The agrobacterium tumefaciens diseases

1.0 INTRODUCTION

Agrobacterium Tumefaciens is a rod shaped gram negative bacterium. It is the major causal agent of crown gall diseases in dicotyledon plants from over 600 species belonging to 90 families (De Block et al., 1985). Agrobacterium Tumefaciens induced grown gall disease by integrating a part of its tumor inducing or Ti plasmid into the genome of the plant. The specific segment from the Ti plasmid that was transferred is called transferred or T-DNA (Walden, 1988). Since the last decade, much interest has been showed to turn Agrobacterium Tumefaciens into a vector for transformation of plants.

Chlorella sp is singled-cell green algae from the Chlorophyta division under the Chlorellaceae family (Hoek et al., 1995). These green algae are spherical in shape, with diameter from 2 to 10 µm and have no flagella. It contains contain photosynthetic pigment chlorophyll-a and chlorophyll-b embedded in its chloroplast (DuPont, 2009). Chlorella has recently been taken as food supplement as it contains Chlorella Growth Factor (CGF). CGF consists of all components inside Chlorella sp which become the main factor to its amazing healing properties (Ley, 2003). In Japan, Chlorella sp has been taken as a supplement in the form of tablets, capsules, liquid extract and food addictive (Halperin et al., 2003).

Centromere in eukaryote chromosome controls the segregation of daughter cells during mitosis and meiosis event by organizing the cells through microtubules connected to their kinetochore (Alberts, 1998). In the yeast such Saccharomyces cerevisiae it is known that only a single microtubule are bound to each centromere (Peterson and Ris, 1976). CEN 4 is a small DNA element from Saccharomyces cerevisiae that can be isolated and included into a plasmid with a chromosomal replicator such as ars 1 or ars 2 which can behave as functional chromosome in yeast, allowed it to replicate successfully in mitotic division and the first meiotic division (Clarke and Carbon, 1980)

This experiment is done to investigate whether a yeast gene, cen4 can be incorporate into plant genome through Agrobacterium-mediated transformation, hence converted it into a chromosome capable of replicating in yeast. Hopefully, if this experiment proves to be successful, a vector can be construct base on algae chromosome bearing cen4 gen which can be use to introduce gene of interest into yeast genome.

1.2 OBJECTIVES

The objectives of these studies are

2.0 LITERATURE REVIEW

2.1 Agrobacterium Tumefaciens

Agrobacterium tumefaciens has long been the source of crown gall disease in dicotyledon plants (Walden, 1988). Crown gall is a plant disease which causes disorganized growth on the stem of the plant near the surface of the soil (Walden, 1988). Since its discovery, crown gall has been described as a neoplastic disease (Nester et al., 1984). The morphology of the tumor may determined by the formation of unorganized callus structure or teratomas (Levine, 1919). Host plant species and also the origin of infection are among the factors affecting the morphology of crown gall to be form (Gelvin, 1990). At the beginning of the century, it was found that a pure culture of bacteria which has been isolated from tumorous tissue can be use to induce tumor formation in a healthy dicotyledon plant (Binns, 2008). Further observation confirmed that crown gall tumors can be transplanted from plant to plant similar to what occur in animal tumors (Walden, 1988). During 1990's, Bacterium tumefaciens has been determined as the earliest bacteria known to cause crown gall diseases in plant by (Smith and Townsend, 1907). A thorough characterization of the bacterium including their shape, size and growth has also been characterized. Since then, the name Bacterium tumefaciens has been changed to Agrobacterium tumefaciens as we know today.

2.2 Ti-Plasmid

Since 1970's, there were many hypothesis regarding the mechanism of DNA transfer from Agrobacterium tumefaciens to the plant genome. It was strongly suggested then, that a bacterial plasmid might play a major role in causing this disease (Watson et al., 1975). A comparison between 11 virulent Agrobacterium tumefaciens with 8 avirulent strain showed that the virulent strain have a large plasmids whereas, avirulent lack such plasmids (Zaenen et al., 1974),. Transfer of this large plasmid from virulent to avirulent strain of Agrobacterium tumefaciens can be done by genetic exchanges between these two strains (Kerr, 1969). It was confirmed that the avirulence strain acquired virulence after receiving plasmids from the virulence strain (Kerr et al., 1977; Kerr, 1971).Interestingly, a detail look at the difference between avirulence strain with virulence strain revealed that a lot of genes involve in tumorigenicity located on a plasmid called Ti-plasmid, which avirulence strain lack such genes on that plasmid (Hooykaas et al., 1977; Zaenen et al., 1974).

