Endy:Screening plasmid/Inverter characterization: Difference between revisions
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==Already measured with SP1.0 (I13534.pSB1A3)== | ==Already measured with SP1.0 (I13534.pSB1A3)== | ||
*Q04400.007 - mutated Q04400 (tetR QPI), point mutation in RBS. Works well, switches later than Q04400. | *[http://parts.mit.edu/registry/index.php/Part:BBa_Q04401 Q04400.007] - mutated Q04400 (tetR QPI), point mutation in RBS. Works well, switches later than Q04400. | ||
[[Image:tetR QPI.png]] | [[Image:tetR QPI.png]] | ||
*Mnt inverters from enterobacteriophage p22 <cite>Knight Stormo</cite> | *Mnt inverters from enterobacteriophage p22 <cite>Knight Stormo</cite> | ||
**Note: The [http://parts.mit.edu/registry/index.php/Part:BBa_R0073 promoter] used in these inverters is missing base 13 in its 17-bp operator site <cite>Stormo</cite>, 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. | **Note: The [http://parts.mit.edu/registry/index.php/Part:BBa_R0073 promoter] used in these inverters is missing base 13 in its 17-bp operator site <cite>Stormo</cite>, 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 ([http://openwetware.org/wiki/Image:June29_2006_Q04720_MOFLO.png picture]) | **[http://parts.mit.edu/registry/index.php/Part:BBa_Q04720 Q04720] - mnt inverter, strong repressor - output not very high, and doesn't appear to be doing much ([http://openwetware.org/wiki/Image:June29_2006_Q04720_MOFLO.png picture]) | ||
**Q04730 - mnt inverter, weak repressor - output not very high, and doesn't appear to be doing much ([http://openwetware.org/wiki/Image:June29_2006_Q04730mut_MOFLO.png picture]) | **[http://parts.mit.edu/registry/index.php/Part:BBa_Q04730 Q04730] - mnt inverter, weak repressor - output not very high, and doesn't appear to be doing much ([http://openwetware.org/wiki/Image:June29_2006_Q04730mut_MOFLO.png picture]) | ||
==Measured with older versions of screening plasmid (I13537.pSB1A3, I13538.pSB1A3, or I13534.pSB4A3)== | ==Measured with older versions of screening plasmid (I13537.pSB1A3, I13538.pSB1A3, or I13534.pSB4A3)== |
Revision as of 09:27, 12 July 2006
Already measured with SP1.0 (I13534.pSB1A3)
- Q04400.007 - mutated Q04400 (tetR QPI), point mutation in RBS. Works well, switches later than Q04400.
- 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.pSB1A3, I13538.pSB1A3, or I13534.pSB4A3)
- Q04400 - tetR inverter [3, 4], works reliably (quantitatively), switches at low input.
- Q03400 - tetR inverter, weaker RBS driving tetR. Looks like it starts to switch, but very high GFP gives very high RFP numbers. Unclear what's happening.
- Q01400 - tetR inverter, weakest RBS. Generally looks like it's stuck in the high-output stage. Some evidence suggests it might be switching, however.
- 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.
- Q04740 - penI inverter [5, 6] with strongest RBS. Works well, switches at higher input than Q04400.
- Q02740 - penI inverter, strong RBS. Looks like it begins to switch at high input.
- Q03740 - penI inverter, medium strength RBS. Stuck on.
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 - 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
- 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.
- 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 |
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Elowitz, M. B. Transport, Assembly, and Dynamics in Systems of Interacting Proteins. Thesis, Princeton Univ., Princeton (1999)
- 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. DOI:10.1093/nar/25.6.1203 |
- 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. DOI:10.1128/jb.168.3.1128-1132.1986 |