Part:BBa_K1152013:Experience

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Applications of BBa_K1152013

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).

Figure 2: OD measurement of indigoidine producing E. coli liquid cultures
We used a Tecan infinite M200 plate reader for OD measurements. 180 ul of LB media were inoculated with 20 ul of an E. coli TOP10 overnight culture expressing both indC (K1152013) and sfp (K1152009) and 20 ul of an untransformed E. coli TOP10 overnight culture. Absorbance scan was performed in intervals of 10 nm with 10 reads per well every 30 minutes.
a OD spectrum of an E. coli TOP10 liquid culture expressing indC and the PPTase sfp (K1152009). Each graph depicts the OD spectrum at a certain time ranging from 0 (dark blue) to 24 hours (light blue) after inoculation. The spectrum shows a local maximum absorption at 590 nm, indicating the production of the blue pigment indigoidine by sfp-activated indC.
b E. coli TOP10 negative control. The plot shows a regular E. coli absorption spectrum for comparison with Figure 2a. c This graph shows the OD590 - which depicts the maximal absorbance of indigoidine - at different levels of OD800 - which depicts the absorbance of the liquid culture without the influence of the indigoidine absorption. The red line indicates a standard curve of an untransformed E. coli liquid culture. The indigoidine producing liquid culture differs from that standard curve due to indigoidine production.
d Indigoidine absorption. Since the ratio OD590/OD800 equals a constant value delta over time in a native E. coli liquid culture (data not shown), we determined the OD590 of the pure indigoidine in liquid culture by substracting OD800*delta from the OD590 of the measurement. The graph illustrates the production of indigoidine in a time range of 24 hours starting from an OD590 (indigoidine) equal 0.

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. 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. 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 ODS = 590 nm: <math> OD_{S,+P} </math> [Indigoidine]= 〖〖OD〗_(S,+P)-OD〗_(S,-P) with 〖OD〗_(S,+P) being the overall OD measurement and 〖OD〗_(S,-P) being the scattering contribution of the cellular components at the sensitive OD. The scattering contribution of the cellular compenents at ODS (ODS,-P ) can be calculated from the scattering contribution measured at the robust wave length according to the following formula: [[File: The correction factor δ is be determined by measuring the OD of pure cellular culture without indigoidine at both the wavelength 〖OD〗_(S,-P) and 〖OD〗_R and calculating their ratio. Finally, the indigoidine production can be determined as File:Heidelberg 5.png For the calculation of the cellular component when measuring indigoidine producing liquid cell cultures, OD measurement at 800 nm as robust wavelength is recommended. By the approach described above, quantitative observation of the indigoidine production in a liquid culture over time as well as the indigoidine production in relation to the cell growth can be conducted. Background correction i. e. the contribution of the culture medium to the OD measurement is achieved by subtracting the mean of pure culture medium replicates from all OD values measured.

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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