Ti-plasmid is a double stranded circular DNA of approximately 200 kilobases (kb) in size (Walden, 1988). It is known now that, Ti-plasmid is involved in causing crown gall disease in plant. The transfer of Ti-plasmid is affected by temperature (thermosensitive) (Tempé et al., 1977). Ti-plasmid can be transfer through conjugation between avirulent strains and virulent strain (Tempé et al., 1977). However, when conjugation was done at temperature above 30°C, the transfer frequency decrease significantly (Tempé et al., 1977). This was due to the fact that Ti-plasmid was loss at temperature 37°C (Watson et al., 1975). Ti-plasmid is made up two major regions. They are transfer or T-DNA region and Virulence region. In T-DNA region, there are two 25-bp direct repeat sequences which flank the T-DNA (Bevan, 1984; Gelvin, 2000; Gheysen et al., 1991; Hernalsteens et al., 1980; Hoekema et al., 1983). They are called Right Border and Left Border. The right border is crucial as it is required in cis for T-DNA transfer, while the left border only act as a divider between transferred and non-transferred DNA.(Gheysen et al., 1991)

Ti-plasmid can be divided base on the type of opines been produce by their genes. Opines is a substances produce by Agrobacterium tumefaciens which supply the bacteria with nitrogen as well as energy (Kaper and Hacker, 1999). Opines can be found inside the tumor which induced by Agrobacterium tumefaciens in plants (Walden, 1988; Kaper and Hacker, 1999). Genes responsible for producing opines are located in a DNA segment, called transfer or T-DNA (Kaper and Hacker, 1999). The type of opines been produce are strain-dependent. So far, 30 opines has been discovered. Nopaline, octopine,succinomapine and leucinopine are example of opines been produced by Agrobacterium tumefaciens (Walden, 1988).

Aside from gene for opine synthesis and the borders, T-DNA also have genes responsible for the synthesis of auxin and cytokine. These two compounds will be produced inside transformed plant which will result in the formation of tumor (Zupan and Zambryski, 1995)

The virulence region is where the genes that responsible of secreting enzymes which will facilitate the integration of T-DNA region into plant genome (Gelvin, 2000). The virulence region is made up of an operon system consisting of virA, virB, virC, virD, virE virF and virG (Schrammeijer et al., 2000).

2.3 Transfer of T-DNA

The mechanism involving the transfer of T-DNA from Agrobacterium tumefaciens into plant and the integration of that T-DNA into plant genome required chemical interaction between the pathogen (Agrobacterium tumefaciens) and the host (plant) (Gelvin, 2000). This series of interaction involved chemical signals such as, neutral or acidic sugars, phenolic compounds, opines, vir proteins and T-DNA (Gelvin, 2000; Winans, 1992).

2.3.1 Initiation of T-DNA transfer process

Wounded plant tissue releases several phenolic compounds, such as acetosyringone. This phenolic compound acts as inducer of the vir genes (Dye et al., 1997; Messens et al., 1990; Xu et al., 1993). At the same moment, nopaline-base Agrobacterium tumefaciens will secrete trans-zeatin compound which will prepare plant cells for the probability of transformation process (Walden, 1988). This was done by initiating cell division (Walden, 1988). Agrobacterium tumefaciens sense phenolic compound released by wounded plant by virA gene product, VirA sensory protein (Table 2.1). virA have the autophosphorylating capability. This allowed virA to phosphorylate itself. An autophosphorylated virA will then phosphorylated virG by donating phosphate to virG (Jin et al., 1987; Jin et al., 1990). Phosphorylated virG bind to consensus sequence before activate vir genes transcription process (Table 2.1).

2.3.2 Preparation of T-DNA for transfer

Prior to the expression of vir genes, virD1 and virD2 will begin to prepare T-DNA for transfer (Table 2.1). At this stage, T-DNA is still inside Ti-plasmid. virD2 nicks 25-bp direct repeat borders of T-DNA to separate T-DNA from Ti-plasmid (Filichkin and Gelvin, 1993; De Vos and Zambryski, 1989; Stachel et al., 1986; Steck et al., 1990; Yanofsky et al., 1986). This was followed by the binding of virD2 with 5' end of T-DNA through the formation of covalent bond with tyrosine (Gelvin, 2000). However, when nicking a double stranded DNA, virD2 will need the help of virD1 (Jayaswal et al., 1987).

2.3.3 Transfer of T-DNA to plant cell

As virD2 bind to 5' end of T-DNA, it will guide the DNA from Agrobacterium tumefaciens to plant genome. A "bridge" from Agrobacterium tumefaciens to plant cells is constructed by the expression of virB (Table 2.1). virB coded for pilus like structure which will allowed T-DNA, as well as virE2 move to plant cells. virE2 is very important in establishing transformation process. The complex which consists of single stranded T-DNA and virD2 is called T-complex, and this structure is coated with virE2 (Howard and Citovsky, 1990). During the transfer of T-complex from Agrobacterium tumefaciens, virE1 is needed to assists virE2 (Deng et al., 1999).

2.3.4 Integaration of T-DNA into plant genome

T-complex is protected by nucleolytic digestion by virE2. virE2 and virD2 facilitate integration of T-complex with plant genome (Table 2.1). The integration of T-complex with plant chromosomes is known as illegitimate recombination (Gheysen et al., 1991). The single stranded T-complex is first converted into a double stranded DNA form. virD2 then insert the double stranded T-complex into plant genome(Gelvin, 2000). This ligation proceeds efficiently due to the fact that virD2 bind to 5' end of T-complex hence protecting it from any nucleolytic attack (Gelvin, 2000; Kumar and Fladung, 2002). virE2 may involve indirectly in integration process. It was suggested that virE2 may protected 3' end from nucleolytic attack, hence enable a precise integration process (Gelvin, 2000).

2.4 Ti-Plasmid as a vector

Since the discovery of Ti-plasmid ability to transfer T-DNA into plant cells, hence transforming the plant, interest of turning Ti-plasmid into transformation vector has mounted. Scientist view Agrobacterium tumefaciens as a natural genetic engineer of plants (Walden, 1988). Ti-plasmid has a mechanism which enables the integration of foreign DNA into plant genome in much more easier way (Walden, 1988).

