Agrobacterium Tumefaciens is a rod shaped gm negative bacteria. It is the major causal agent of crown saddle sore diseases in magnoliopsid workss from over 600 species belonging to 90 households ( De Block et al. , 1985 ) . Agrobacterium Tumefaciens induced grown gall disease by incorporating a portion of its tumour inducement or Ti plasmid into the genome of the works. The specific section from the Ti plasmid that was transferred is called transferred or T-DNA ( Walden, 1988 ) . Since the last decennary, much involvement has been showed to turn Agrobacterium Tumefaciens into a vector for transmutation of workss.
Chlorella sp is singled-cell green algae from the Chlorophyta division under the Chlorellaceae household ( Hoek et al. , 1995 ) . These green algae are spherical in form, with diameter from 2 to 10 µm and have no scourge. It contains contain photosynthetic pigment chlorophyll-a and chlorophyll-b embedded in its chloroplast ( DuPont, 2009 ) . Chlorella has late been taken as nutrient addendum as it contains Chlorella Growth Factor ( CGF ) . CGF consists of all constituents inside Chlorella sp which become the chief factor to its astonishing healing belongingss ( Ley, 2003 ) . In Japan, Chlorella sp has been taken as a addendum in the signifier of tablets, capsules, liquid infusion and nutrient addictive ( Halperin et al. , 2003 ) .
Centromere in eukaryote chromosome controls the segregation of girl cells during mitosis and miosis event by forming the cells through microtubules connected to their centromere ( Alberts, 1998 ) . In the barm such Saccharomyces cerevisiae it is known that merely a individual microtubule are bound to each kinetochore ( Peterson and Ris, 1976 ) . CEN 4 is a little DNA component 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 act as functional chromosome in barm, allowed it to retroflex successfully in mitotic division and the first meiotic division ( Clarke and Carbon, 1980 )
This experiment is done to look into whether a yeast cistron, cen4 can be integrated into works genome through Agrobacterium-mediated transmutation, therefore converted it into a chromosome capable of retroflexing in barm. 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 present cistron of involvement into yeast genome.
The aims of these surveies are
- To transform Agrobacterium tumefaciens with PVT 200
- To set up an axenic civilization of Chorella sp
- To transform Chlorella sp by Agrobacterium tumefaciens transporting the centromere cistron
2.0 LITERATURE REVIEW
2.1 Agrobacterium Tumefaciens
Agrobacterium tumefaciens has long been the beginning of crown saddle sore disease in magnoliopsid workss ( Walden, 1988 ) . Crown saddle sore is a works disease which causes disorganised growing on the root of the works near the surface of the dirt ( Walden, 1988 ) . Since its find, Crown saddle sore has been described as a neoplastic disease ( Nester et al. , 1984 ) . The morphology of the tumour may determined by the formation of unorganised callosity construction or teratomas ( Levine, 1919 ) . Host works species and besides the beginning of infection are among the factors impacting the morphology of crown saddle sore to be form ( Gelvin, 1990 ) . At the beginning of the century, it was found that a pure civilization of bacterium which has been isolated from tumorous tissue can be use to bring on tumour formation in a healthy magnoliopsid works ( Binns, 2008 ) . Further observation confirmed that Crown saddle sore tumours can be transplanted from works to works similar to what happen in carnal tumours ( Walden, 1988 ) . During 1990 ‘s, Bacterium tumefaciens has been determined as the earliest bacteriums known to do crown saddle sore diseases in works by ( Smith and Townsend, 1907 ) . A thorough word picture of the bacteria including their form, size and growing has besides been characterized. Since so, the name Bacterium tumefaciens has been changed to Agrobacterium tumefaciens as we know today.
Since 1970 ‘s, there were many hypothesis sing the mechanism of DNA transportation from Agrobacterium tumefaciens to the works genome. It was strongly suggested so, that a bacterial plasmid might play a major function in doing this disease ( Watson et al. , 1975 ) . A comparing between 11 deadly Agrobacterium tumefaciens with 8 avirulent strain showed that the virulent strain have a big plasmids whereas, avirulent deficiency such plasmids ( Zaenen et al. , 1974 ) , . Transportation of this big plasmid from virulent to avirulent strain of Agrobacterium tumefaciens can be done by familial exchanges between these two strains ( Kerr, 1969 ) . It was confirmed that the avirulence strain acquired virulency after having plasmids from the virulency strain ( Kerr et al. , 1977 ; Kerr, 1971 ) .Interestingly, a item expression at the difference between avirulence strain with virulency strain revealed that a batch of cistrons involve in tumorigenicity located on a plasmid called Ti-plasmid, which avirulence strain deficiency such cistrons on that plasmid ( Hooykaas et al. , 1977 ; Zaenen et al. , 1974 ) .
