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 (Fritsch 1935; L.Barsanti 2006). 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).
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 (Figure 5.1).
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 (Peberdy and Ferenczy 1985; Black 2008). 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 (Chichester 1980; Nickoloff 1995). 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