Among the characteristic that Ti-plasmid had which makes it a suitable plant transformation vector are:

Ti-plasmid can be converted into a useful vector by deleting all the oncogenic functions that can be found in the T-DNA, without affecting the vir region (Zambryski et al., 1983). During 1983, a nopaline-based Ti-plasmid, pGV3850 was modified by deleting oncogenic components inside its T-DNA region and replaced by pBR322. pBR322 is a artificial plasmid which contained a replicon region, the ampR gene which encoded for Ampicillin resistant and tetR gene which encoded for Tetracycline resistant gene. This plasmid also has restriction sites for 40 restriction enzymes which lie within tetr and ampr (Zambryski et al., 1983). It was then proved that reengineering Ti-plasmid such as pGV3850 by removing all the oncogenic components in the T-DNA region will still maintain the Ti-plasmid ability to transformed plant cells (Zambryski et al., 1983).

The deletion of oncogenic genes resulted in non-oncogenic Ti-plasmid or disarmed Ti-plasmid. Due to the fact that, deletion of oncogenic genes left transformed plant cells to appear in normal morphology, some kind of detection method is needed (Argawal, 1998). In order to achieve a desired gene expression, suitable promoters, termination and translation region need to be included in Ti-plasmid vector. Nowadays, a wide range of selectable marker genes are included in Ti-plasmid vector. For example, kanR gene which code for an enzyme called Neomycin Phosphotransferase Type II or NPTII is often use as a selectable gene marker in transformed plant cells (Chawla, 2002). In order for this gene to be express, it has to be provided with plant promoter and terminator. Ti-plasmid as a vector can be either trans or cis. The differences between these two type of Ti-plasmid vector is than in the 25-bp repeat borders is constructed form the same or different replicon sequences from the vir region (Walden, 1988). Different sequences will produce binary vector while, a same order of sequences will produce co-integrative vector (Argawal, 1998).

2.5 Binary and Co-Integrative Vector

Binary vector consist of intact vir region as well disarmed T-DNA. T-DNA is located on a plasmid called miniplasmid (Razdan, 2003), which also bears genes such as selectable marker. This allowed the selectable marker and T-DNA to be inserted as a whole. Another plasmid holds vir genes. vir genes will be supplied in trans on the miniplasmid to transfer T-DNA as well as the selectable marker genes into plant cells (Walden, 1988). The benefits of using binary vector are it able to replicate both in Agrobacterium tumefaciens and Esherichia coli (Argawal, 1998; Razdan, 2003; Walden, 1988). Transfromation of plants by using binary vector usually take place by inserting gene of interest into Escherichia coli before transferring the gene of interest into Agrobacterium tumefaciens by tri-parental mating or electroporation. The famous ‘Bin' series of plasmids and pBi121 is among examples of binary vector (Razdan, 2003).

Co-integrative vector lacks the ability to replicate inside Agrobacterium tumefaciens. Hence, this plasmid needs to be co-integrated with an endogenous Ti-plasmid (Razdan, 2003). The integration proceeds through a segment of DNA common to both plasmids. pBR322 discussed earlier is one of an example of co-integrative vector. A distinctive feature of a co-integrative vector is that it retain the orientation of vir genes and T-DNA functions in a cis configuration (Walden, 1988).

2.6 Agrobacterium Tumefaciens-mediated Transformation

Agrobacterium tumefaciens nowadays have been studied enough to allowed scientist to use their unique capability to transform plants. However, there were several factors affecting Agrobacterium-mediated transformation process. Naturally, Agrobacterium tumefaciens can only cause crown gall in dicotyledons plants. Hence, the use of Agrobacterium tumefaciens to transform economical monocots plants such as wheat (Oryza sativa) and maize (Zea mays) are limited. The reason why Agrobacterium tumefaciens cannot stably incorporate their T-DNA into monocots plants is perhaps monocots plant had a better proofreading system, which may restrict the T-DNA integration into plant genome (Tinland, 1996). However, nowadays Agrobacterium tumefaciens capability to transform monocots plant has been reported. In 2002, Agrobacterium-mediated transformation of maize embryo have successfully achieved (Frame et al., 2002). Besides that, it is now no longer limited to plants transformation. It was reported by (Soltani et al., 2008) that Agrobacterium tumefaciens were able to transform at least 80 non-plant, which included fungi, algae and even mammalian cells. In 2004, (Hooykaas, 2004) reported that Agrobacterium-mediated transformation has been reported in cereal and fungi. Agrobacterium tumefaciens was also able to stably transfer and integrate genetic materials into mammalian cell in a way similar to the mechanisms occurred in plants (Kunik et al., 2001).

2.7 Algae

Algae can divided into two major group, either macroalgae or microalgae (Anne E. Osbourn, 2009). Algae are plant-like organism, in a way that they does not possess true roots, leaves, stems or even vascular tissues. They have simple reproduction systems usually consist of alternate generation (L.Barsanti, 2006). They usually exists as a photosynthetic and aquatic organism which come in various size from microscopic to as large as 50m in length (Joseph, 2005). Algae have become a prized organism due to its vast benefits that include as biodiesel to replace the needs of fossil fuel, food supplement and fertilizer (Chisti, 2008; Mulbry et al., 2005).

