Team:Stuttgart/Collaborations

Collaboration

Validation of our new algae based PhyCoVi medium.

Besides our own experiments with the algae extract, we wanted to have a cross-validation from other iGEM teams and their organisms. Through this, we can make sure, that everybody can work with our medium and also improve the product itself, if problems occur. Kindly, the iGEM teams of Hamburg, Tübingen, TU Darmstadt, Freiburg, and TU Dresden offered their help. Therefore, we prepared 15 ml tubes with two variations of PhyCoVi and a LB control dry medium and send them to the teams, respectively. Additionally, they received a SOP for their growth curve including an Excel sheet to plot their results directly. Before we discuss their results individually, it can be said, that we are happy with every result and already improved the product concerning the issues that occurred for some teams.

Hamburg (E. coli DH5-α)

As seen in Fig. 1, the E. coli grew on all three mediums. The PhyCoVi medium including additional soy peptone and the high salt LB medium show similar growth curves after 45 minutes. The PhyCoVi medium without supplements shows a very long lag phase, growth occurs after 135 minutes. The DH5-α strain is very robust and optimized for optimal transformation 1. These optimizations also seem to have a positive effect on high salt concentrations.

Tübingen (E. coli Nissle 1917)

Fig. 2 shows the attempted growth curves by Team Tübingen. Probably due to algae lipid residues in the PhyCoVi medium samples, the OD values are corrupted and appear in negative values. But still, a growth of the Nissle 1917 strain can be observed in the medium with soy peptone additive. As described 2, this wildtype strain has a low tolerance for high salt medium, therefore the reduced growth is expected.

TU Dresden (E. coli GB05)

The results of the iGEM Team TU Dresden shows very low growth for all three mediums, possibly caused by the high salt concentrations (Fig. 3). After the long lag phase, only the LB medium manages a nice growth curve. Also, the PhyCoVi medium with additional soy peptone shows high deviations, probably due to the already discussed problem with insoluble medium components. The used strain is able to take up linear DNA fragments but seems not to be able to cope with high salt mediums 3.

TU Darmstadt (E. coli BL21 (DE3))

The BL21 (DE3) strain shows a growth pattern strongly influenced by the PhyCoVi medium (Fig. 4). While the LB medium reaches high OD values, both algae-based mediums show very slow growth and long lag phases. Also, the SD values show high differences during the measurement. These are, as already discussed, remnants of the lipids and cell wall, which had not been removed properly. The strain is one of the most frequent used E. coli strains and as stated in growths best on low salt medium 4.

Freiburg (E. coli DH5-α)

This measurement was conducted in a 96-well plate reader (Fig. 5). Both algae-based mediums are affected by the lipid and cell wall remnants and therefore show negative values. But highly appreciated is the measurement length of 15 hours.

Conclusion

In conclusion, the collaboration was a full success, as it showed us that bacteria do indeed grow on our medium. Also, we increased our clean-up efforts for lipids and cell wall aggregates by including an additional step of autoclaving and centrifugation of the medium, as many aggregates formed during an initial sterilization by autoclaving.

The result is visible as seen in Fig. 7 with left being the old medium version, while on the right, our new advanced medium can be seen.

References