Ti-plasmid is a dual isolated round Deoxyribonucleic acid of about 200 kilobases ( kilobit ) in size ( Walden, 1988 ) . It is known now that, Ti-plasmid is involved in doing crown saddle sore disease in works. The transportation of Ti-plasmid is affected by temperature ( thermosensitive ) ( Temp & A ; eacute ; et al. , 1977 ) . Ti-plasmid can be transfer through junction between avirulent strains and virulent strain ( Temp & A ; eacute ; et al. , 1977 ) . However, when junction was done at temperature above 30 & A ; deg ; C, the transportation frequence lessening significantly ( Temp & A ; eacute ; et al. , 1977 ) . This was due to the fact that Ti-plasmid was loss at temperature 37 & A ; deg ; C ( Watson et al. , 1975 ) . Ti-plasmid is made up two major parts. They are transfer or T-DNA part and Virulence part. In T-DNA part, there are two 25-bp direct repetition 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 boundary line is important as it is required in Commonwealth of Independent States for T-DNA transportation, while the left boundary line merely act as a splitter 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 cistrons. Opines is a substances produce by Agrobacterium tumefaciens which supply the bacterium with N every bit good as energy ( Kaper and Hacker, 1999 ) . Opines can be found inside the tumour which induced by Agrobacterium tumefaciens in workss ( Walden, 1988 ; Kaper and Hacker, 1999 ) . Genes responsible for bring forthing opines are located in a Deoxyribonucleic acid section, called transportation or T-DNA ( Kaper and Hacker, 1999 ) . The type of opines been green goodss are strain-dependent. So far, 30 opines has been discovered. Nopaline, octopine, succinomapine and leucinopine are illustration of opines been produced by Agrobacterium tumefaciens ( Walden, 1988 ) .
Aside from cistron for opine synthesis and the boundary lines, T-DNA besides have cistrons responsible for the synthesis of auxin and cytokine. These two compounds will be produced inside transformed works which will ensue in the formation of tumour ( Zupan and Zambryski, 1995 )
The virulency part is where the cistrons that responsible of releasing enzymes which will ease the integrating of T-DNA part into works genome ( Gelvin, 2000 ) . The virulency part is made up of an operon system dwelling of virA, virB, virC, virD, virE virF and virG ( Schrammeijer et al. , 2000 ) .
2.3 Transportation of T-DNA
The mechanism affecting the transportation of T-DNA from Agrobacterium tumefaciens into works and the integrating of that T-DNA into works genome required chemical interaction between the pathogen ( Agrobacterium tumefaciens ) and the host ( works ) ( Gelvin, 2000 ) . This series of interaction involved chemical signals such as, impersonal or acidic sugars, phenolic compounds, opines, vir proteins and T-DNA ( Gelvin, 2000 ; Winans, 1992 ) .
2.3.1 Initiation of T-DNA transportation procedure
Wounded works tissue releases several phenolic compounds, such as acetosyringone. This phenolic compound acts as inducer of the vir cistrons ( Dye et al. , 1997 ; Messens et al. , 1990 ; Xu et al. , 1993 ) . At the same minute, nopaline-base Agrobacterium tumefaciens will release trans-zeatin compound which will fix works cells for the chance of transmutation procedure ( Walden, 1988 ) . This was done by originating cell division ( Walden, 1988 ) . Agrobacterium tumefaciens sense phenolic compound released by hurt works by virA cistron merchandise, VirA sensory protein ( Table 2.1 ) . virA have the autophosphorylating capableness. This allowed virA to phosphorylate itself. An autophosphorylated virA will so phosphorylated virG by donating phosphate to virG ( Jin et al. , 1987 ; Jin et al. , 1990 ) . Phosphorylated virG bind to consensus sequence before activate vir cistrons written text procedure ( Table 2.1 ) .
2.3.2 Preparation of T-DNA for transportation
Prior to the look of vir cistrons, virD1 and virD2 will get down to fix T-DNA for transportation ( Table 2.1 ) . At this phase, T-DNA is still inside Ti-plasmid. virD2 nicks 25-bp direct repetition boundary lines of T-DNA to divide 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 ‘ terminal of T-DNA through the formation of covalent bond with tyrosine ( Gelvin, 2000 ) . However, when nicking a dual isolated Deoxyribonucleic acid, virD2 will necessitate the aid of virD1 ( Jayaswal et al. , 1987 ) .
2.3.3 Transportation of T-DNA to works cell
As virD2 bind to 5 ‘ terminal of T-DNA, it will steer the Deoxyribonucleic acid from Agrobacterium tumefaciens to works genome. A “ span ” from Agrobacterium tumefaciens to works cells is constructed by the look of virB ( Table 2.1 ) . virB coded for hair like construction which will let T-DNA, every bit good as virE2 move to works cells. virE2 is really of import in set uping transmutation procedure. The complex which consists of individual stranded T-DNA and virD2 is called T-complex, and this construction is coated with virE2 ( Howard and Citovsky, 1990 ) . During the transportation of T-complex from Agrobacterium tumefaciens, virE1 is needed to aids virE2 ( Deng et al. , 1999 ) .
2.3.4 Integaration of T-DNA into works genome
T-complex is protected by nucleolytic digestion by virE2. virE2 and virD2 facilitate integrating of T-complex with works genome ( Table 2.1 ) . The integrating of T-complex with works chromosomes is known as illicit recombination ( Gheysen et al. , 1991 ) . The individual isolated T-complex is foremost converted into a dual isolated Deoxyribonucleic acid signifier. virD2 so insert the dual isolated T-complex into works genome ( Gelvin, 2000 ) . This ligation proceeds expeditiously due to the fact that virD2 bind to 5 ‘ terminal of T-complex hence protecting it from any nucleolytic onslaught ( Gelvin, 2000 ; Kumar and Fladung, 2002 ) . virE2 may affect indirectly in integrating procedure. It was suggested that virE2 may protected 3 ‘ terminal from nucleolytic onslaught, hence enable a precise integrating procedure ( Gelvin, 2000 ) .
