Difference between revisions of "Part:BBa K1152007"

Revision as of 01:39, 5 October 2013

Helper construct for NRP-Indigoidine-tagging

This part serves as helper plasmid for cloning custom NRPSs and thereby labeling the corresponding non-ribosomal peptides with indigoidine blue pigment. NRPS modules are introduced by Giboson cloning in a desired order, thereby replacing the ccdB suicide gene located in front of the indigoidine module. Note: as ccdB is replaced during cloning, only constructs bearing the desired custom NRPS are successfully propagated in convential E. coli cloning strains (e.g. Top10 or DH5alpha). Thereby, cloning background is reduced alsmost to 0 %. Since the blue pigment Indigoidine can be seen without auxillary means, analytical procedures can be faciliated a lot. Instead of complicated Mass spectrometry analysis, a simple thin-layer chromatography can be used for analysing the peptide produced by the corresponding NRPS.

To verify the genotype of our construct colony-PCRs, and restriction digests with Pst1 and EcoRI were conducted. Assembly overhang regions were sequenced as well.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 3061
Illegal BamHI site found at 891
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 341
Illegal SapI.rc site found at 4324

@@ Line 2: / Line 2: @@
 <partinfo>BBa_K1152007 short</partinfo>
-This part serves as helper plasmid for cloning custom NRPSs and thereby labeling the corresponding non-ribosomal peptides with indigoidine blue pigment. NRPS modules are introduced by Giboson cloning in a desired order, thereby  replacing the ccdB suicide gene located in front of the indigoidine module. Note: as ccdB is replaced during cloning, only constructs bearing the desired custom NRPS are successfully propagated in convential E. coli cloning strains (e.g. Top10 or DH5alpha). Thereby, cloning background is reduced alsmost to 0 %. Since the blue pigment Indigoidine can be seen without auxillary means, analytical procedures can be faciliated a lot. Instead of complicated MassSpec analysis, a simple thin-layer chromatography can be used for analysing the peptide produced by the corresponding NRPS.
+This part serves as helper plasmid for cloning custom NRPSs and thereby labeling the corresponding non-ribosomal peptides with indigoidine blue pigment. NRPS modules are introduced by Giboson cloning in a desired order, thereby  replacing the ccdB suicide gene located in front of the indigoidine module. Note: as ccdB is replaced during cloning, only constructs bearing the desired custom NRPS are successfully propagated in convential E. coli cloning strains (e.g. Top10 or DH5alpha). Thereby, cloning background is reduced alsmost to 0 %. Since the blue pigment Indigoidine can be seen without auxillary means, analytical procedures can be faciliated a lot. Instead of complicated Mass spectrometry analysis, a simple thin-layer chromatography can be used for analysing the peptide produced by the corresponding NRPS.
 To verify the genotype of our construct colony-PCRs, and restriction digests with Pst1 and EcoRI were conducted. Assembly overhang regions were sequenced as well.