Difference between revisions of "Part:BBa K1152007"

Revision as of 01:47, 5 October 2013

Helper construct for NRP-Indigoidine-tagging

This part serves as helper plasmid for efficient cloning of custom non-ribosomal peptide synthetases (NRPSs) that enable labeling of the synthesized non-ribosomal peptides with the blue pigment Indigoidine.

NRPS modules can be introduced by Giboson cloning in any desired order. When inserted, they are 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 sequences are successfully propagated in E. coli strains such as Top10 or DH5alpha. Thereby, false-positive-rate of clones is reduced to almost to 0 %.
Since the blue pigment Indigoidine can be seen by the naked eye, analytical procedures can be facilitated. Blue colonies indicate synthesis of the synthetic peptide fused to Indigoidine. A thin-layer chromatography can be easily applied to confirm those observations.

To verify the sequence of our construct, colony PCRs, and analytical restriction digests with Pst1 and EcoRI were performed. Assembly overhang regions were correctly 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

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 <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 Mass spectrometry 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 efficient cloning of custom non-ribosomal peptide synthetases (NRPSs) that enable labeling of the synthesized non-ribosomal peptides with the blue pigment Indigoidine.
-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.
+NRPS modules can be introduced by Giboson cloning in any desired order. When inserted, they are replacing the ccdB suicide gene located in front of the indigoidine module.<br> '''Note''': as ccdB is replaced during cloning, only constructs bearing the desired custom NRPS sequences are successfully propagated in ''E. coli'' strains such as Top10 or DH5alpha. Thereby, false-positive-rate of clones is reduced to almost to 0 %.
+<br>Since the blue pigment Indigoidine can be seen by the naked eye, analytical procedures can be facilitated. Blue colonies indicate synthesis of the synthetic peptide fused to Indigoidine. A thin-layer chromatography can be easily applied to confirm those observations.
+<br><br>
+To verify the sequence of our construct, colony PCRs, and analytical restriction digests with Pst1 and EcoRI were performed. Assembly overhang regions were correctly sequenced as well.
 <!-- Add more about the biology of this part here