2.4 Ti-Plasmid as a vector
Since the find of Ti-plasmid ability to reassign T-DNA into works cells, therefore transforming the works, involvement of turning Ti-plasmid into transmutation vector has mounted. Scientist position Agrobacterium tumefaciens as a natural familial applied scientist of workss ( Walden, 1988 ) . Ti-plasmid has a mechanism which enables the integrating of foreign DNA into works genome in much more easy manner ( Walden, 1988 ) .
Among the characteristic that Ti-plasmid had which makes it a suited works transmutation vector are:
- The vir part of Ti-plasmid map in trans
- Foreign DNA which inserted between man-made or natural 25-bp direct repetition boundary lines of Thursday the T-DNA will be transferred to the works cell.
- There will be no rearrangements or alterations occur of the DNA located between the T-DNA boundary lines during the transportation procedure
- The foreign DNA which transferred to the works cell will be stably integrated into works genome and can be inherited in a Mandelian mode
Ti-plasmid can be converted into a utile vector by canceling all the oncogenic maps that can be found in the T-DNA, without impacting the vir part ( Zambryski et al. , 1983 ) . During 1983, a nopaline-based Ti-plasmid, pGV3850 was modified by canceling oncogenic constituents inside its T-DNA part and replaced by pBR322. pBR322 is a unreal plasmid which contained a replicon part, the ampR cistron which encoded for Ampicillin resistant and tetR cistron which encoded for Tetracycline immune cistron. This plasmid besides has limitation sites for 40 limitation enzymes which lie within tetr and ampr ( Zambryski et al. , 1983 ) . It was so proved that reengineering Ti-plasmid such as pGV3850 by taking all the oncogenic constituents in the T-DNA part will still keep the Ti-plasmid ability to transformed works cells ( Zambryski et al. , 1983 ) .
The omission of oncogenic cistrons resulted in non-oncogenic Ti-plasmid or disarmed Ti-plasmid. Due to the fact that, omission of oncogenic cistrons left transformed works cells to look in normal morphology, some sort of sensing method is needed ( Argawal, 1998 ) . In order to accomplish a coveted cistron look, suited boosters, expiration and interlingual rendition part demand to be included in Ti-plasmid vector. Nowadays, a broad scope of selectable marker cistrons are included in Ti-plasmid vector. For illustration, kanR cistron which codification for an enzyme called Neomycin Phosphotransferase Type II or NPTII is frequently use as a selectable cistron marker in transformed works cells ( Chawla, 2002 ) . In order for this cistron to be express, it has to be provided with works booster and eradicator. Ti-plasmid as a vector can be either trans or Commonwealth of Independent States. The differences between these two type of Ti-plasmid vector is than in the 25-bp repetition boundary lines is constructed form the same or different replicon sequences from the vir part ( Walden, 1988 ) . Different sequences will bring forth binary vector while, a same order of sequences will bring forth co-integrative vector ( Argawal, 1998 ) .
2.5 Binary and Co-Integrative Vector
Binary vector consist of integral vir part as good disarmed T-DNA. T-DNA is located on a plasmid called miniplasmid ( Razdan, 2003 ) , which besides bears cistrons such as selectable marker. This allowed the selectable marker and T-DNA to be inserted as a whole. Another plasmid holds vir cistrons. vir cistrons will be supplied in trans on the miniplasmid to reassign T-DNA every bit good as the selectable marker cistrons into works cells ( Walden, 1988 ) . The benefits of utilizing binary vector are it able to retroflex both in Agrobacterium tumefaciens and Esherichia coli ( Argawal, 1998 ; Razdan, 2003 ; Walden, 1988 ) . Transfromation of workss by utilizing binary vector normally take topographic point by infixing cistron of involvement into Escherichia coli before reassigning the cistron of involvement into Agrobacterium tumefaciens by tri-parental coupling or electroporation. The celebrated & A ; lsquo ; Bin ‘ series of plasmids and pBi121 is among illustrations of binary vector ( Razdan, 2003 ) .
Co-integrative vector lacks the ability to retroflex inside Agrobacterium tumefaciens. Hence, this plasmid needs to be co-integrated with an endogenous Ti-plasmid ( Razdan, 2003 ) . The integrating returns through a section of DNA common to both plasmids. pBR322 discussed earlier is one of an illustration of co-integrative vector. A typical characteristic of a co-integrative vector is that it retain the orientation of vir cistrons and T-DNA maps in a Commonwealth of Independent States constellation ( Walden, 1988 ) .
