Difference between revisions of "Part:BBa K1152007"
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This part serves as helper plasmid for efficient cloning of custom [http://2013.igem.org/Team:Heidelberg non-ribosomal peptide synthetases] (NRPSs) that enable labeling of the synthesized non-ribosomal peptides with the blue pigment Indigoidine. | This part serves as helper plasmid for efficient cloning of custom [http://2013.igem.org/Team:Heidelberg 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.<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 %. | 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>Since the blue pigment Indigoidine can be seen after induction with Isopropyl-β-D-thiogalactopyranosid 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> | <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. | 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. | ||
Revision as of 01:56, 5 October 2013
Helper construct for NRP-Indigoidine-tagging
This part serves as helper plasmid for efficient cloning of custom [http://2013.igem.org/Team:Heidelberg 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 after induction with Isopropyl-β-D-thiogalactopyranosid 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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 3061
Illegal BamHI site found at 891 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 341
Illegal SapI.rc site found at 4324