Microalgae has emerge the most dependable biofuel that can completely replace fossil fuel in transportation without affecting our food source or crop products (Chisti, 2008). Microalgae are believed to be a better biofuel than any other suggested renewable fuel such as oil palm and bioethanol from sugarcane, in a way that algae can be grown in the area where there will be no competition with food crops (Chisti, 2008; Neltner, 2008). Algae also have been suggested to be able to replace chemical fertilizer as algae can be converted into fertilizer to supply Phosphorus and Nitrogen (Mulbry et al., 2005). Algae biomass that has been undergone pretreatment from anaerobically digested dairy manure can be use as commercial fertilizer to provide Phosphorus and Nitrogen (Mulbry et al., 2005).

2.7.1 Chlorella sp

Chlorella sp is singled-cell green algae from the Chlorophyta division under the Chlorellaceae family (Hoek et al., 1995). These green algae are spherical in shape with no flagella, with size ranging from 2-10 µm (DuPont, 2009). It contains photosynthetic pigment chlorophyll-a and chlorophyll-b embedded in its chloroplast (DuPont, 2009). There are three species under this genus. They are; Chlorella minutissima, Chlorella pyrenoidosa and Chlorella vulgaris. Chlorella sp has recently been taken as food supplement as it contains Chlorella Growth Factor (CGF), which is a water-soluble extract which contains high concentration of vitamins, minerals, nucleic acids, amino acids and enzymes that are needed by human (Ley, 2003; Merchant and Andre, 2001). It has been reported that by taking Chlorella as supplement has extensively promote healing and growth, stimulate immune system and also have anticancer properties (Merchant and Andre, 2001).

Algae has greater potential than other alternative fuels, in that algae based biodiesel is completely compatible with existing biodiesel (Neltner, 2008). Diesel engines can be alternatively run by pulverized coal but the particles involve have to be in the size ranging from 5-8µm in order for the engine to combust properly. A biomass slurry fuel can be made with the mixture of biodiesel extracted from rapeseed oil with Chlorella vulgaris which can run on ordinary diesel engine (Scragg et al., 2003).

2.8 Yeast Centromere Plasmid (YCp)

Centromere in eukaryote chromosome controls the segregation of daughter cells during mitosis and meiosis event by organizing the cells through microtubules connected to their kinetochore (Alberts, 1998). In the yeast such Saccharomyces cerevisiae it is known that only a single microtubule are bound to each centromere (Peterson and Ris, 1976). Cen4 is a small DNA element from Saccharomyces cerevisiae that can be isolated and included into a plasmid with a chromosomal replicator such as ars 1 or ars 2 which can behave as functional chromosome in yeast, allowed it to replicate successfully in mitotic division and the first meiotic division (Clarke and Carbon, 1980). Plasmids contain yeast centromer gene are now called Yeast Centromere Plasmid (YCp), and widely use as a vector to introduce gene of interest in molecular works (Verachtert and Mot, 1990). This plasmid an ARS-based plasmid containing basic chromosomal elements which can provide a rate of a copy number of one cell per cell, hence very useful in low copy numbers situation, such as when trying to cloning genes which become lethal when excessively expressed (Chawla, 2002). This plasmid usually have multiple cloning site which can be digest with restriction enzyme to insert gene of interest and also contain antibiotic resistant gene such as Kanamycin resistant gene, nptII and Ampicillin resistant gene, bla (Verachtert and Mot, 1990).

3.0 MATERIALS AND METHOD

3.1 General Method

3.1.1 Sterilization

All glassware and media were autoclaved at 15 psi (121°C) for 15 minutes.

3.2 Culturing Media

3.2.1 Luria Bertani Agar (LA) and Broth (LB)

LA was prepared by adding 10.0 g/L tryptone, 5.0 g/L yeast extract, 5.0 g/L NaCl and 1.5% (w/v) of agar powder and top up till 1L with distilled water. LB was prepared by adding same ingredients as used in making LA with the exception of agar powder. Both LA and LB were autoclaved before use.

3.2.2 Agrobacterium Tumefaciens Culture

Single colony of Agrobacterium Tumefaciens GV3101 is inoculated into LB medium. Appropriate antibiotics are included when necessary. The culture is left to grow at 28°C with 150rpm shaking. It will take approximately 16 hours (overnight) to grow Agrobacterium Tumefaciens GV3101 in 100 mL LB.

Single colony of Agrobacterium Tumefaciens GV3101 is streak onto LA plate. Appropriate antibiotics are included when necessary. The culture is then left to incubate at 28°C in the lab. It will take approximately 2 days for the colonies to appear.

3.2.3 Walne's Medium for Chlorella sp culture

Walne's medium was prepare as described by (Walne, 1970). Walne's medium is made up of, Trace Metal Solution (TMS), Vitamin Solution and Nutrient Solution.

