Endy:Screening plasmid/Inverter characterization

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Already measured with SP1.0 (I13534.pSB1A2)

  • Q04400.007 - mutated Q04400 (tetR QPI), point mutation in RBS. Works well, switches later than Q04400.

Image:tetR QPI.png

  • mnt inverters from enterobacteriophage p22 [1, 2]
    • Note: The promoter used in these inverters is missing base 13 in its 17-bp operator site [2], though I think they meant to include it (they reference the article and mention it in the registry). It's also missing a -35 site.
    • Q04720 - mnt inverter, strong repressor - output not very high, and doesn't appear to be doing much (picture)
    • Q04730 - mnt inverter, weak repressor - output not very high, and doesn't appear to be doing much (picture)

Measured with older versions of screening plasmid (I13537.pSB1A2, I13538.pSB1A2, or I13534.pSB4A3)

  • tetR inverters [3, 4]
    • Q04400 - works reliably (quantitatively), switches at low input.
    • Q03400 - weaker RBS driving tetR. Looks like it starts to switch, but very high GFP gives very high RFP numbers. Unclear what's happening.
    • Q01400 - weakest RBS. Generally looks like it's stuck in the high-output stage. Some evidence suggests it might be switching, however.
  • p22 cII inverters
    • Q01530 - p22 cII inverter. Appears to be stuck in the low-output stage, perhaps due to crosstalk with CW2553/lambda.
    • Q04530 - Same as Q01530, but with a stronger RBS driving the cII. Also stuck in low-output.
  • penI inverters [5, 6] from bacillus licheniformis
    • Q02740 - strong RBS, looks like it begins to switch at high input.
    • Q03740 - medium strength RBS, stuck on.
    • Q04740 - strongest RBS, works well, switches at higher input than Q04400.
      Image:PenI_QPI.png

To be measured/constructed/etc

  • I14103 (Q04740-Q04400.007) - bistable switch one way
  • I14104 (Q04400.007-Q04740) - bistable switch the other way - having trouble getting it cloned in this direction (tetR-penI) - weird stuff happening with growth rate and trying to prep the final plasmid, though colony PCR looks fine - load issues?
  • Q04510 - lambda cI inverter, ligated into the screening plasmid, but somehow missed being characterized. Seems unlikely to work, but hasn't been tested.
  • Q07400/Q08400/Q09400 - tetR inverters using RBS's from the Voigt lab. Not BB'd, and previous attempts were unsuccessful.

References

  1. Knight KL and Sauer RT. . pmid:2668272. PubMed HubMed [Knight]
  2. Stormo GD, Strobl S, Yoshioka M, and Lee JS. . pmid:8445649. PubMed HubMed [Stormo]
  3. Elowitz, M. B. Transport, Assembly, and Dynamics in Systems of Interacting Proteins. Thesis, Princeton Univ., Princeton (1999)

    [Elowitz]

  4. Lutz R and Bujard H. . pmid:9092630. PubMed HubMed [Lutz]
  5. Himeno T, Imanaka T, and Aiba S. . pmid:3096969. PubMed HubMed [Himeno]
  6. corrections to Himeno et al., J. Bact. 168:1128 (1986)

    [Himeno_errata]

All Medline abstracts: PubMed HubMed
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