Difference between revisions of "Part:BBa K1088017"

Latest revision as of 12:14, 28 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." For more info go to [http://ecocyc.org/ECOLI/NEW-IMAGE?type=GENE&object=EG10054 Ecocyc's page]

The gene is expressed from a constitutivly active promoter, has a strong RBS and is followed by an efficient terminator to prevent transcription of downstream elements.

To test if overexpression of AraC improved expression control of the arabinose promoter, devices with and without this regulatory device were assayed. Duplicates of MG1655 strains carrying either pSB1C3-P.ara-HRT2 (BBa_K1088024; -araC) or pSB1C3-P.con-araC-term-P.ara-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 analyzed by northern blot. First samples were run on a gel, blotted onto a membrane, and hybridized with radioactive probes specific for HRT2 mRNA and 5S rRNA (loading control), respectively.

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 this. Within 15 min of induction the expression levels were 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.

We haven't tested if the lack of improvement is due to a fault in the design so that no AraC is expressed. Instead we have tested devices of similar design which we proved to be functional (BBa_K1088019 and BBa_K1088020).

Sequence and Features

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
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

@@ Line 2: / Line 2: @@
 <partinfo>BBa_K1088017 short</partinfo>
-From ECOCYC:
+'''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 "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."
+For more info go to [http://ecocyc.org/ECOLI/NEW-IMAGE?type=GENE&object=EG10054 Ecocyc's page]
-The gene is expressed from a constitutivly active promoter, has a strong RBS and is followed by a terminator.
+'''The gene is expressed''' from a constitutivly active promoter, has a strong RBS and is followed by an efficient terminator to prevent transcription of downstream elements.
-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 improved expression control of the arabinose promoter, devices with and without this regulatory device were assayed. Duplicates of MG1655 strains carrying either pSB1C3-P.ara-HRT2 ([https://parts.igem.org/Part:BBa_K1088024 BBa_K1088024]; -araC) or pSB1C3-P.con-araC-term-P.ara-HRT2 ([https://parts.igem.org/Part:BBa_K1088016 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 analyzed by northern blot. First samples were run on a gel, blotted onto a membrane, and hybridized with radioactive probes specific for HRT2 mRNA and 5S rRNA (loading control), respectively.
-<nowiki>
+https://static.igem.org/mediawiki/2013/e/ee/SDU2013_Part_BBa_K1088017.png
-<img src="https://static.igem.org/mediawiki/2013/e/ee/SDU2013_Part_BBa_K1088017.png" width="300">
-</nowiki>
+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 this. Within 15 min of induction the expression levels were 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.
+We haven't tested if the lack of improvement is due to a fault in the design so that no AraC is expressed. Instead we have tested devices of similar design which we proved to be functional ([https://parts.igem.org/Part:BBa_K1088019 BBa_K1088019] and [https://parts.igem.org/Part:BBa_K1088020 BBa_K1088020]).
-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.
 <!-- Add more about the biology of this part here