3.3 Preparation of Glycerol Stock

A single colony of Agrobacterium tumefaciens was inoculated into 3 mL LB medium. 50 µg/mL of Gentamicin and 25 µg/mL of Rifampicin were included in the LB medium. The culture was grown for 16 hours (overnight) at temperature 28°C with 150 rpm shaking. 675 µL of the overnight culture was pipetted into a sterile microcentrifuge tube. A volume of 325 µL of autoclaved 40% glycerol was pippeted into the eppednorf tube. The mixture was mix thoroughly by tapping several times. The microcentrifuge tube was then stored at -70°C. Glycerol stock of Agrobacterium tumefaciens was prepared as described by (Sambrook et al., 1989)

3.4 Construction of pVT200

Plasmid vector, pVT200 are constructed by the combination of two separate plasmid; pVT101 and pYAC-RC. pVT101 is derived from pBI121.

pBI121 is a expression vector commonly use in plant transformation.pVT101 size is approximately 9 kb. pVT101 contained multiple cloning site (MCS) such as HindIII, SalI, XmaI and EcoRI.

pYAC-RC is derived from its ancestor, pYAC3. pYAC-RC carryied ampicillin resistance gene as well as the artificial centromere gene, cen4. This yeast artificial centromere plasmid, have multiple cloning site (MCS) such as SmaI and XhoI. pYAC-RC can be included into Escherichia coli or into Saccharomyces cerevisiae as an artificial centromere. The overall size of this plasmid is approximately 16 kb.

The construction of pVT200 starts by digesting pVT101 with SalI restriction enzyme. Due to the fact that pVT101 only contained one site for SalI, the plasmid will linearize without any changes in its size. pYAC-RC is digested with XhoI restriction enzyme. This digestion will produce two separate plasmids with the size of 13 kb and 3 kb, respectively. The 13 kb pYAC-RC contained gene cassettes consists of, cen4, ars1, trp1, ampR and oriColE1. This plasmid was then ligated with pVT101 by using ligase to form pVT200. pVT200 is a plasmid carrying Kanamycin and Ampillin resistance gene, cen4 gene as well as oriColE1 gene. pVT200 is approximately 21kb. Figure 3.1 showed the construction scheme of pVT200.

3.5 Transformation of pVT200 Into Agrobacterium tumefaciens

3.5.1 Preparation of competent cells

A single colony of Agrobacterium Tumefaciens was inoculated into 5 mL of LB. 50 µg/mL of Gentamycin and 25 µg/mL of Rifampicin was included in the LB medium. The culture was incubated at 28°C with 150rpm shake for 16 hours (overnight). 3 mL of the overnight culture are then inoculated into 100 mL of LB. 50 µg/mL of Gentamycin and 25 µg/mL of Rifampicin are included in the LB medium. The culture is then left for 5 hours at 28°C with 150 rpm shaking. The culture are the transferred to four sterile 50 mL tubes, with each 50 mL tubes containing 20 ml of the culture. The cultured was centrifuged at 4,000xg at 4°C for 5 minutes. The supernatant was discarded before all the tubes were inverted for 60 seconds. Each tube containing pellet was resuspended with 20 ml of iced-cold 10% glycerol, before they were centrifuged again at 4,000xg at 4°C for 5 minutes. Supernatants were discarded from all the 50 mL tubes. The pellets were resuspended again with 4 ml iced-cold 10 % glycerol. All four 50 mL tubes were combined and aliquot into two new sterile 50 mL tubes before they were centrifuged again at 4,000g at 4°C for 5 minutes. Supernatants from all 50 mL tubes were discarded. The culture were wash again by resuspended them in 2 ml iced-cold 10 % glycerol before poured into a new 50 mL tube. The 50 mL tube containing all the pellets from all four previous 50 mL tubes were then centrifuged 4,000xg at 4°C for 5 minutes. The pellet was wash again with 1.5 ml iced-cold 10 % glycerol after discarding its supernatant. All of the culture was aliquot into fifteen pre-chilled microcentrifuge tubes, each containing 100 µl of competent cells. All eppedorf were stored at -20°C. This method was a modification from a method described by (Main et al., 1995)

3.5.2 Electroporation

Tubes containing competent cells were left to thaw slowly on ice. 50 µL of competent cells were transferred into a sterile microcentrifuge tube. 2 µL of pVT200 are added to microcentrifuge tube containing the competent cells. The competent cells were allowed to mix with pVT200 by tapping. The mixture was transferred to a pre-chilled 0.2 cm electroporated cuvette. The Micropulser ™ (Bio-Rad) was set at "agr" which stand for Agrobacterium Tumefaciens. The cuvette containing the mixture was then place in the chamber slide. The cuvette are push into the chamber until the cuvette are placed between the contact in the base of the chamber. The mixture of competent cells and pVT200 was pulse once.1 ml of LB medium was transferred immediately into the 0.2 electroporated cuvette. The electroporated Agrobacterium Tumefaciens are then left to incubate on shaker at 28°C for 3 hours. 100 µL of the electroporated Agrobacterium tumefacies are then plate onto selective LA medium. 50 µg/mL Kanamycin, 50 µg/mL Gentamicin and 25 µg/mL Rifampicin are included in the LA medium. A negative plate is also prepared. A negative plate was plated with competent cells which was not electroporated with pVT200. For each colony that was formed on the plate, was isolated and spread onto a new LA plate with the addition of antibiotics. The antibiotics that were added on each plate were 50 µg/mL of Kanamycin and 25 µg/mL of Rifampicin. All plates were left to incubate for three days in the dark room at 28°C.

