Difference between revisions of "Part:BBa K1152007"

Revision as of 00:28, 29 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/Project non-ribosomal peptide synthetases] (NRPSs) that enable labeling of the synthesized non-ribosomal peptides with the blue pigment indigoidine in-vivo. Peptides labelled this way can easily be purified and characterized by thin-layer chromatography, since peptide production can be seen with the naked eye or quantified photometrically. Therefore, the helper-construct can be applied for high-throughput cloning of custom NRPSs for production of synthetic peptides ( [http://2013.igem.org/Team:Heidelberg/RFCs RFC 100] for details).

NRPS modules can be introduced by Giboson ([http://www.synbio.org.uk/gibson/downloads/files/RFC57.pdf BBF RFC 57]) or CPEC cloning ([http://hdl.handle.net/1721.1/81332 BBF RFC 99]) in any desired order. When inserted, they are replacing the ccdB suicide gene located in front of the indigoidine module. The ccdB gene acts as a negative selection marker (death cassette) and is lost in recombined (with insert) form. During cloning the gene is replaced with an cloning efficiency close to 100 %.

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 (IPTG) by the naked eye, analytical procedures can be facilitated. Blue colonies indicate synthesis of the synthetic peptide fused to Indigoidine. A thin-layer chromatography (TLC) 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 usually performed.

We characterized this construct by cloning different NRPS module compositions into this helper construct, leading to the production of different peptides bearing the indigoidine tag. Please refer to the experience page for charaterization of this part. Also refer to [http://hdl.handle.net/1721.1/81333 BBF RFC 100] for further details on how to engineer synthetic NRPSs.

Sequence and Features