2.6 Agrobacterium Tumefaciens-mediated Transformation
Agrobacterium tumefaciens presents have been studied adequate to let scientist to utilize their alone capableness to transform workss. However, there were several factors impacting Agrobacterium-mediated transmutation procedure. Naturally, Agrobacterium tumefaciens can merely do crown saddle sore in magnoliopsids workss. Hence, the usage of Agrobacterium tumefaciens to transform economical liliopsids workss such as wheat ( Oryza sativa ) and maize ( Zea Mayss ) are limited. The ground why Agrobacterium tumefaciens can non stably integrate their T-DNA into monocots workss is possibly monocots works had a better proofreading system, which may curtail the T-DNA integrating into works genome ( Tinland, 1996 ) . However, presents Agrobacterium tumefaciens capableness to transform liliopsids works has been reported. In 2002, Agrobacterium-mediated transmutation of corn embryo have successfully achieved ( Frame et al. , 2002 ) . Besides that, it is now no longer limited to workss transmutation. It was reported by ( Soltani et al. , 2008 ) that Agrobacterium tumefaciens were able to transform at least 80 non-plant, which included Fungis, algae and even mammalian cells. In 2004, ( Hooykaas, 2004 ) reported that Agrobacterium-mediated transmutation has been reported in cereal and Fungi. Agrobacterium tumefaciens was besides able to stably transportation and integrate familial stuffs into mammalian cell in a manner similar to the mechanisms occurred in workss ( Kunik et al. , 2001 ) .
Algae can split into two major group, either macroalgae or microalgae ( Anne E. Osbourn, 2009 ) . Algae are plant-like being, in a manner that they does non possess true roots, foliages, stems or even vascular tissues. They have simple reproduction systems normally consist of surrogate coevals ( L.Barsanti, 2006 ) . They normally exists as a photosynthetic and aquatic being which come in assorted size from microscopic to every bit big as 50m in length ( Joseph, 2005 ) . Algae have become a prized being due to its huge benefits that include as biodiesel to replace the demands of dodo fuel, nutrient addendum and fertiliser ( Chisti, 2008 ; Mulbry et al. , 2005 ) .
Microalgae has emerge the most reliable biofuel that can wholly replace fossil fuel in transit without impacting our nutrient beginning or harvest merchandises ( Chisti, 2008 ) . Microalgae are believed to be a better biofuel than any other suggested renewable fuel such as oil thenar and bioethanol from sugar cane, in a manner that algae can be grown in the country where there will be no competition with nutrient harvests ( Chisti, 2008 ; Neltner, 2008 ) . Algae besides have been suggested to be able to replace chemical fertiliser as algae can be converted into fertiliser to provide Phosphorus and Nitrogen ( Mulbry et al. , 2005 ) . Algae biomass that has been undergone pretreatment from anaerobically digested dairy manure can be use as commercial fertiliser to supply 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 household ( Hoek et al. , 1995 ) . These green algae are spherical in form with no scourge, with size runing 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 late been taken as nutrient addendum as it contains Chlorella Growth Factor ( CGF ) , which is a water-soluble infusion which contains high concentration of vitamins, minerals, nucleic acids, aminic acids and enzymes that are needed by human ( Ley, 2003 ; Merchant and Andre, 2001 ) . It has been reported that by taking Chlorella as addendum has extensively promote healing and growing, excite immune system and besides have anticancer belongingss ( Merchant and Andre, 2001 ) .
Algae has greater possible than other alternate fuels, in that algae based biodiesel is wholly compatible with bing biodiesel ( Neltner, 2008 ) . Diesel engines can be instead run by powdered coal but the atoms involve have to be in the size runing from 5-8µm in order for the engine to burn decently. 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 girl cells during mitosis and miosis event by forming the cells through microtubules connected to their centromere ( Alberts, 1998 ) . In the barm such Saccharomyces cerevisiae it is known that merely a individual microtubule are bound to each kinetochore ( Peterson and Ris, 1976 ) . Cen4 is a little DNA component 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 act as functional chromosome in barm, allowed it to retroflex successfully in mitotic division and the first meiotic division ( Clarke and Carbon, 1980 ) . Plasmids contain yeast centromer cistron are now called Yeast Centromere Plasmid ( YCp ) , and widely usage as a vector to present cistron of involvement in molecular plants ( Verachtert and Mot, 1990 ) . This plasmid an ARS-based plasmid incorporating basic chromosomal elements which can supply a rate of a transcript figure of one cell per cell, therefore really utile in low transcript Numberss state of affairs, such as when seeking to cloning cistrons which become deadly when overly expressed ( Chawla, 2002 ) . This plasmid normally have multiple cloning site which can be digest with limitation enzyme to infix cistron of involvement and besides contain antibiotic immune cistron such as Kanamycin immune cistron, nptII and Ampicillin immune cistron, bla ( Verachtert and Mot, 1990 ) .
3.0 MATERIALS AND METHOD
3.1 General Method
All glasswork and media were autoclaved at 15 pounds per square inch ( 121 & A ; deg ; C ) for 15 proceedingss.
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 barm infusion, 5.0 g/L NaCl and 1.5 % ( w/v ) of agar pulverization and exceed up till 1L with distilled H2O. LB was prepared by adding same ingredients as used in doing LA with the exclusion of agar pulverization. Both LA and LB were autoclaved earlier usage.