3.6 Plasmid extraction

A single colony of transformed Agrobacterium Tumefaciens was inoculated into a 10 mL LB medium. 50 µg/mL of Kanamycin and 50 µg/mL of Rifampicin were added into the LB medium. The culture was incubated at 28°C with shaking of 150 rpm for 16 hours (overnight). The culture was then transferred into a sterile 50 mL tube. It was then centrifuged at 14,000xg for 1 minute. All supernatant was discarded by using pipette. The pellet was resuspended with 100 µL ice-cold Solution I (Refer Appendix A). All the contents inside the 50 mL tube were transfer into a sterile microcentrifuge tube using pipette. The mixture was left to incubate at room temperature for 5 minute. 200 µL of Solution II (Refer Appendix B) was added to the mixture. The mixture is mixed by inverting the microcentrifuge tube several times. The mixture was then left to incubate at room temperature for 5 minute. A volume of 150 µL of Solution III (Refer Appendix C) was added. The mixture was then mix thoroughly by vortex. The mixture was centrifuged for 15 minute at 12,000xg. The supernatant was then transferred into a new sterile microcentrifuge tube by using pipette. A volume of 1 mL of iced-cold 95% ethanol was added to the supernatant. The mixture was mixed by invert. The mixture was centrifuged at 12,000xg for 15 minute. The supernatant was removed from the mixture by pipette. A volume of 1 mL of 70% ethanol was added and centrifuged again at 12,000xg for 10 minutes. The supernatant was then discarded. The leftover supernatant was the left to be aspirated by air drying the pellet. A volume 0f 30 µL of deionized distilled water (ddH20) was added and the plasmid was stored at -20°C for future use. Plasmid extraction was done as described by (Sambrook et al., 1989)

3.7 Plasmid Purification

The extracted plasmid was purified using the Wizard®Plus, SV Minipreps DNA Purification System kit (Promega, USA). The purification process is essential to ensure the quality results will be obtained from gel electrophoresis.

One to five mL of overnight bacterial culture was harvested and centrifuged at 10,000xg for 5 minute. The supernatant was discarded and blotted on towel paper to remove excess media. Then, 1250 µL of Cell Resuspension Solution was added and resuspended. After that, 250 µL of Cell Lysis Solution was added and thoroughly mixed by inverting 4 times. Then, 10 µL of Alkaline Protease was added and mixed by inverting. The mixture was centrifuged at 14,000xg for 10 minutes. The clear lysate was transferred into spin column and centrifuged at maximum speed for 1 minute. Then 750 µL of Column Wash Solution previously diluted with 95% ethanol was added and centrifuged at maximum speed for 1 minute. The same process was repeated by adding 250 µL of the Column Wash Solution. Centrifuged at maximum speed for 2 minute and the spin column were transferred into a microcentrifuge tube. Finally, 100 µL of Nuclease-Free water was added and centrifuged at maximum speed for 1 minute. The purified plasmid was stored at -20°C

3.8 Gel Electrophoresis

3.8.1 Preparation of Agarose gel

0.7% agarose gel was prepared by weighed 0.14g of agarose powder. 20 mL of TAE buffer was then added to the agaros powder. The mixture was heat until all agarose was dissolved. The mixture was allowed to cool for 5 minutes. A wedge was then placed inside the casting tray. An 8-well comb was placed firmly onto the casting tray. The cooled agarose then poured into the casting tray.

3.8.2 Loading samples

3 µL of DNA samples was added into a 2 µL of blue bromophenol dye. The mixture was resuspend several times. 3 µL of Lambda HindIII restriction enzyme DNA marker was added into another 2 µL of blue bromophenol dye. The comb is taken out was the agarose gel have cooled. The wedge containing the agarose gel was then put inside the tank. The tank was filled with TAE buffer until the agarose gel submerges. The first lane was then loaded with DNA marker. The rest of the lane was loaded with DNA samples. The leads were then connected to the power supply. The current was set at 90v before it was turn on. The gel was run until the ¾ across the agarose gel.

3.8.3 Staining

The wedge was taken out and submerge it the Ethidium Bromide (EtBr) staining solution. The agarose gel was left to be stain for 10 minutes. Remove the agarose gel and submerge it into distilled water for 5 minutes. The gel was then observe under UV light.

3.9 Axenic culture of Chlorella sp

A single colony of Chlorella sp was streak on LA medium added with antibiotic. The plates were incubated for two weeks. The antibiotic used was Ampicillin (25µg/mL), Chloramphenicol (20µg/mL), Straptomycin (25µg/mL) and Tetracycline (30µg/mL). Only plates with viable Chlorella sp and no contamination are considered as a positive result.

4.0 Results

4.1 Chlorella sp colonies grown on Conway medium

For the first two weeks, it appears that all plates were contaminated with a lawn of whitish unidentified bacteria. All efforts to achieve aseptic conditions were done when culturing a new batch of Chlorella sp plates. However, the plates were still contaminated with the same whitish unidentified bacteria. After two more weeks, the lawn of that unknown bacterium seems to periodically vanish and Chlorella sp appears to grow on top or near the bacteria.

Out of ten plates, only two plates appear to have colonies of Chlorella sp. It was expected that the Chlorella sp will take 2 weeks to grow on Walne's medium, however it took approximately one month for the first colony to appear.

