Difference between revisions of "Part:BBa K1075026"

Revision as of 19:26, 3 October 2013

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

a

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 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 proteins 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 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.

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 7: / Line 7: @@
 ===construction===
-The part contains the promoter plac, a ribosomal binding site, the red fluorescent protein mCherry fused to the ssrA (DAS+4) tag and a double terminator.
+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.
@@ Line 15: / Line 15: @@
 The ribosomal binding side is a sequence of mRNA where the ribosome binds to start translation.
-The ssrA 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 proteins SspB binding and delivering substrates to the proteases in order to make the process more efficient. The mutated ssrA (DAS+4) weakens a direct binding between proteases and ssrA and increases the dependance of sspB. [http://www.ncbi.nlm.nih.gov/pubmed/16762842]
+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 proteins 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]
@@ Line 23: / Line 23: @@
 ===application===
-As we want to control protein degradation by controlling the function of SspB, we tag the red fluorescent protein mCherry with ssrA (DAS+4) to measure the degradation rate.
+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.