3.2.2 Agrobacterium Tumefaciens Culture
Single settlement of Agrobacterium Tumefaciens GV3101 is inoculated into LB medium. Appropriate antibiotics are included when necessary. The civilization is left to turn at 28 & A ; deg ; C with 150rpm agitating. It will take about 16 hours ( nightlong ) to turn Agrobacterium Tumefaciens GV3101 in 100 milliliter LB.
Single settlement of Agrobacterium Tumefaciens GV3101 is streak onto LA home base. Appropriate antibiotics are included when necessary. The civilization is so left to incubate at 28 & A ; deg ; C in the lab. It will take about 2 yearss for the settlements to look.
3.2.3 Walne ‘s Medium for Chlorella sp civilization
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 individual settlement of Agrobacterium tumefaciens was inoculated into 3 milliliters LB medium. 50 µg/mL of Gentamicin and 25 µg/mL of Rifampicin were included in the LB medium. The civilization was grown for 16 hours ( nightlong ) at temperature 28 & A ; deg ; C with 150 revolutions per minute shaking. 675 µL of the nightlong civilization was pipetted into a unfertile microcentrifuge tubing. A volume of 325 µL of autoclaved 40 % glycerin was pippeted into the eppednorf tubing. The mixture was mix exhaustively by tapping several times. The microcentrifuge tubing was so stored at -70 & A ; deg ; 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 look vector normally usage in works transformation.pVT101 size is about 9 kilobits. pVT101 contained multiple cloning site ( MCS ) such as HindIII, SalI, XmaI and EcoRI.
pYAC-RC is derived from its ascendant, pYAC3. pYAC-RC carryied Principen opposition cistron every bit good as the artificial centromere cistron, cen4. This yeast artificial kinetochore 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 unreal kinetochore. The overall size of this plasmid is about 16 kilobits.
The building of pVT200 starts by digesting pVT101 with SalI limitation enzyme. Due to the fact that pVT101 merely contained one site for SalI, the plasmid will linearise without any alterations in its size. pYAC-RC is digested with XhoI limitation enzyme. This digestion will bring forth two separate plasmids with the size of 13 kilobits and 3 kilobit, severally. The 13 kilobits pYAC-RC contained cistron cassettes consists of, cen4, ars1, trp1, ampR and oriColE1. This plasmid was so ligated with pVT101 by utilizing ligase to organize pVT200. pVT200 is a plasmid carrying Kanamycin and Ampillin opposition cistron, cen4 cistron every bit good as oriColE1 cistron. pVT200 is about 21kb. Figure 3.1 showed the building strategy of pVT200.
3.5 Transformation of pVT200 Into Agrobacterium tumefaciens
3.5.1 Preparation of competent cells
A individual settlement of Agrobacterium Tumefaciens was inoculated into 5 milliliter of LB. 50 µg/mL of Gentamycin and 25 µg/mL of Rifampicin was included in the LB medium. The civilization was incubated at 28 & A ; deg ; C with 150rpm shingle for 16 hours ( nightlong ) . 3 milliliter of the nightlong civilization are so inoculated into 100 milliliter of LB. 50 µg/mL of Gentamycin and 25 µg/mL of Rifampicin are included in the LB medium. The civilization is so left for 5 hours at 28 & A ; deg ; C with 150 revolutions per minute shaking. The civilization are the transferred to four sterile 50 milliliter tubings, with each 50 milliliter tubings incorporating 20 milliliter of the civilization. The cultured was centrifuged at 4,000xg at 4 & A ; deg ; C for 5 proceedingss. The supernatant was discarded before all the tubings were inverted for 60 seconds. Each tubing incorporating pellet was resuspended with 20 milliliters of iced-cold 10 % glycerin, before they were centrifuged once more at 4,000xg at 4 & A ; deg ; C for 5 proceedingss. Supernatants were discarded from all the 50 milliliter tubings. The pellets were resuspended once more with 4 milliliters iced-cold 10 % glycerin. All four 50 milliliter tubings were combined and aliquot into two new unfertile 50 milliliter tubings before they were centrifuged once more at 4,000g at 4 & A ; deg ; C for 5 proceedingss. Supernatants from all 50 milliliters tubings were discarded. The civilization were wash once more by resuspended them in 2 ml iced-cold 10 % glycerin before poured into a new 50 milliliter tubing. The 50 milliliter tubing incorporating all the pellets from all four old 50 milliliter tubings were so centrifuged 4,000xg at 4 & A ; deg ; C for 5 proceedingss. The pellet was wash once more with 1.5 milliliters iced-cold 10 % glycerin after flinging its supernatant. All of the civilization was aliquot into 15 pre-chilled microcentrifuge tubings, each incorporating 100 µl of competent cells. All eppedorf were stored at -20 & A ; deg ; C. This method was a alteration from a method described by ( Main et al. , 1995 )
Tubes incorporating competent cells were left to dissolve easy on ice. 50 µL of competent cells were transferred into a unfertile microcentrifuge tubing. 2 µL of pVT200 are added to microcentrifuge tubing incorporating the competent cells. The competent cells were allowed to blend with pVT200 by tapping. The mixture was transferred to a pre-chilled 0.2 centimeter electroporated cuvette. The Micropulser ™ ( Bio-Rad ) was set at “ agr ” which stand for Agrobacterium Tumefaciens. The cuvette incorporating the mixture was so topographic point 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 milliliter of LB medium was transferred instantly into the 0.2 electroporated cuvette. The electroporated Agrobacterium Tumefaciens are so left to incubate on shaker at 28 & A ; deg ; C for 3 hours. 100 µL of the electroporated Agrobacterium tumefacies are so 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 home base is besides prepared. A negative home base was plated with competent cells which was non electroporated with pVT200. For each settlement that was formed on the home base, was isolated and spread onto a new LA home base with the add-on of antibiotics. The antibiotics that were added on each home base were 50 µg/mL of Kanamycin and 25 µg/mL of Rifampicin. All home bases were left to incubate for three yearss in the dark room at 28 & A ; deg ; C.