4.2 Transformation of Agrobacterium tumefaciens

After two days of incubation, there were three single colonies observed on plate which was spread with electroporated Agrobacterium tumefaciens. There was no single colony form on the negative plate. Both plates were left to incubate for another day. On the third day of incubation, the number of single colony has increased from three colonies to five colonies, and no single colony form on negative control plate. Both plates were left for another day in the dark room. On the fourth day, the number of colonies formed on the plate spread with electroporated Agrobacterium tumefaciens has not change. Hence, the total of colonies of successful electroporated Agrobacterium tumefaciens observed on the LA plate is five colonies, with no observed growth detected in the negative plate

4.3 Plasmid Extraction

The size of the original pVT200 was determined by running the plasmid through agarose gel and observed it under the UV light. The size was determined to be approximately 23 kb (Figure 4.8)

The extracted plasmids from electroporated Agrobacterium tumefaciens was then run through agarose gel and observed under the UV light. It showed the presence of a plasmid in each colony. Figure 4.9 shows the result of electrophoresis of pVT200, plasmids extracted from Agrobacterium tumefaciens and respected DNA marker.

4.4 Axenic culture of Chlorella sp

All 10 plates showed no growth of Chlorella sp after two weeks of incubation. Moreover, six out ten plates were contaminated with fungi and whitish unidentified bacteria. The other four plates were left for another two weeks to incubate. After one month of incubation, all four plates showed no sign of Chlorella sp colonies

5.0 Discussion

5.1 Growth of Chlorella sp

Chlorella sp were initially grown on a set of ten plates of Conway medium. The plates were then left to incubate at 25°C and were illuminated by cool florescence lamps at 1450-1490 lux for 24 hours. It was expected that the culture were able to showed initial growth by the end of two weeks incubation period. However, it appears that all plates were contaminated for the first batch. For the subsequent two more batches, aseptic techniques were far more stringent as well as the source of Chlorella sp for the initial inoculum were purified to ensure no contamination. However, contamination still exists. The contaminations consist of either fungi or unknown whitish bacteria.

As mention earlier, all Chlorella sp were left to incubate for two weeks, but due to unexpected event of contamination and also repetition of culturing a new batch of Chlorella sp under strict aseptic conditions does not resulted in any changes, all contaminated plates were left for another two weeks. Surprisingly, after approximately three weeks the lawn of bacteria who initially dominated the plate appears do periodically diminished in numbers. Moreover, Chlorella sp colonies begin to grow on top or near the dead cells of those unknown bacteria. Hence, at the end of the extended one month incubation period, two out of ten plates manage to produce a lawn of Chlorella sp.

Hence, a relationship between algae and bacteria might exist. In many cases, an axenic culture of algae is needed in order to ensure reliable results can be achieved. Axenic culture is a culture which is free of any bacteria or any microorganisms' contamination. In reality, it is quite difficult to achieve axenic culture, although strict aseptic measurements should always be taken to decrease bacterial contamination, but it is quite acceptable to accept a degree of contamination (L.Barsanti, 2006; Fritsch, 1935). The relationship between algae and bacteria might not be species specific. This relationship proved to be beneficial for the growth of algae, if the associated bacteria produce growth factors or C02 needed by algae (Lange et al., 1971). The relationship might also appear essential to the growth of algae. In growing pelagic diatoms, Phaeodactylum triconutum and Coscinodiscus concinnus it was observed that in the absence of associated bacteria, a successful culture can be established (Droop and Elson, 1966).

5.2 Transformation

5.2.1 Electroporation

Transformation is any changes in organism's characteristics through the transfer of naked DNA (Black, 2008). Transformation in this experiment has to occur in two stages. The first one is to transform Agrobacterium tumefaciens with pVT200. From there, we use Agobacterium tumefaciens to naturally transform Chlorella sp. Nowadays, transformation can be done in numerous ways.

Protoplast fusion is a technique which required enzymatically removing the cell walls of two organisms to allow the fusion of their genetic materials (Black, 2008). Protoplast fusion will allowed two organisms to fuse together their entire genomes, hence avoiding difficulty in transferring the right promoters and also produced a hybrid organism with all desirable characteristics (Black, 2008; Peberdy and Ferenczy, 1985). Chemically-induced fusion use chemical substance such as Polyethylene Glycol (PEG) which cause the membrane cell to shrink due to the removal of water by hydrophilic region of PEG, this will caused an increase in the fluidity of the membrane bilayer resulting in the tendency to fuse with other membrane plasmid (Altman and Colwell, 1998). Microinjection is a molecular technique which use glass micropipette to inject desired substances or even genetic material such as gene directly into a living cell (Pawley and Masters, 2008). Agroinfection, is a method which combine the ability of Agrobacterium and virus such as Maize Streak Virus (MSV) to transform plant. This method is achieved by incorporating viral genome into T-DNA section of Agrobacterium prior to infection. One the advantage of using Agrobacterium-mediated virus infection to transform plant is that, transformation can be done on wide range of plants, not restricted only dicotyledons plants which the traditional Agrobacterium-mediated transformation cannot transform (Chawla, 2002).

To transfer pVT200 which carried cen4 gene is transfer into Agrobacterium tumefaciens is done by electroporation. Electroporation is a method which exposes the cells to brief exposure of electric field, which enable the transfer of material across the membrane (Main et al., 1995). For this experiment, transformation step using electroporation is successful. However, the number of colonies that have been transformed should be higher. An efficient transformation of Agrobacterium tumefaciens can be as high 1x108 to 3x108 transformants per microgram (µg) DNA (Mersereau et al., 1990).

