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Team:BostonU/Workflow
From 2014.igem.org
Phase I - Build and test basic parts.Key software tools: TASBE Tools, Eugene (optional), Raven (optional) | |
|---|---|
General Chimera Workflow |
Case Study: BU Priority Encoder |
| general I |
• Add parts to MoClo library. These 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 • X MoClo level 0 fusion proteins:
• TetR_GFP • X MoClo level 0 tandem promoters:
• pTet_pLac |
Phase II - Build and characterize TU behavior.Key software tools: TASBE Tools, Eugene, Raven | |
General Chimera Workflow |
Case Study: BU Priority Encoder |
| general II |
• Run one-pot Multiplexing MoClo reaction. We initially multiplexed 5' UTIs and Terminators • 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 |
| general III |
• Test individual TU regulatory arcs: • ... • 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. |
