Part:BBa_M36698:Design
Pyruvate Sensor v2
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 679
Illegal BamHI site found at 1196 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
We chose to use a medium level 5' UTR meaning the ribosome binds the DNA roughly 50% of the time because IclR inhibits the aceBAK promoter without pyruvate so having extremely high levels of IclR would mean that regardless of the pyruvate concentrations, the aceBAK promoter will always be inhibited. With IclR at a medium level in the cell, the presence of pyruvate should stabilize the ability of IclR to bind the DNA and inhibit the ribosome binding. The terminator following the IclR gene ensures that the test actuator, has an independent 5' UTR site. The aceBAK promoter regulates the expression of test actuator gene.
We chose to use the aceBAK promoter and IclR because of the relationship of pyruvate to IclR and the aceBAK promoter. The latter half of the experiment is the relationship between pressure and the expression of the test actuator downstream of the aceBAK promoter. Lactate Dehydrogenase, LDH, converts lactate into pyruvate under aerobic, non-acidic conditions. Pressure inhibits the activity of LDH so less pyruvate was present in the cell so the aceBAK promoter is less inhibited so there are higher levels of the test actuator downstream of the aceBAK promoter in the cell.
The reason we had to alter the part from M36708 was because of a missing part of the promoter. In the M36707 version, the -10 region of the promoter was missing, as well as a sequence of what we believe is spacing DNA. When using multiple sources to figure out the sequence we needed, there was a mis-numbering in which the full promoter was included on the one website (biocyc.org) while the numbering on the website we actually copied the sequence from, (genome.jp) had a slightly different numbering system. Without the -10 region of the promoter, and the spacer following it, not only would the polymerase not bind, but without the geographic spacing between the binding site and the start codon, there may not be adequate space for the gene downstream to be synthesized. Thus we concluded that without the -10 region of the promoter, the polymerase will never bind and thus the downstream gene will never made even in the absence of IclR and pyruvate. As for without the spacer, even if the polymerase can bind the DNA, the downstream gene still will not be made because the ribosome does not have adequate space to bind and find the start codon.
Source
See BBa_M36708
References
Information regarding the nature of IclR, pyruvate, and the aceBAK promoter were found using these articles:
http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0002042
http://www.jbc.org/content/282/22/16476.long
http://www.jbc.org/content/282/22/16476/F4.expansion.html
http://onlinelibrary.wiley.com/doi/10.1111/j.1574-6968.1998.tb12855.x/full
Information about the IclR binding sites on the aceBAK promoter, and the aceBAK promoter itself was found using biocyc.org, specifically found on this page:
http://biocyc.org/ECOLI/NEW-IMAGE?type=NIL&object=TU00001&redirect=T
The sequence of the IclR binding sites on the aceBAK promoter and the IclR gene was found using genome.jp
aceBAK promoter: http://www.genome.jp/kegg-bin/cut_sequence_genes.pl?FROM=5092392&TO=5094223&VECTOR=1&ORG=ece
IclR gene(highlighted in blue): http://www.genome.jp/kegg-bin/cut_sequence_genes.pl?FROM=5092368&TO=5094223&VECTOR=1&ORG=ece&CHR=c