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 ( B0032 )

Fluorescent Protein

 * Color?
 * previous work ( E0040 )
 * Tagged?

Terminator

 * Do we need one?
 * Which one?
 * Previous work ( B0015 )

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 - E0240
 * 2 - R0040
 * 3 - I13401
 * 4 - J04650
 * 5 - B0032

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.