Cryo Stock preparation
For the glycerol stock, a preculture of the required organism was incubated overnight in 5 mL LB medium with the appropriate antibiotic in a test tube. Subsequently, 1 mL of the preculture was centrifuged for 10 minutes at 6000 x g and 4 °C (Heraeus Fresco 17, Thermo Fisher Scientific Inc.). The supernatant was discarded, and the pellet was resuspended in 500 µL of fresh medium containing the antibiotic. The culture was transferred to a sterile 1.5 mL reaction tube or a sterile -80 °C 1.5 mL reaction tube. Approximately 500 µL of autoclaved 99% glycerol was added. The glycerol stocks were stored at -20 °C or -80 °C.
Primer design
The primers were designed using SnapGene. All primers were designed for an annealing temperature of 60°C and provided with an overhang of 15 to 20 base pairs to enable ligation of backbone plasmid with insert using Gibson Assembly. A cytosine or guanine was also added to the end of each primer to increase the stability of the annealing.
Plasmid isolation
The EasyPure® Plasmid MiniPrep Kit from TransGen Biotech was used for this purpose in accordance with the protocol provided. The concentration of the purified plasmids was determined using the NanoDrop (NanoPhotometer NP80; Implen).
Polymerase chain reaction (PCR)
PCR follows a 3-step structure that uses denaturation, primer annealing and elongation with the goal of exponentially amplifying specific DNA fragments. To perform PCR a DNA template, a fitting primer pair, and depending on the provider either a master mixture or singular contents of a polymerase, free nucleotides and a polymerase specific buffer. The PCR samples used in this project all apply the following ratio with a total volume of 25 µl unless stated otherwise.
The following PCR method was used as a general structure and adjusted if necessary.
Agarose gel electrophoresis
To verify the success of the PCR, the PCR products obtained were analyzed using agarose gel electrophoresis. For this purpose, a 1% agarose gel was prepared in 1x TAE buffer (40 mM Trizma base, 20 mM acetic acid, 1 mM EDTA, pH 8.5). The gel was additionally stained with a 1:7000 dilution of Serva DNA Stain G. For sample preparation, 10 µL of the PCR product was mixed with 1 µL of 10x FastDigest Green Buffer (Thermo Fisher Scientific, Inc.). The GeneRuler 1 kb DNA Ladder (Thermo Fisher Scientific, Inc.) was used to determine the fragment size. Electrophoresis was performed at 100 V for 45 minutes in 1x TAE buffer. The Compact Multi-Wide System from Biometra and the Consort EV 231 power supply (Consort) were used for this purpose. The DNA bands were detected in the UVP GelStudio System from Analytik Jena GmbH & Co.
Gibson assembly
To combine DNA fragments that are not inherently compatible a Gibson assembly can be utilized. Following a PCR the mixture can be subjected to a DpnI digestion to remove the sample of methylated template DNA. Afterwards the Gibson sample can be prepared with the following structure Samples were incubated for 1h at 50°C or at room temperature 30°C, 37°C overnight. Samples were subsequently kept on ice and stored at -20°C for later use during heatshock transformation.
Restriction digestion
Similar to a Gibson Assembly a restriction digestion with a follow up ligation can be utilized to introduce new genes and DNA fragments into recipient plasmids. For this approach NdeI and Bam-HI were used as restriction enzymes, if not told otherwise. The following sample preparation method was used for all restriction digestions unless stated otherwise.
After adding all components together, the sample is incubated at 37°C either for 1h, or 15 minutes depending on slow or fast digestion enzymes. After the incubation the samples were loaded onto a 1% agarose gel to verify the digestion.
Heatshock transformation
For the transformation, 2 µL of either the plasmid pET16b-11ß-fold, pBlueScript-1-10ß-fold or the cloned pNit plasmid were mixed with the cloning strain E. coli DH5α. The cells were then incubated with the plasmid on ice for 30 minutes. Heat shock was performed at 42°C for 60 seconds. The cells were then placed on ice again for 10 minutes and subsequently incubated in 950 µL LB medium for 1 hour at 37°C. The cells were centrifuged for 7 minutes at 6000 x g (Heraeus Fresco 17, Thermo Fisher Scientific Inc.), resuspended in 50 to 100 µL LB medium, and plated on agar plates with 100 µg/mL ampicillin .
CAS-Assay
The CAS-reagent was prepared as described below. For the assay, the desired amount of sample was mixed with an equal amount of CAS-reagent. The mixture was then incubated in the dark at room temperature for 4h. After incubation, the absorption was measured at 630 nm. This assay is based on the paper of Alexander and Zuberer from 1991.
Alexander, D. B.; Zuberer, D. A. (1991): Use of chrome azurol S reagents to evaluate siderophore production by rhizosphere bacteria. In: Biology and Fertility of Soils 12 (1), S. 39–45. DOI: 10.1007/BF00369386.
Desferrioxamine extraction
A preculture of Streptomyces coellicolor was prepared in 5 ml LB medium, inoculated to an initial OD600 of 0.01 and incubated at 30°C for two days. Subsequently, a 1 L main culture was initiated with the same starting OD600 and incubated at 30 °C for one week. After incubation, the culture was centrifuged at 4°C for 30 min at 4000 xg. The pellet was discarded and the supernatant adjusted to pH 6.0. Then, 10 g each of XAD-4 and XAD-16 resins were added to the supernatant and the mixture was incubated at 4 °C for 24h. The resins were removed by filtration and the bound DFOB was eluted by stepwise addition of up to 50 ml Methanol with vortexing. The DFOB extraction based on the paper of Hofmann et al. 2021.
Hofmann, L.; Scherlach, K.; Kloss, F.; Roth, M.; Hertweck, C. (2021): Desferrioxamine E Siderophores Mediate Iron Acquisition and Virulence in the Fish Pathogen Streptococcus iniae. In: Applied and Environmental Microbiology 87 (7), e02549-20. DOI: 10.1128/AEM.02549-20.
Growth curve
Samples with a total volume of 1 mL were prepared and filled in a well plate. Each sample contained 900 µL of LB medium and was supplemented with one of the following: 100 µL of Iron solution containing 95 nmol DFOB , 100 µl 5 mM Ascorbic acid with 95 nmol DFOB, 100 µl of 95 nmol DFOB alone, or 100 µl ddH2O (control). The well plate was incubated at 30°C for 96 h. Measurements were taken at 12 h, 24 h, 36 h, 48 h, 60 h, 72 h and 96 h. After 36 h a 1:10 dilution of each sample was prepared and monitored in subsequent measurement.
Material
In this section you can find an overview of all the materials we used during our project.