3.6 Plasmid extraction
A individual settlement of transformed Agrobacterium Tumefaciens was inoculated into a 10 milliliter LB medium. 50 µg/mL of Kanamycin and 50 µg/mL of Rifampicin were added into the LB medium. The civilization was incubated at 28 & A ; deg ; C with shaking of 150 revolutions per minute for 16 hours ( nightlong ) . The civilization was so transferred into a unfertile 50 milliliter tubing. It was so centrifuged at 14,000xg for 1 minute. All supernatant was discarded by utilizing pipette. The pellet was resuspended with 100 µL ice-cold Solution I ( Refer Appendix A ) . All the contents inside the 50 milliliter tubing were transfer into a unfertile microcentrifuge tubing utilizing 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 tubing several times. The mixture was so 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 so mix exhaustively by whirl. The mixture was centrifuged for 15 minute at 12,000xg. The supernatant was so transferred into a new unfertile microcentrifuge tubing by utilizing pipette. A volume of 1 milliliters of iced-cold 95 % ethyl alcohol was added to the supernatant. The mixture was assorted 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 milliliter of 70 % ethyl alcohol was added and centrifuged once more at 12,000xg for 10 proceedingss. The supernatant was so discarded. The remnant supernatant was the left to be aspirated by air drying the pellet. A volume 0f 30 µL of deionized distilled H2O ( ddH20 ) was added and the plasmid was stored at -20 & A ; deg ; C for future usage. Plasmid extraction was done as described by ( Sambrook et al. , 1989 )
3.7 Plasmid Purification
The extracted plasmid was purified utilizing the Wizard®Plus, SV Minipreps DNA Purification System kit ( Promega, USA ) . The purification procedure is indispensable to guarantee the quality consequences will be obtained from gel cataphoresis.
One to five milliliters of nightlong bacterial civilization was harvested and centrifuged at 10,000xg for 5 minute. The supernatant was discarded and blotted on towel paper to take extra media. Then, 1250 µL of Cell Resuspension Solution was added and resuspended. After that, 250 µL of Cell Lysis Solution was added and exhaustively assorted 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 proceedingss. The clear lysate was transferred into spin column and centrifuged at maximal velocity for 1 minute. Then 750 µL of Column Wash Solution antecedently diluted with 95 % ethyl alcohol was added and centrifuged at maximal velocity for 1 minute. The same procedure was repeated by adding 250 µL of the Column Wash Solution. Centrifuged at maximal velocity for 2 minute and the spin column were transferred into a microcentrifuge tubing. Finally, 100 µL of Nuclease-Free H2O was added and centrifuged at maximal velocity for 1 minute. The purified plasmid was stored at -20 & A ; deg ; C
3.8 Gel Electrophoresis
3.8.1 Preparation of Agarose gel
0.7 % agarose gel was prepared by weighed 0.14g of agarose pulverization. 20 milliliter of TAE buffer was so added to the agaros pulverization. The mixture was heat until all agarose was dissolved. The mixture was allowed to chill for 5 proceedingss. A cuneus was so placed inside the casting tray. An 8-well comb was placed steadfastly onto the casting tray. The cooled agarose so poured into the casting tray.
3.8.2 Loading samples
3 µL of DNA samples was added into a 2 µL of bluish bromophenol dye. The mixture was resuspend several times. 3 µL of Lambda HindIII limitation enzyme DNA marker was added into another 2 µL of bluish bromophenol dye. The comb is taken out was the agarose gel have cooled. The cuneus incorporating the agarose gel was so put inside the armored combat vehicle. The armored combat vehicle was filled with TAE buffer until the agarose gel submerges. The first lane was so loaded with DNA marker. The remainder of the lane was loaded with DNA samples. The leads were so connected to the power supply. The current was set at 90v before it was turn on. The gel was run until the ? across the agarose gel.
The cuneus was taken out and submerse it the Ethidium Bromide ( EtBr ) staining solution. The agarose gel was left to be stain for 10 proceedingss. Remove the agarose gel and submerse it into distilled H2O for 5 proceedingss. The gel was so observe under UV visible radiation.
3.9 Axenic civilization of Chlorella sp
A individual settlement of Chlorella sp was streak on LA medium added with antibiotic. The home bases were incubated for two hebdomads. The antibiotic used was Ampicillin ( 25µg/mL ) , Chloramphenicol ( 20µg/mL ) , Straptomycin ( 25µg/mL ) and Tetracycline ( 30µg/mL ) . Merely plates with feasible Chlorella sp and no taint are considered as a positive consequence.
