Difference between revisions of "Part:BBa K1075026"

Latest revision as of 01:29, 5 October 2013

AraC-pBad-RBS34-mCherry-(Ec)ssrA(DAS+4)-TT

The part contains the AraC-pBad promoter system, a ribosomal binding site, the red fluorescent protein mCherry fused to the E. coli(ec)ssrA (DAS+4) tag and a double terminator.

biology

The (ec)ssrA tag is a short peptide sequence that can be fused to the C-terminus of proteins which should be degraded. The adaptor protein SspB binds to it and delivers the substrates to the protease ClpXP complex in E. coli. The mutation (DAS+4) in (ec)ssrA prevents the degradation of the ssrA-tagged protein without the presence of sspB and therefore makes the system more specific and controllable. [http://www.ncbi.nlm.nih.gov/pubmed/16762842]

mCherry is a red fluorescent protein with the excitation maximum at 587 nm and the Emission maximum at 610 nm. [http://www.ncbi.nlm.nih.gov/pubmed/15558047]

application

As we want to control protein degradation by controlling the function of ecSspB, we tagged the red fluorescent protein mCherry with ecssrA(DAS+4) to measure the degradation rate.

The part was designed for proof of principle. Application as a bacterial fotographic film might be possible as well.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 2726
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 1144
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 979
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI site found at 961

@@ Line 3: / Line 3: @@
+The part contains the AraC-pBad promoter system, a ribosomal binding site, the red fluorescent protein mCherry fused to the E. coli(ec)ssrA (DAS+4) tag and a double terminator.
-<!-- Add more about the biology of this part here-->
-===construction===
-The part contains the promoter plac, a ribosomal binding site, the red fluorescent protein mCherry fused to the E.coli(ec)ssrA(DAS+4) tag and a double terminator.
 ===biology===
-AraC-pbad is an arabinose inducible regulatory promoter/repressor unit.
+The (ec)ssrA tag is a short peptide sequence that can be fused to the C-terminus of proteins which should be degraded. The adaptor protein SspB binds to it and delivers the substrates to the protease ClpXP complex in E. coli. The mutation (DAS+4) in (ec)ssrA prevents the degradation of the ssrA-tagged protein without the presence of sspB and therefore makes the system more specific and controllable. [http://www.ncbi.nlm.nih.gov/pubmed/16762842]
-The ribosomal binding side is a sequence of mRNA where the ribosome binds to start translation.
-The ecssrA tag is a short peptide sequence, which is fused to the C-terminus of proteins, which should be degraded. It relates to the protease ClpXP complex in E.coli and it also allows the adaptor protein ecSspB binding and delivering substrates to the proteases in order to make the process more efficient. The mutated ecssrA(DAS+4) weakens a direct binding between proteases and ecssrA(DAS+4) and increases the dependance of ecSspB. [http://www.ncbi.nlm.nih.gov/pubmed/16762842]
 mCherry is a red fluorescent protein with the excitation maximum at 587 nm and the Emission maximum  at 610 nm. [http://www.ncbi.nlm.nih.gov/pubmed/15558047]
-The double terminator stops the transcription at this point.
+===application===
+As we want to control protein degradation by controlling the function of ecSspB, we tagged the red fluorescent protein mCherry with ecssrA(DAS+4) to measure the degradation rate.
-===application===
-As we want to control protein degradation by controlling the function of ecSspB, we tag the red fluorescent protein mCherry with ecssrA(DAS+4) to measure the degradation rate.
-Application as a bacterial fotographic film might be possible as well.
+The part was designed for proof of principle. Application as a bacterial fotographic film might be possible as well.