Team:Manchester-Graz/Project/Results
Results
Validation of pCERI
via SDS-Page
To show how E. coli BL21 and E. coli Nissle 1917 deal with our synthetic vector pCERI we started a fermentation in 300 ml cultures. For each E. coli strain seven shaking flasks were inoculated with an ONC of E. coli BL21 and E. coli Nissle 1917, both transformed with pCERI, to start the fermentation with an OD600 of 0.05. In addition, negative (wild type E. coli BL21 and E. coli Nissle 1917) and positive controls were set up. The positive controls comprise recombinant E. coli BL 21 pSB3C5_J04421 and pSB3C5_J04450. The transformed plasmids contain a lac promoter, leading to the expression of mRFP and CFP after the induction with IPTG. All samples were cultivated under the same conditions (37°C, 100 rpm). Every hour one fermentation of E. coli BL21_pCERI and E. coli Nissle 1917_pCERI was stopped on ice. One culture was fermented over night to reach 16 h. The positive, as well as the negative controls were stopped after 6h. After fermentation, all samples were centrifuged and prepared for SDS analysis (LINK). Regarding to protein concentrations measured by spectrophotometry (Nanodrop™) we loaded 10µg of soluble protein from the E. coli BL21_pCERI samples (1h-6h) as well as from the negative and positve controls on a SDS gel (Fig 1). For the E. coli Nissle 1917_pCERI and the E. coli Nissle 1917 negative control 20 µg of soluble protein were used for SDS-PAGE (Fig 2).
The SDS-PAGE of the soluble protein fractions of E. coli BL21 samples shows well separated but quite weak bands. In comparison to the negative control, the E. coli BL21_pCERI samples only show one further band at about 22 kDa. None of the proteins of our regulatory system refer to a band of this size. However, one of our proteins could be expressed in a truncated form. The band at about 24 kDa that is visible in the samples from hour 1 to 6 on the gel picture is also slightly observable in the negative control on the gel itself.
The positive controls show, next to some E. coli BL21 specific bands, typical bands for CFP (26.89 kDa) and mRFP (25.42 kDa). For the mRFP positive control two further bands at 40 kDa and 20 kDa are conspicuous. Those again, are observable on the gel itself in all other E. coli BL21 samples as well.
Figure 1 SDS-PAGE of soluble protein fractions of recombinant E. coli BL21 fermentations. Std = Standard, 1h-6h = E. coli BL21_pCERI
fermentations that were stopped on ice after the different time points, - = negative control: wild type E. coli BL21, +CFP/+mRFP =
positive controls: E. coli BL21 cultures with IPTG inducible gen expression of CFP and mRFP.
The SDS-PAGE for the E. coli Nissle 1917 as well showed quite separated but even weaker bands as the E. coli BL21 samples. For this SDS gel we tried to load as much soluble protein as possible into 10µl of SDS-PAGE sample volume. Still, not more than 20 µg, regarding spectrophotometric measurements, were possible to be prepared for the SDS analysis gel. It has to be considered, that protein concentration measurements at 280 nm by Nanodrop™ can differ from the actual yields. As E. coli Nissle 1917 is not an engineered protein expression stain, like E. coli BL21 is, the protein concentration stays lower due to protease activity.
Figure 2 SDS-PAGE of soluble protein fractions of recombinant E. coli Nissle 1917 fermentations. Std = Standard, 1h-6h = E. coli Nissle 1917_pCERI fermentations that were stopped on ice after the different time points, - = negative control: wild type E. coli Nissle 1917.
This second try of SDS-PAGE of all E. coli BL21 samples showed much weaker bands as the first try even though much more protein was loaded. That might indicate that our soluble protein fractions are not stable in 20 mM sodium phosphate buffer. The samples from the E. coli Nissle 1917 cultures were prepared for SDS-PAGE straight after fermentation. Still the final gel only showed very weak bands. A possible reason for that is that the E. coli Nissle 1917 strain still expresses several proteases, which would degrade our proteins and hence lower the total protein yield.
Both SDS-PAGE results are thus inconclusive. Hence, for more accurate characterization further experiments are performed.