Deleted
Sample Discontinued
Experience: None
Not Used
Get This Part
Tag
ccdB_Ind

Part:BBa_K1152007

Designed by: Joshua Sachs   Group: iGEM13_Heidelberg   (2013-09-20)
Revision as of 23:01, 28 October 2013 by Philipp.walch (Talk | contribs)

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. 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.

Heidelberg Registry 2007 main fig1.png


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


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


[edit]
Categories
//proteindomain
Parameters
n/aHelper construct for NRP-Indigoidine-tagging