Coding

Part:BBa_K1152009:Experience

Designed by: Ralf Beer, Konrad Herbst, Nikolaos Ignatiadis   Group: iGEM13_Heidelberg   (2013-09-19)
Revision as of 23:15, 5 October 2013 by Herbstk (Talk | contribs)


For validation and characterization of sfp as functional PPtase we used the indigoidine producing NRPS indC from P. luminiscense. This one module NRPS is able to convert glutamine into the bright blue pigment indigoidine. We used this part in a pSB3K3-derived plasmid with a lac promoter (BBa_R0010) and the RBS BBa_B0029 for coexpression with pSB1C3 derived plasmids with following engineered indC deivatives:

indC derivative T domain
BBa_K1152015 T8
BBa_K1152016 T10
BBa_K1152017 T12
BBa_K1152018 T13
BBa_K1152019 T14

With indC as measuring device it is possible to determine effiency of phosphopantetheinyl transferase reaction of sfp. The following figures show indigoidine production derived from OD measurements. Because indigoidine has its absorbance maxima around 600 nm we had to take an additional OD value into account. To summarize measurements at OD800 and OD590 are used for calculation of indigoidine production. Detailed methodology is described at the iGEM 2013 Heidelberg project website ([http://2013.igem.org/Team:Heidelberg/Indigoidine|Heidelberg 2013 - Indigoidine]).

  • Heidelberg2013 spectra pRB23-T8+pRB15.png

    File 1: Absorbtion spectra measurements over time for an indC construct (pRB23_T8) which does not produce indigoidine

  • Heidelberg2013 spectra pRB23-T12+pRB15.png

    File 2: Absorbtion spectra measurements over time for an indC construct (pRB23_T12) which does produce indigoidine

  • Heidelberg2013 ind pRB23-T8+pRB15.png

    File 3: Derived indigoidine production for an indC construct with T8 domain

  • Heidelberg2013 ind pRB23-T12+pRB15.png

    File 4: Derived indigoidine production for an indC construct with T12 domain

Applications of BBa_K1152009

This part can be used in order to manufacture NRPS activating constructs. (Examples can be seen at our project site: [http://2013.igem.org/Team:Heidelberg/Indigoidine|Heidelberg 2013 - Indigoidine])

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