Part:BBa_K1152006:Design
Valine-Indigoidine fusion - NRPS
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 4118
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 962
Illegal SapI.rc site found at 1158
Illegal SapI.rc site found at 5381
Design Notes
BBa_K1152006 was assembled via Gibson Cloning out of four fragments. The modules that were required for the NRPS were amplified from B. parabrevis and P. luminescens. There were no illegal restiction sites, neither in TycC4dC, nor in the C-domain of TycC2. However in indC, the gene that encodes for the Indigoidine synthetase, two illegal restriction sites (one for EcoRI, one for SpeI) had to be removed by mutagenesis. In order to make TycC4 an appropriate initiation module, the C-domain was not included in the costruct. In order to change the Indigoidine-module from an initiation module to a subsequent module, it has to be put behind a C-domain. We selected the C-domain of TycC4-module, as it is specific for Glutamine, which is the subtrate for Indigoidine.
Source
Coding regions were amplified from genomes of B. parabrevis that was sent to us by the lab of Prof. Marahiel from Marburg and P. luminescens that we bought from DSMZ.
References
1. Marahiel MA, Stachelhaus T, Mootz HD (1997) Modular Peptide Synthetases Involved in Nonribosomal Peptide Synthesis. Chem Rev 97:2651–2674.
2. Mootz HD, Schwarzer D, Marahiel MA (2000) Construction of hybrid peptide synthetases by module and domain fusions. Proc Natl Acad Sci USA 97: 5848–5853.
3. Mootz HD, Marahiel MA (1997) The tyrocidine biosynthesis operon of Bacillus brevis: complete nucleotide sequence and biochemical characterization of functional internal adenylation domains. JBacteriol 179: 6843–6850.
4. Finking R, Marahiel MA (2004) Biosynthesis of nonribosomal peptides. Annu Rev Microbiol 58: 453–488.