4.1 Chlorella sp settlements grown on Conway medium
For the first two hebdomads, it appears that all home bases were contaminated with a lawn of milky unidentified bacteriums. All attempts to accomplish sterile conditions were done when culturing a new batch of Chlorella sp home bases. However, the home bases were still contaminated with the same milky unidentified bacteriums. After two more hebdomads, the lawn of that unknown bacteria seems to sporadically vanish and Chlorella sp appears to turn on top or near the bacteriums.
Out of 10 home bases, merely two home bases appear to hold settlements of Chlorella sp. It was expected that the Chlorella sp will take 2 hebdomads to turn on Walne ‘s medium, nevertheless it took about one month for the first settlement to look.
4.2 Transformation of Agrobacterium tumefaciens
After two yearss of incubation, there were three individual settlements observed on home base which was spread with electroporated Agrobacterium tumefaciens. There was no individual settlement signifier on the negative home base. Both home bases were left to incubate for another twenty-four hours. On the 3rd twenty-four hours of incubation, the figure of individual settlement has increased from three settlements to five settlements, and no individual settlement signifier on negative control home base. Both home bases were left for another twenty-four hours in the dark room. On the 4th twenty-four hours, the figure of settlements formed on the home base spread with electroporated Agrobacterium tumefaciens has non change. Hence, the sum of settlements of successful electroporated Agrobacterium tumefaciens observed on the LA home base is five settlements, with no ascertained growing detected in the negative home base
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 visible radiation. The size was determined to be about 23 kilobits ( Figure 4.8 )
The extracted plasmids from electroporated Agrobacterium tumefaciens was so run through agarose gel and observed under the UV visible radiation. It showed the presence of a plasmid in each settlement. Figure 4.9 shows the consequence of cataphoresis of pVT200, plasmids extracted from Agrobacterium tumefaciens and respected DNA marker.
4.4 Axenic civilization of Chlorella sp
All 10 home bases showed no growing of Chlorella sp after two hebdomads of incubation. Furthermore, six out 10 home bases were contaminated with Fungis and milky unidentified bacterium. The other four home bases were left for another two hebdomads to incubate. After one month of incubation, all four home bases showed no mark of Chlorella sp settlements
5.1 Growth of Chlorella sp
Chlorella sp were ab initio grown on a set of 10 home bases of Conway medium. The home bases were so left to incubate at 25 & A ; deg ; C and were illuminated by cool blossoming lamps at 1450-1490 lx for 24 hours. It was expected that the civilization were able to demo initial growing by the terminal of two hebdomads incubation period. However, it appears that all home bases were contaminated for the first batch. For the subsequent two more batches, sterile techniques were far more rigorous every bit good as the beginning of Chlorella sp for the initial inoculant were purified to guarantee no taint. However, taint still exists. The taints consist of either Fungi or unknown milky bacteriums.
As reference earlier, all Chlorella sp were left to incubate for two hebdomads, but due to unexpected event of taint and besides repeat of culturing a new batch of Chlorella sp under rigorous sterile conditions does non resulted in any alterations, all contaminated home bases were left for another two hebdomads. Surprisingly, after about three hebdomads the lawn of bacteriums who ab initio dominated the home base appears do sporadically diminished in Numberss. Furthermore, Chlorella sp settlements begin to turn on top or near the dead cells of those unknown bacteriums. Hence, at the terminal of the drawn-out one month incubation period, two out of 10 home bases manage to bring forth a lawn of Chlorella sp.
Hence, a relationship between algae and bacteriums might be. In many instances, an axenic civilization of algae is needed in order to guarantee dependable consequences can be achieved. Axenic civilization is a civilization which is free of any bacteriums or any micro-organisms ‘ taint. In world, it is rather hard to accomplish axenic civilization, although rigorous sterile measurings should ever be taken to diminish bacterial taint, but it is rather acceptable to accept a grade of taint ( L.Barsanti, 2006 ; Fritsch, 1935 ) . The relationship between algae and bacteriums might non be species specific. This relationship proved to be good for the growing of algae, if the associated bacteriums produce growing factors or C02 needed by algae ( Lange et al. , 1971 ) . The relationship might besides look indispensable to the growing of algae. In turning oceanic diatoms, Phaeodactylum triconutum and Coscinodiscus concinnus it was observed that in the absence of associated bacteriums, a successful civilization can be established ( Droop and Elson, 1966 ) .
Transformation is any alterations in being ‘s features through the transportation of bare DNA ( Black, 2008 ) . Transformation in this experiment has to happen in two phases. The first 1 is to transform Agrobacterium tumefaciens with pVT200. From at that place, we use Agobacterium tumefaciens to of course transform Chlorella sp. Nowadays, transmutation can be done in legion ways.
