Difference between revisions of "Part:BBa K1088017"
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To test if overexpression of AraC is necessary for expression control of the arabinose promoter, devices with and without this regulatory device was assayed. Duplicates of MG1655 strains carrying either pSB1C3-Para-HRT2 (BBa_K1088024; -araC) or pSB1C3-Pcon-araC-term-Para-HRT2 (BBa_K1088016; +araC) were grown to late-exponential phase: OD<sub>600</sub>=0.8. At this OD the strains were induced with 0.2 % arabinose at time t=0 min, and samples were taken at times: -2 min, 15 min, and 30 min. Total RNA purified from the samples were run on a gel, blotted onto a membrane, and hybridized with probes specific for HRT2 mRNA and 5S rRNA (loading control), respectively. | To test if overexpression of AraC is necessary for expression control of the arabinose promoter, devices with and without this regulatory device was assayed. Duplicates of MG1655 strains carrying either pSB1C3-Para-HRT2 (BBa_K1088024; -araC) or pSB1C3-Pcon-araC-term-Para-HRT2 (BBa_K1088016; +araC) were grown to late-exponential phase: OD<sub>600</sub>=0.8. At this OD the strains were induced with 0.2 % arabinose at time t=0 min, and samples were taken at times: -2 min, 15 min, and 30 min. Total RNA purified from the samples were run on a gel, blotted onto a membrane, and hybridized with probes specific for HRT2 mRNA and 5S rRNA (loading control), respectively. | ||
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Revision as of 19:08, 3 October 2013
AraC device (constitutive promoter, RBS and terminator)
From ECOCYC: The "arabinose regulator," AraC, is a transcription factor that regulates transcription of several genes and operons involved in arabinose catabolism and transport. It coregulates with another transcriptional regulator, CRP; both are transcription factors involved in l-arabinose degradation. These regulators bind cooperatively to activate transcription of five operons related to transport, catabolism, and autoregulation of l-arabinose. Transcription of these operons is induced when E. coli is grown in the absence of glucose and when the physiological inducer, l-arabinose, binds to the AraC regulator. In the absence of glucose, cellular cyclic AMP levels are high and cyclic AMP forms a dimeric complex with CRP to coregulate with AraC.
The gene is expressed from a constitutivly active promoter, has a strong RBS and is followed by a terminator.
To test if overexpression of AraC is necessary for expression control of the arabinose promoter, devices with and without this regulatory device was assayed. Duplicates of MG1655 strains carrying either pSB1C3-Para-HRT2 (BBa_K1088024; -araC) or pSB1C3-Pcon-araC-term-Para-HRT2 (BBa_K1088016; +araC) were grown to late-exponential phase: OD600=0.8. At this OD the strains were induced with 0.2 % arabinose at time t=0 min, and samples were taken at times: -2 min, 15 min, and 30 min. Total RNA purified from the samples were run on a gel, blotted onto a membrane, and hybridized with probes specific for HRT2 mRNA and 5S rRNA (loading control), respectively.
<img src="
" width="300">
A) Normalized intensity of HRT2 mRNA using intensity of 5S rRNA as reference. The normalized intensity of sample -araC -2min were set to 1 and the other samples are relative to that. Within 15 min of induction the expression levels are at its maximum in both strains, and overexpression of AraC does not seem to be necessary for expression control of the arabinose promoter.
B) Northern blot result reflecting diagram.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]