Culturing

2.1.1 Growth and maintenance of E. coli strains

The genotype of the three E. coli strains used in this thesis is described in Table 2.1.

Luria Broth (LB) was used for the routine growth and maintenance of E. coli strains. The composition of LB medium is 10 g L-1 tryptone, 5 g L-1 yeast extract and 10 g L- 1

NaCl. The pH was adjusted to 7.0 before sterilisation by autoclaving at 121°C for 15 min. The LB medium was supplemented with antibiotics, as described, when applicable.

SOC medium was used for recovery growth of E.coli strains after electroporation in the clone library experiments. The composition of SOC is 20 g L-1 tryptone, 5 g L-1 yeast extract, 0.5 g L-1 NaCl, 0.1875 g L-1 KCl, 1 g L-1 MgCl2, 1.2 g L-1 MgSO4 and 3.6 g L-1 glucose.

Table 2.1. Genotype of the three E. coli strains Top10F’, JM109 and DH10B T1 Phage Resistant used in this study.

Strain Genotype Supplier

Top10F’ F´{lacIq Tn10 (TetR)} mcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 ΔlacX74 recA1

araD139 Δ(ara-leu)7697 galU

galK rpsL endA1 nupG

Invitrogen SOLAS clone library

JM109 recA1, endA1, gyrA96, thi,

hsdR17 (rK–,mK+),

relA1, supE44, Δ(lac-proAB), [F´, traD36, proAB,

lacIqZΔM15]

Promega AMT clone library DH10B T1 Phage Resistant F- mcrAΔ(mrr-hsdRMS- mcrBC) Φ80lacZΔM15

ΔlacX74 recA1 endA1

araΔ139 Δ(ara, leu)7697 galU

galK λ- rpsL nupG tonA

Invitrogen cDNA library construction

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2.1.2 Isolation of photosynthetic picoeukaryote (PPE) cultures

2.1.2.1 Collection of seawater samples for culturing during the AMT18 cruise

During the AMT18 cruise (October–November 2008), water samples for cultures were taken from the 55% light level and the deep chlorophyll maximum (DCM) during a pre-dawn CTD, using 20L Niskin bottles. The 55% light level was calculated from the previous day’s noon CTD cast. Cells were concentrated, whilst on board, using two different methods:

2.1.2.2 PPE cell sorting using flow cytometry

PPEs were isolated from seawater samples using a FacSort flow cytometer (Becton Dickinson, US) at various stations during AMT 18. Using endogenous chlorophyll fluorescence (FL3) and side scatter (SSC) to discriminate the photosynthetic picoeukaryotes, between 5000 and 50000 cells were sorted into capped glass test tubes. K artificial seawater medium (see section 2.1.2.4) was added to each tube. Vials were then incubated as described below. All isolation tubes were immediately incubated at 21°C, under a constant light intensity of 30 µmol m-2 s-1.

2.1.2.3 Concentration from natural seawater samples

In order to target the pico size fraction of the plankton, cells from pre-filtered seawater samples (through 10 µm and 5 µm pore size polycarbonate filters (PALL)) were further filtered onto a 0.22 µm Supor filter (PALL). Cells were washed off the filter using 2 ml of 0.22 µm filtered deep water and then diluted, using K medium, into plastic 50 ml culture flasks with 0.22 µm filter lids (BD Biosciences). All isolation flasks were immediately incubated at 21°C, under a constant light intensity of 30 µmol m-2 s-1.

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2.1.2.4 Growth of PPEs

To enhance the growth of different PPEs in enrichment cultures, sub cultures were established into different media. The composition of the media used is described in Table 2.2. Nutrients were added to artificial seawater (Sigma) and autoclaved for 15 min at 121°C. The media were allowed to cool before filter sterilised trace metals and vitamins were added. In the case of the soil extract medium (Andersen, 2005) the soil extract was added after autoclaving.

2.1.3 Maintenance of PPE cultures

Routinely PPE cultures were maintained at 21°C, under a light intensity which was set at 30 µmol m-2 s-1 with a 12 h light/dark cycle. A replicate set of the culture collection was maintained in ambient laboratory conditions, at room temperature and under room lights.

2.1.4 Purification of agar

Commercial Bacto agar (Becton Dickinson) (250 g) was suspended in 5 L of MilliQ water and stirred for 30 min. The agar was allowed to settle and the water was poured off. The agar was then filtered through 3MM Whatman (Whatman) filter paper, using a Buchner funnel over a side arm flask with the aid of a vacuum pump. Repeated washes with water were undertaken until the flow through of water was clear. The cleaned agar was then stirred for 30 min in 5 L of ethanol before being filtered through 3MM Whatman paper and then further washed for 30 min in 5 L acetone. The washed agar was filtered through 3MM Whatman paper and left to dry completely in a fume hood. Cleaned agar was stored in an air tight container until required.

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Table 2.2. Composition of PPE growth media. Final concentrations of nutrients are given. Component K Medium (Keller et al, 1987) f/2 Medium (Guillard and Ryther, 1962) PC Medium (Andersen, 2005) Soil Extract Medium (Andersen, 2005) Seawater (Sigma) 993.5ml 996.5ml 997ml 990ml NaNO3 882µM 882µM 882µM NaH2PO4·H2O 36.2µM 36.2µM NH4Cl 26.7µM 50µM Na2β-glycerophosphate 10µM 10µM H2SeO3 100nM 1nM Tris-base (pH7.2) 1mM Urea 50µM

Soil water extract (see section 5.2.1) 10ml FeCl3·6H2O 11.7µM 11.7µM 11.7µM Na2EDTA·2H2O 11.7µM 11.7µM 112µM MnCl2·4H2O 910nM 910nM 99nM ZnSO4·7H2O 76.5nM 76.5nM 7.65nM CoCl2·6H2O 42.0nM 42.0nM 4.20nM CuSO4·5H2O 39.3nM 39.3nM Na2MoO4·2H2O 26.0nM 26.0nM 2.6nM NiSO4·6H2O 1nM Thiamine·HCl (vitamin B1) 296nM 296nM 29.6nM Biotin (vitamin H) 2.05nM 2.05nM 0.205nM Cyanocobalamin (vitamin B12) 0.369nM 0.369nM 0.0369nM

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2.1.5.1 Preparation of agar

Agar plates were prepared by supplementing liquid media (Table 2.2) with 9 g L-1 of purified agar. The agar was sterilised by autoclaving for 15 min at 121°C and allowed to cool. Filter sterilised trace metals and vitamins (Table 2.2) were added separately to the cooled media. Cultures were plated using two different methods.

2.1.5.2 Culturing of picoeukaryotes using pour plates

An aliquot of liquid culture was mixed gently in a Petri dish with warm medium (~40°C), supplemented with agar. Once the medium set, the plates were incubated at 21°C with a 12 h light/dark cycle under a light intensity of 30 µmol m-2 s-1. Cells were retrieved from the agar using a modified glass pasteur pipette. The pipette was flamed and stretched until a thin tube was obtained, in which a single colony could be isolated. The single colony was transferred into 50 ml plastic culture flasks containing 10 ml of the appropriate medium.

2.1.5.3 Culturing of picoeukaryotes using solid agar.

Solid medium, supplemented with 0.9% (w/v) agar, was prepared in Petri dishes. Cultures were streaked onto the surface of the agar plate using sterile spreading loops. Plates were incubated at 21°C with a 12 h light/dark cycle under a light intensity of 30 µmol m-2 s-1. Single colonies were retrieved from the surface of the plate and placed into 50 ml culture flasks containing the appropriate medium.

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