Coding

Part:BBa_K1152012:Experience

Designed by: Ralf Beer, Konrad Herbst, Nikolaos Ignatiadis   Group: iGEM13_Heidelberg   (2013-09-20)


For validation and characterization of delC as functional PPtase we used the indigoidine producing NRPS indC from P. luminescence. This one module NRPS is able to convert L-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

Detecting the amount of the NRP expressed by the bacterial host strain is desirable e.g. in order to determine the efficiency of phosphopantetheinyl transferase reaction of delC. By tagging the NRP with indigoidine, the amount of the fusion peptide can be determined by quantifying the amount of blue pigment present in the cells. Quantification of the pure indigoidine pigment can be easily achieved by optical density (OD) measurements at its maximum wavelength of about 590 nm.

Figure 1: applied from Myers 2012

In cellular culture, indigoidine quantification by OD measurements is impaired. Cellular density of liquid cultures is standardly measured as the optical density (OD) at a wave length of 600 nm, i. e. the absorption peak of indigoidine interferes with the measurement of cell density at the preferred wave length (compare to Figure 1, grey dashed line). Thus, for measurement of NRP expression without time consuming a priori purification of the tagged-protein, a method to separate the cellular and pigment-derived contributions to the OD is required (compare to Figure 1, brown and blue lines, respectively). The method of choice, as described by Myers et al.[2013], requires the OD measurement of cell culture at two distinct wavelengths: the robust wave length ODR and the sensitive wave length ODS. The concentration of indigoidine will have to be deducted from measurements at OD800 (ODR) and OD590 (ODS).

Detailed methodology is described at the iGEM 2013 Heidelberg project website ([http://2013.igem.org/Team:Heidelberg/Project/Indigoidine Heidelberg 2013 - Indigoidine]).

Figure 2: Absorbtion spectra measurements over time for two indC constructs (left: pRB23_T10; right: pRB23_T12) with various PPtase helper constructs.

Applications of BBa_K1152012

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/Project/Indigoidine Heidelberg 2013 - Indigoidine])

References

  • Myers, J. a, Curtis, B. S., & Curtis, W. R. (2013). Improving accuracy of cell and chromophore concentration measurements using optical density. BMC Biophysics, 6(1), 4. doi:10.1186/2046-1682-6-4

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