Design considerations for the expression measurement kit
- Relative strength
- Location of transcription initiation (outside of or within the promoter?)
- Previous work
Ribosome Binding Site
- Relative Strength
- Previous work (<bbpart>B0032</bbpart>)
- previous work (<bbpart>E0040</bbpart>)
- 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)
- 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.