Registry/Measurement kit/Notebook/Design

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Design considerations for the expression measurement kit

Contents

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.
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