonies that incorporated the new plasmid. We have decided to begin by picking 10 colonies per plate to increase the possibility of picking at least one colony per RBS. Each colony that we screen must be sequence verified to link its flow cytometry results to the ribosomal binding site used.

Testing of multiplex reactions

Testing of our multiplex reactions will follow our flow cytometry workflow and our testing scheme with the appropriate single and double expression fluorescence controls for use with the TASBE Tools. For testing of the initial RBS multiplexing experiment, we will measure fluorescence induced with varying concentrations of arabinose and note the changes in expression observed across TUs due to changed RBSs.

When screening for function, we expect to see a range of transfer curves based on the strength of the RBS used in the device. Each colored line is meant to represent the same construct with a different RBS.


Following characterization of our RBS multiplexing, we will carry out multiplexing reactions for our remaining individual parts: tandem promoters, fusion proteins, and vector backbones. These reactions will be similarly planned with the Eugene software, which will provide a preliminary visualization of the transcriptional unit variety possible by changing individual parts. Our multiplexed TUs will be assembled into a library of variants, and the transfer curves will be used to predictively assemble the priority encoder as part of Phase III.

References


[1] B. Stanton, A. Nielsen, A. Tamsir, K. Clancy, T. Peterson & C. Voigt (2014). "Genomic mining of prokaryotic repressors for orthogonal logic gates." Nature Chemical Biology 10: 99-105. doi:10.1038/nchembio.1411







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