Team:BostonU/Workflow

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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

• Break down large device into TUs and further break down into individual genetic parts (promoters, RBS, CDS, terminator)

• Decide which parts will be necessary that don't yet exist in your parts collection

• If new to synbio/wetlab work, use Raven to design primers and generate steps for building

• If unsure, read through literature for ideas of how to design part

• If experienced, design primers while keeping in mind you'll have to combine new part with other genetic parts to test it

• After building part: test for function

 • 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
• p15A
• pSC101

• 4 MoClo level 0 fusion proteins:

• TetR_GFP
• TetR_YFP
• AraC_YFP
• AraC_GFP


• X MoClo level 0 tandem promoters:

• pTet_pBad
• pBad_pTet

These parts were cloned into a E. coli Bioline strain using our MoClo and transformation protocols. They were purified, sequenced, and tested using our FACS Workflow. These parts were then cloned into appropriate vectors and tested in our Fortessa flow cytometer. The TASBE Tools were then employed to characterize their expression.

Phase II - Build and characterize TU behavior.

Key software tools: TASBE Tools, Eugene, Raven

General Chimera Workflow

Case Study: BU Priority Encoder

• Use Eugene and Raven to generate all possible combinations of genetic parts and narrow down based on rules

• Create TUs and test using Flow Cytometry

• Analyze transfer curves and choose combinations based on resulting transfer curves and desired function

 • Run one-pot Multiplexing MoClo reaction. We initially multiplexed 5' UTIs and Terminators

• Eugene was employed to visualize all possible part substitutions.
• Raven was employed to optimize the assembly of these combinations.



• 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 every possible combination of the transcriptional units chosen from Phase 2 to test individual regulatory arcs using flow cytometry

• Use Raven and Eugene in this phase to allow for efficient design of the complex device and decide which building strategy would work best to test the relationship between two or more transcriptional units

• Testing several pairs of units with varying concentrations of small molecule induction is always a good idea so that a large range of data can be collected. This data will then be analyzed to gauge the efficacy of the device. 		• 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.







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