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.

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.
      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. Identification of functionally important residues in the DNA binding region of the mnt repressor. J Biol Chem. 1989 Aug 15;264(23):13706-10. PubMed ID:2668272 | HubMed [Knight]
  2. Stormo GD, Strobl S, Yoshioka M, and Lee JS. Specificity of the Mnt protein. Independent effects of mutations at different positions in the operator. J Mol Biol. 1993 Feb 20;229(4):821-6. DOI:10.1006/jmbi.1993.1088 | PubMed ID:8445649 | 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. Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements. Nucleic Acids Res. 1997 Mar 15;25(6):1203-10. PubMed ID:9092630 | HubMed [Lutz]
  5. Himeno T, Imanaka T, and Aiba S. Nucleotide sequence of the penicillinase repressor gene penI of Bacillus licheniformis and regulation of penP and penI by the repressor. J Bacteriol. 1986 Dec;168(3):1128-32. PubMed ID:3096969 | HubMed [Himeno]
  6. corrections to Himeno et al., J. Bact. 168:1128 (1986) [Himeno_errata]
All Medline abstracts: PubMed | HubMed