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Team:BostonU/Workflow
From 2014.igem.org
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| - | These parts were cloned into a <i>E. coli</i> Bioline strain using our MoClo and transformation protocols. They were purified and sequenced. Additionally, we built testing devices for each of the new parts. Details can be found on the <a href="https://2014.igem.org/Team:BostonU/FusionProteins">fusion proteins | + | These parts were cloned into a <i>E. coli</i> Bioline strain using our MoClo and transformation protocols. They were purified and sequenced. Additionally, we built testing devices for each of the new parts. Details can be found on the <a href="https://2014.igem.org/Team:BostonU/FusionProteins">fusion proteins </a>, <a href="https://2014.igem.org/Team:BostonU/ProjectTandemPromoters">tandem promoters </a>, and <a href="https://2014.igem.org/Team:BostonU/Backbones">origin of replication</a> pages. We tested these using our <a href="https://static.igem.org/mediawiki/2014/7/7c/Flow_Cytometer_WorkflowYABU.xls">FACS Workflow</a> and our BD LSRFortessa flow cytometer.The TASBE Tools were then employed to characterize their expression. |
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Revision as of 00:10, 18 October 2014
For a detailed example of our Chimera Characterization Workflow, please check out the Chimera Example page. Below, we present a brief outline of the major steps involved in each stage (Design, Build, Test) of the Chimera workflow, along with a few high level examples. We also define what we consider Phase I, II, and III to be for our workflow.
Phase I - Build and test basic parts.Key software tools: TASBE Tools, Eugene (optional), Raven (optional) | |
General Chimera Workflow |
Case Study: BU Priority Encoder |
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• Add parts to MoClo library. The following parts were found to be necessary for our priority encoder: • 3 MoClo level 1 and 3 MoClo level 2 backbones, each with a different origin of replication:
• ColE1 • 4 MoClo level 0 fusion proteins:
• tetR_GFP • X MoClo level 0 tandem promoters:
• pTet_pBad |
Phase II - Build and characterize TU behavior.Key software tools: TASBE Tools, Eugene, Raven | |
General Chimera Workflow |
Case Study: BU Priority Encoder |
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• Run one-pot Multiplexing MoClo reaction. We initially multiplexed RBSs.
• Eugene was employed to visualize all possible part substitutions. • Clone multiplexed reactions into Pro strain of E. coli using Pro Transformation protocol. • Pick 20 colonies per plate, purify, and sequence. • Test using flow cytometry workflow and analyze data using the TASBE Tools. |
Phase III - Test regulatory arcs and assemble final device.Key software tools: TASBE Tools, Eugene, Raven | |
General Chimera Workflow |
Case Study: BU Priority Encoder |
 |
• Test individual TU regulatory arcs • Use Eugene to plan final device topology. • Use Raven to guide MoClo assembly of encoder. • Clone multiplexed reactions into Pro strain of E. coli using Pro Transformation protocol. • Pick colonies, purify, and sequence. • Test using flow cytometry workflow and analyze data using the TASBE Tools. |
