**2. Methodology**

*Synthetic Biology - New Interdisciplinary Science*

pressure [9].

Many planktonic organisms can form mass occurrences in the water column.

Microscopic marine algae can be vectors of microbial communities because they are universally associated in the ocean. In nature, most microbial communities are found adhered to microalgae, organism and inanimate surfaces. These interactions are dynamic and are important factors in microbial proliferation and survival. Aquatic algae in situ as well as in laboratory culture condition are often found to be associated with a variety of bacterial strains [10]. Bacteria community can be defined as multi – species of bacteria assemblages in which organisms live together in a contiguous environment (host) and interact with other [11]. Bacteria reproduce asexually, are sized between 0.1 to 20 μm, and can be rod, cocci or comma shaped. For marine microorganisms (bacterioplankton), there are approximately 106 bacterial cells per ml of surface seawater throughout the world's oceans [12]. While this number has been known for at least 30 years, how many bacterial species are actually present in the bacterioplankton are still unknown. Bacterioplankton commonly found in marine environment are mainly from bacteria group of Proteobacteria, Cytophaga-Flexibactar-Bacteroides (CFB), marine Archaea and other groups of bacteria, where bacteria group from Proteobacteria is the largest. Proteobacteria group are divided to some class, which are Alpha (α-), Beta (β-), Delta (δ-), Epsilon (ε-) and Gamma (γ-) Proteobacteria [13]*.* Up until now, the estimated abundance and genetic diversity of bacterioplankton are based on the data in the GenBank database. Hagström et al. [14] had analyzed on all of the 16S rDNA sequences sent to GenBank to get the estimation of marine bacterioplankton species that were available in the GenBank database. Their studies show that the richness of

When the cell densities reach values considerably higher than their general background distribution, they are called blooms [3]. Blooms can be almost mono specific, others are formed by a combination of species [4, 5]. Many prominent blooms can be traced back to high nutrient loads [6–8], but they can occur whenever a species is able to outgrow its competitors while partially reducing grazer

marine bacterioplankton species in the GenBank database was low relatively.

tion by bacteria associated with dinoflagellates cultures.

The ecology of bacterioplankton and phytoplankton is widely recognized to be tightly coupled. Interactions between bacteria and phytoplankton such as dinoflagellates may play an important role in regulating dinoflagellate toxin production. Previous studies on the interactions between bacteria and dinoflagellates have been shown to be highly variable and are sometimes specific. Effects of bacteria on toxic dinoflagellates include negative effects such as cell lysis and death [15] and positive effects such as growth enhancement with an addition of bacteria to cultures [16]. Examples of factors which may cause stimulation or inhibition by bacteria include production of co-factors and secretion of signaling molecules controlling cellular processes of dinoflagellates [17]. In addition, bacterial influences on nutrient availability may result in stimulation or inhibition of the growth or toxin production of dinoflagellates. Both toxin production by dinoflagellates and bacteria associated with toxic or non-toxic dinoflagellates have been documented. For example, Gallacher et al. [18] described evidence of paralytic shellfish toxin (PST) produc-

Cultures of dinoflagellates contain a considerable amount of bacteria which probably accompanied the dinoflagellates in the original sample. Bacterial assemblage found in the phycosphere of dinoflagellates may play an important role in regulating dinoflagellate toxin production. While several studies have suggested that bacteria-phytoplankton interactions have the potential to dramatically influence harmful algal bloom dynamics, little is known about how bacteria and phytoplankton communities interact at the species composition level. Other studies have indicated that inside a phytoplankton bloom, α-Proteobacteria overwhelm

**40**

**Dinoflagellate Culture.** Clonal culture of *Alexandrium tamiyavanichii* were obtained from UKM Microalgae Culture Collections and maintained in ES-DK medium [23] and growth in a light–dark cycle (14:10 hour) incubator at 26°C (model 2015 Shelab, USA).

**DNA Extraction.** Bulk genomic DNA were directly extracted from a 2.0 L of mid exponential growth phase dinoflagellate [24]. Firstly, culture medium was filtered through 0.2 μm nitrate cellulose membrane (Whatmann, England). Cell pellets were then concentrated and resuspended in buffer (100 mM EDTA, 10 mM Tris–HCl [pH 8.0]) and treated with proteinase K (0.5 mg/mL)-1% sodium dodecyl sulfate (SDS) for 1 h at 37°C. Lysates were further treated by CTAB extraction (0.5 M NaCl, 1% CTAB) for 10 min at 65°C. Then, DNA was extracted once with equal volume of chloroform-isoamyl alcohol (24:1) and phenol-chloroformisoamyl alcohol (25:4:1) and spin at 21000 x g for 5 min at 4°C. After that, DNA was precipitated with 0.6 volume of isopropanol by centrifugation at 21000 x g for 15 min. The DNA pellet was then washed with one volume of 70% ethanol and spin at 21000 x g for 10 min. Finally, DNA was resuspended in 50 μL of ddH2O and stored at −20°C.

**Metagenomic Library Construction and End-sequences Analysis.** Sheared DNA with sizes ranged from 30 kilo bases to 40 kilo bases were used to construct the metagenomic library. Metagenomic library was constructed using CopyControl pCC1FOS library construction kit (Epicenter, USA) following the manufacturer's protocol and fosmids were then purified using Millipore 96-well prep BAC purification kit (Millipore, USA) following the manufacturer's protocol and end-sequenced using T7 universal primer (5′- TAATACGACTCACTATAGGG-3′). End-sequences were edited by using Staden Package software [25]. Low quality DNA sequences were identified and trimmed using Pregap4. The resulting high-quality sequences were assembled into contigs by using Gap4. All dataset was then analyzed by BLASTX [26]. The taxonomical analysis of sequence matches was performed using MEGAN version 4.0 [27] and gene ontology analysis was carried out using Blast2GO suite [28].
