Contribution

iGEM team Heidelberg 2023 aims to introduce Pseudomonas fluorescens as a new chassis organism for the iGEM competition. We established standardized protocols and characterized certain growth limitations. We chose to use P. fluorescens as it is an excellent organism to conduct plastic degradation experiments as it is naturally capable of degrading polyurethane (Wilkes and Aristilde, 2017), polythene (Thomas et al., 2015), polyethylene (Mouafo Tamnou et al., 2021; Lakshmi and Selvi, 2021). Furthermore, the organism is facultative anaerobes and capable of utilizing inorganic compounds such as nitrates as terminal electoral receptors. This is beneficial in terms of scalability for industrialization purposes as the production rate is not dependent on local oxygen supplies (Kampers et al., 2021). An important aspect for usage in the context of iGEM is that P. fluorescens is classified as S1 and can be cultivated in the laboratory. Despite these benefits, P. fluorescens is an under utilized model organism. We tested various factors necessary for the cultivation of this organism in order to identify the optimal growth conditions and antibiotic resistance.

Growth rate assays were conducted on M9 minimal media with different carbon sources. As seen in figure 1, 18 different carbon sources were tested. P. fluorescens could grow on nine of them. For our experiments, LB broth was primarily used. The antibiotic resistances of P. fluorescens were also tested using ampicillin (amp), chloramphenicol (cm), kanamycin (kan), and streptomycin (strep). The 1x concentration of each antibiotic are as followed: amp (100 µg/mL), cm (20 µg/mL), kan (25 µg/mL), and strep (100 µg/mL). An antibiotic dilution series of each antibiotic were prepared and plated in a 96-well plate along with P. fluorescens. An OD₆₀₀ nm measurement was taken every 10 min for 20 h. Figure 2 shows the growth of P. fluorescens in the different antibiotics. Amp does nothing to prevent cell growth, which is supported by the literature (BacDrive DSM50900, n.d.).The other three antibiotics reduce the cell growth depending on the used antibiotic concentration. Cm prevented some cell growth, however, kan and strep were best at preventing cell growth. For our experiment, strep was used at a concentration of 200 µg/mL. A concentration of 100 µg/mL should be enough according to our initial results. However, based on our following experiments it was not sufficient to cause selection pressure. We recommend using kan as an alternative for strep, because we observed spontaneous mutations against strep after around 16 h of cultivation.