Difference between revisions of "Part:BBa M36708:Design"

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===Design Notes===
 
===Design Notes===
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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.   
+
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.
 
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.
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Information regarding the nature of IclR, pyruvate, and the aceBAK promoter were found using these articles:
 
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.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.long
 +
 
http://www.jbc.org/content/282/22/16476/F4.expansion.html
 
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
 
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
+
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
 
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
 
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
 
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

Latest revision as of 21:44, 5 May 2011

Pyruvate Sensor


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 679
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE 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.

Source

Katie Lund and Wyatt Woodson parts obtained from Stanford BIOE44 - S11

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