Part:BBa_K1152004
Expression cassette for NRPS that synthesizes a Pro-Leu-Dipeptide
BBa_K1152004 is a Non-Ribosomal Peptide Synthetase that synthesizes a dipeptide consisting of Proline and Leucine. It was created by Module Shuffling within the Tyrocidine-cluster from B. parabrevis.
Validation and Biology
pIK03 was amplified out of three fragments via Gibson Cloning: Modules TycB1dComdC and TycC6 from B. parabrevis, as well as a backbone. We verified the genotype by restriction digest with various restriction enzymes, among them EcoRI and PstI, and sequencing of the assembly overhang regions. Furthermore, SDS-PAGE was performed after induction of cells in order to prove expression – the expected band at 212 kDa was clearly visible after Coomassie staining. Finally, Mass-Spec analysis was performed to prove the functionality of the expressed NRPS.
(Figures 1, 2 and 3: Verification of the genotype, the expression and the functionality of BBa_K1152004)
In restriction digest, as one can see in the first figure, the cutting pattern was as expected. For restriction digest with EcoRI alone, one can nicely see that only linearized vector at the size of 8200 bp is present. If the digest ist conducted with EcoRI and PstI, as expected two bands show up, one at around 2000 bp (backbone) and one at around 6200 bp (insert).
In the SDS-PAGE, expression of the NRPS could be verified, as the expected band at 212kDa is clearly visible, 12 hours after induction with IPTG (see figure 2). In order to show functionality of BBa_K1152004, we performed ornithine screening and Mass-Spectrometry.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 2138
Illegal SapI.rc site found at 3255
Illegal SapI.rc site found at 3492
| n/a | Expression cassette for NRPS that synthesizes a Pro-Leu-Dipeptide |