Expression Cassette

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Work in progress - just some basic notes:

Basics

  • As short as possible (keep in mind that all cDNAs from Twist will have their 25nt barcodes on either end - this is an issue for assembly).
  • Multiple cloning site(s)?
  • Contains T7 promoter
  • Contains E. coli RBS - do we want three or four RBS strengths so people can try to dial in protein levels? (so as not to hog resources?)
  • Contains E. coli promoter - how about the Phage T5 flanked by lac operators? See: "A phage T5 derived promoter which is recognized by E.coli RNA polymerase. The promoter is controlled by two flanking lac operator sequences that allow induction by addition of IPTG. (www.wiley-vch.de/books/sample/3527327290_c01.pdf)"
  • Hexa-his or FLAG tag at end?
  • Standard E. coli stop codon?
  • Transcription terminator

Open Questions

Do we want to be able to turn ON/OFF the second E. coli based boot phase? If so, we would want the E. coli promoter to be small-molecule regulatable. Then we can do mass spec +/- small molecule, and see to which extent the "second" boot phase based on gradually accumulating E. coli machinery is relevant/changes things. Related to that - shall we have two different types of promoter system, one of then T7+E.coli and the other E. coli only, so we could actually follow the two boot phases?