Sample purification methods

5.4.1 Fish toxins sample purification

Streptomyces griseus strains were inoculated on sterile cellophane membranes on

starch casein agar medium in a Petri plate and incubated at 30°_{C for 7 days. Biomass} was collected and weighed. After removing the membrane the agar was divided into two halves. Biomass and one half of the agar were soaked into 50 mL of distilled water and the second half of agar into 50mL of ethyl acetate. Agar was crushed using spatula into small pieces. The mixtures were left at 4°C for two days before filtering off the biomass and agar. The filtrate left from biomass filtration was identified as ‘aqueous filtrate’, ethyl acetate filtrate as ‘organic filtrate’, while the filtrate from the agar soaked into water was then subjected to organic extraction using ethyl acetate. After extraction the organic phase was identified as ‘organic extract’ and aqueous phase as ‘aqueous extract’. Organic filtrate and organic extract were dried under vacuum while aqueous fractions were frozen in liquid nitrogen and lyophilized on a freeze dryer.

5.4.1.1 Sample preparation for fish toxin experiment

Based on the weight of the biomass, calculations were made to dissolve dried stock samples into specific quantity of solvent (water in case of aqueous sample and ethyl acetate in case of organic samples) to prepare three dilutions of the samples, I) equivalent to 6mg of biomass, II) 12mg of biomass and III) 18mg of biomass. Required quantities of stock samples were transferred into glass vials and dried (organic samples under vacuum and aqueous samples on freeze dryer). When dry they were shipped for fish-exposure experiments in London.

5.4.2 Foroxymithine purification

5.4.2.1 Growth of Streptomyces narbonensis in iron deficient medium

For foroxymithine purification Streptomyces narbonensis G3P3/110 and Streptomyces

nabonensis type strain DSM 40016 were grown in 500mL iron deficient medium with

Innoculation on starch casein agar medium

Incubation at 30 °C for 7 days

Biomass

(collected and weighed)

Agar Soaked in dH2O at 4 °C for 2 days Filter Aqueous filtrate Soaked in dH2O at 4 °C for 2 days Soaked in ethyl acetate

at 4 °C for 2 days Filter Organic filtrate Filter Extraction with ethyl acetate

Organic extract Aqueous extract

5.4.2.2 Preliminary purification on HP-20 resin

Iron deficient medium was centrifuged after incubation at 3000 rpm for 10 minutes. Spent media were filtered through Whatman No. 1 filter paper. TFA was added into spent media at final conc. of 0.05% (i.e. 0.05mL or 50µL in 100mL). HP-20 resin was

put into a glass chromatography column. The column was eluted with 100% MeOH (3 volumes of resin). Solvent was then replaced with distilled water containing 0.05% TFA gradually by adding and the resin was washed with 3 volume of distilled water containing 0.05%TFA. Spent media containing 0.05%TFA was loaded. It was then washed with distilled water containing 0.05%TFA (3 volumes of resin). Finally 20% MeOH (no TFA) was used to elute foroxymithine. After freezing in liquid nitrogen the fraction containing forxymithine was subjected to freeze drying.

5.4.2.3 Purification of Ferric-foroxymithine complex by HPLC

Freeze-dried sample was then dissolved in FeCl3 to a final concentration of 10 mM (of FeCl3). The solution turned dark red. The mixture was then centrifuged at 13,000 rpm for 5 minutes and the supernatant was separated to be injected on HPLC (Agilent 100) for purification.

UV detector on HPLC was set to show absorbance at 435 nm and flow rate was set at 5 mL/min using C18 RP column. The method set was as below

Table 5.6 HPLC method for ferri-foroxymithine purification

Time A (%) B (%) 0 100 0 20 90 10 25 0 100 35 0 100 40 100 0 50 100 0

Flow rate 5mL/min, UV = 435nm A= water + 0.1% TFA;

Peak fractions were analysed on Bruker microTOF ESI-MS to see if they contained ferri-foroxymithine. The retention time of ferri-foroxymithine was around 10 minutes. Collected fraction were put together in a round bottom flask and after putting on rotary vacuum for 15-20 minutes to remove any acetonitrile left, put on freeze dryer after freezing in liquid nitrogen.

5.4.2.4 Removal of Iron from ferri-foroxymithine

All glassware used were rinsed with 6N HNO3 and MilliQ water. Using an established protocol249 _{freeze-dried ferri-foroxymithine dry powder was solubilised in 25 mL} deionized water. To this solution 145 mg (1 mmole) of 8-hydroxyquinoline dissolved in 25 mL MeOH was added. Solution turned from yellowish-red to dark green as Fe(II) complexed to 8-hydroxyquinoline. It was left in shaker for 1 hour at room temperature. Fe-quinoline complex was then extracted by using 20 mL DCM (dichloromethane). Desferri-foroxymithine was in aqueous phase. The extraction was repeated five times. Excess organic solvents were evaporated under vacuum. The flask was snap-frozen in liquid N2 and lyophilized overnight.

5.4.2.5 Preparation and purification of gallium-foroxymithine complex

The freeze-dried sample of desferri-foroxymithine was dissolved in 5 mL of deionised water and mixed with the gallium sulphate solution (0.151 g of gallium sulphate in 5 mL of 0.05 M sulphuric acid). The solution was left for 30 minutes on stirrer. After 30 minutes pH neutralized and solution was frozen in liquid nitrogen and put on freeze- dryer. Freeze-dried gallium-foroxymithine complex was diluted into 5 mL of deionised water. Supernatant was separated after centrifugation for 8 minutes at 3000 rpm.

The gallium-foroxymithine complex was separated by RP-HPLC using a C18 RP semi-preparative column. The column was eluted with 100% 10 mM ammonium

rate of 5mL/min and detector wavelength set at 210 nm. The peak at approx 4-5 minutes was collected and freeze-dried.

5.4.3 Genomic DNA purification

Streptomyces narbonensis strain spore stock (10 µL) was inoculated in 25 mL TSB

medium in 250 mL baffled conical flask. The flask was incubated on shaker (180 rpm) for 4 days at 30°C. After incubation 1 volume of water (25 mL) was added and then centrifuged for 10 min. at 2000 rpm. The tube was then washed in 50 mL of EDTA (10mM pH 8.0) and centrifuged again for 10 min. at 2000 rpm. Mycelia in the pellet were then resuspended in 5mL SET buffer and 100 µL lysozyme aqueous

solution (50mg/mL) was added to it and then incubated for 1h at 37°C. 140 µL of

proteinase K aqueous solution (20 mg/mL) was then added followed by 600 µL 10% SDS and mixed by inversion. It was incubated for 2 h at 55ºC. After incubation 2 mL of 5 M NaCl was added and mixed thoroughly by inversion, let to cool to 37ºC and then 5mL chloroform was added and mixed by inversion for 30 min at RT. It was centrifuged for 15 min at 4000 rpm. Supernatant was transferred to fresh 15 mL tube (about 6mL) then added 0.6 vol. isopropanol (about 3.6 mL), mixed by inversion, after about 3 min. DNA was fished out with plastic loop, rinsed in 5 mL 70% ethanol and was transferred in a 1.5 mL eppendorf to dry for 30 min. It was then resolubilised in 1 mL TE (pH 8.0) O/N at 4°C, then prepared 300 µL aliquots and stored at -20°C.