Part:BBa_K1152016:Experience

Applications of BBa_K1152016

Figure 1: ccdB CPEC assembly strategy for exxchanging T-domains in indC

We used this device to exchange T-domains of indC using a CPEC approach. To minimize the background colonies when exchanging the T-domain of the indigoidine synthetase we generated the ccdB-Ind plasmid where we replaced the indC T-domain with the ccdB gene (Modul structure: AoxA-ccdB-TE) which kills E. coli TOP10 cells but not E. coli OneShot ccdB survival cells (see part K1152014. Test-transformation in both E. coli TOP10 and the E. coli OneShot ccdB survival cells showed that background colonies could be eliminated by this strategy (Plattenbild top10 vs survival cells). We used the Ind-ccdB for all further CPEC experiments aiming to swap T-domains (see parts K1152015, K1152016, K1152017, K1152018, K1152019).

Moreover, indC can be used to label nonribosomal peptides (NRP) in the creation of novel NRPS pathways. This enables the user to create NRPS libraries in a high throughput, since the validation of NRP production is simple due to the indigoidine tag. We successfully created a fusion NRPS together with a NRPS module of the tyrocidine pathway from B. parabrevis, producing an indigoidine-tagged valine (see part K1152006).

Detecting the amount of the NRP expressed by the bacterial host strain is desirable. 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. As the amount of blue pigment is proportional to the amount of the NRP of interest, a method for the quantification of the blue pigment will yield information about the expression of the NRP. Quantification of the pure indigoidine pigment can be easily achieved by optical density (OD) measurements at its maximum wavelength of about 590 nm.

Figure 3: 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 3, 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 3, 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).

References

1. Myers JA, Curtis BS, Curtis WR (2013) Improving accuracy of cell and chromophore concentration measurements using optical density. Bmc Biophysics 6:

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