Registry/Measurement kit/Notebook/Design

Design considerations for the expression measurement kit

Promoter

• Constituitive?
• Relative strength
• Location of transcription initiation (outside of or within the promoter?)
• Previous work

Ribosome Binding Site

• Relative Strength
• Previous work (<bbpart>B0032</bbpart>)

Fluorescent Protein

• Color?
• previous work (<bbpart>E0040</bbpart>)
• Tagged?

Terminator

• Do we need one?
• Which one?
• Previous work (<bbpart>B0015</bbpart>)

Preliminary Proposed Kit Designs

Promoter tester with GFP

• E0240 (pSB1A2)

Promoter tester with RFP

• B0032 (pSB1A2) + J04650 (pSB1AK3)

RBS tester with GFP

• R0040 (pSB1A2) + I13401 (pSB1A2)

RBS tester with RFP

• R0040 (pSB1A2) + J04650 (pSB1AK3)

Internal labels

• 1 - <bbpart>E0240</bbpart>
• 2 - <bbpart>R0040</bbpart>
• 3 - <bbpart>I13401</bbpart>
• 4 - <bbpart>J04650</bbpart>
• 5 - <bbpart>B0032</bbpart>

Suggestions from peers

• Using LacZ with a Miller Assay to measure expression instead of an FP
• 3-series plasmid as the final vector (with flanking terminators)
• Ensure appropriate spacing between RBS and coding region

Workaround for the RBS spacing problem

• In the RBS tester plasmid, use a standard biobrick site minus the PstI restriction site. [...Promoter - EcoRI - XbaI - SpeI - Reporter gene...]
• To insert a RBS, the user would cut both the kit plasmid and the RBS BioBrick plasmid at SpeI and either XbaI or EcoRI and then perform the necessary ligation.
• To retrieve the RBS from the kit and standardize it, one would cut it at EcoRI and SpeI, and insert it into a standard BioBrick plasmid cut at EcoR1 and Spe1.