Traditionally, transformation was done by using the freeze-thaw or triparental method. Freeze-thaw method is method which the cells are rapidly freeze then slowly thawed, which caused the membrane to accept DNA through their pores. Triparental method used the conjugation process. This method needed another microorganism, for example Escherichia coli to receive the DNA through transformation, before transferring it to another microorganism (Nickoloff, 1995). Freeze-thaw method tends to be inefficient, with resulting only 103 transformant per microgram (µg) of DNA and also costly (Nickoloff, 1995; Chichester, 1980). While triparental seem to be time consuming as well as, inefficient compared to electroporation. Moreover, triparental required the needs of complex media and selection method (Julio Salinas, 2006).Therefore, electroporation is the best method to use to transform Agribacterium tumefaciens.

5.2.2 Plasmid Extraction

Plasmids which have been extracted from electroporated Agrobacterium tumefaciens were analyze by agarose gel electrophoresis and observed under the UV light. Under the UV light, it appeared that the size of plasmids, pVT200 and extracted plasmid form Agrobacterium tumefaciens was around 23.13 kb. However, further analysis should be done. The most common and accurate method is plasmid digestion analysis. In this method, restriction enzyme is use to digest both the original pVT200 and also extracted plasmids from Agrobacterium tumefaciens. If both plasmids are identical, they should produce the same band(s) of the same size.

5.4 Axenic culture of Chlorella sp

As mention earlier, to establish an axenic culture of algae is difficult due to its relationship with the associated microorganism, primarily bacteria. However, there have been numerous method that can be use to decrease the amount of contamination to the level that can be tolerated. Purification of algae can be achieved either by nutritional enrichment method, mechanical manipulations, antibiotic treatment or the combination of these three methods (Droop, 1969).

Although other methods such as treating algae culture with gamma radiation and UV light can produced axenic culture, but it also can caused possible damage to algal cells. However, some algae are reported to be quite resistant towards gamma radiation and UV light. Blue-green algae can be treated with gamma radiation and UV light to produce an axenic culture (Bowyer and Skerman, 1968).For motile algae, scoring the agar surfaces before incubation can provide an efficient and rapid separation of gliding filamentous algae such as cyanobacteria from bacteria colonies (Vaara et al., 1979). Scoing method involved making a parallel line on the agar surface to provide a way for motile algae to glide away from the contaminants. However, antibiotic treatments provide an alternative way to produce axenic culture. Some methods discuss above, are specific for certain species of algae, however antibiotic treatments can be apply to all algae species. In order to use antibiotics to produce axenic culture, first we have to determine which antibiotics that bacteria are sensitive as well as the concentration of antibiotic to use in order to produce viable algae culture. Moreover, the exposure time of the antibiotics to both algae and contaminants have to be short but effective. This to ensure that, no development of antibiotic resistant algae and bacteria can be formed, but at the same time eliminate all contaminants (Jones et al., 1973).

Although the antibiotic treatments seem to provide a good and easy way to produce an axenic culture, information about the contaminants are needed. This will enable us to formulate a combination of the most effective antibiotics that can eliminate all contaminants but still produce viable algae cells.

An attempt to establish an axenic culture of Chlorella sp failed might due to the wrong combination of antibiotics used. The concentration of antibiotics also may cause the failure in eliminating all contaminants. Hence, further analysis can be done by isolating and identifying all bacteria associated with the algae before and after the antibiotic treatment. Next, the contaminants should undergo antibiotic sensitivity test. This will provide us with the information needed to modify as well as increase the effectiveness of the antibiotics treatments on all contaminants but at the same time produce viable axenic culture of algae.

It was reported that an antibiotics treatment combination consisting of, Benzyl-penicillin-SO4, Tetracycline, Chloramphenicol, Aureomycin, Ceporin and neomycin-SO4 can effectively eliminate all contaminants to produce axenic culture of Chlorella vulgaris (Jones et al., 1973). Hence, it is highly suggested to repeat this experiment by using the antibiotics combination used by Jones.

6.0 CONCLUSION

Agrobacterium tumefaciens GV3101 (pMP90RK) has successfully been transformed with pVT200 by electroporation. All Agrobacterium tumefaciens GV3101 (pMP90RK) transformants was able to grow on LA medium with the presence of Kanamycin. However, further analysis such as digestion of the extracted plasmid from Agrobacterium tumefaciens GV3101 (pMP90RK) should be done to confirm this result.

Cultivation of Chlorella sp on Walne's medium was successful. Nevertheless, the culture was heavily contaminated with unknown microorganisms. It was propose that there was an obligatory relationship between algae and bacteria which seem to prevent from obtaining an axenic culture. Future study should be directed at discovering the length of this obligatory relationship.

An axenic culture of Chlorella sp failed to achieve due to the difficulty to eliminate all contaminants by using antibiotics treatment. A new set of antibiotics combination consist of Benzyl-penicillin-SO4, Tetracycline, Chloramphenicol, Aureomycin, Ceporin and neomycin-SO4 should be use in future attempt to obtain an axenic culture of Chlorella sp.

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