Protoplast merger is a technique which required enzymatically taking the cell walls of two beings to let the merger of their familial stuffs ( Black, 2008 ) . Protoplast merger will let two beings to blend together their full genomes, therefore avoiding trouble in reassigning the right boosters and besides produced a intercrossed being with all desirable features ( Black, 2008 ; Peberdy and Ferenczy, 1985 ) . Chemically-induced merger usage chemical substance such as Polyethylene Glycol ( PEG ) which cause the membrane cell to shrivel due to the remotion of H2O by hydrophilic part of PEG, this will caused an addition in the fluidness of the membrane bilayer ensuing in the inclination to blend with other membrane plasmid ( Altman and Colwell, 1998 ) . Microinjection is a molecular technique which use glass micropipette to shoot coveted substances or even familial stuff such as cistron straight into a life 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 works. This method is achieved by integrating viral genome into T-DNA subdivision of Agrobacterium prior to infection. One the advantage of utilizing Agrobacterium-mediated virus infection to transform works is that, transmutation can be done on broad scope of workss, non restricted merely magnoliopsids workss which the traditional Agrobacterium-mediated transmutation can non transform ( Chawla, 2002 ) .
To reassign pVT200 which carried cen4 cistron 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 transportation of stuff across the membrane ( Main et al. , 1995 ) . For this experiment, transmutation measure utilizing electroporation is successful. However, the figure of settlements that have been transformed should be higher. An efficient transmutation of Agrobacterium tumefaciens can be every bit high 1×108 to 3×108 transformants per mcg ( µg ) DNA ( Mersereau et al. , 1990 ) .
Traditionally, transmutation was done by utilizing the freeze-thaw or triparental method. Freeze-thaw method is method which the cells are quickly freeze so easy thawed, which caused the membrane to accept DNA through their pores. Triparental method used the junction procedure. This method needed another micro-organism, for illustration Escherichia coli to have the DNA through transmutation, before reassigning it to another micro-organism ( Nickoloff, 1995 ) . Freeze-thaw method tends to be inefficient, with ensuing merely 103 transformant per mcg ( µg ) of Deoxyribonucleic acid and besides dearly-won ( Nickoloff, 1995 ; Chichester, 1980 ) . While triparental seem to be clip devouring every bit good as, inefficient compared to electroporation. Furthermore, triparental required the demands of complex media and choice method ( Julio Salinas, 2006 ) .Therefore, electroporation is the best method to utilize to transform Agribacterium tumefaciens.
5.2.2 Plasmid Extraction
Plasmids which have been extracted from electroporated Agrobacterium tumefaciens were analyze by agarose gel cataphoresis and observed under the UV visible radiation. Under the UV visible radiation, it appeared that the size of plasmids, pVT200 and extracted plasmid signifier Agrobacterium tumefaciens was around 23.13 kilobit. However, farther analysis should be done. The most common and accurate method is plasmid digestion analysis. In this method, limitation enzyme is use to digest both the original pVT200 and besides extracted plasmids from Agrobacterium tumefaciens. If both plasmids are indistinguishable, they should bring forth the same set ( s ) of the same size.
5.4 Axenic civilization of Chlorella sp
As reference earlier, to set up an axenic civilization of algae is hard due to its relationship with the associated micro-organism, chiefly bacteriums. However, there have been legion method that can be use to diminish the sum of taint to the degree that can be tolerated. Purification of algae can be achieved either by nutritionary enrichment method, mechanical uses, antibiotic intervention or the combination of these three methods ( Droop, 1969 ) .
Although other methods such as handling algae civilization with gamma radiation and UV visible radiation can bring forth axenic civilization, but it besides can caused possible harm to algal cells. However, some algae are reported to be rather immune towards gamma radiation and UV visible radiation. Bluish green algae can be treated with gamma radiation and UV light to bring forth an axenic civilization ( Bowyer and Skerman, 1968 ) .For motile algae, hiting the agar surfaces before incubation can supply an efficient and rapid separation of gliding filiform algae such as blue-green algaes from bacterium settlements ( Vaara et al. , 1979 ) . Scoing method involved doing a parallel line on the agar surface to supply a manner for motile algae to glide off from the contaminations. However, antibiotic interventions provide an alternate manner to bring forth axenic civilization. Some methods discuss above, are specific for certain species of algae, nevertheless antibiotic interventions can be apply to all algae species. In order to utilize antibiotics to bring forth axenic civilization, foremost we have to find which antibiotics that bacteriums are sensitive every bit good as the concentration of antibiotic to utilize in order to bring forth feasible algae civilization. Furthermore, the exposure clip of the antibiotics to both algae and contaminations have to be short but effectual. This to guarantee that, no development of antibiotic immune algae and bacteriums can be formed, but at the same clip extinguish all contaminations ( Jones et al. , 1973 ) .
Although the antibiotic interventions seem to supply a good and easy manner to bring forth an axenic civilization, information about the contaminations are needed. This will enable us to explicate a combination of the most effectual antibiotics that can extinguish all contaminations but still produce feasible algae cells.
An effort to set up an axenic civilization of Chlorella sp failed might due to the incorrect combination of antibiotics used. The concentration of antibiotics besides may do the failure in extinguishing all contaminations. Hence, farther analysis can be done by insulating and placing all bacteriums associated with the algae before and after the antibiotic intervention. Following, the contaminations should undergo antibiotic sensitiveness trial. This will supply us with the information needed to modify every bit good as addition the effectivity of the antibiotics interventions on all contaminations but at the same clip green goods feasible axenic civilization of algae.
It was reported that an antibiotics intervention combination dwelling of, Benzyl-penicillin-SO4, Tetracycline, Chloramphenicol, Aureomycin, Ceporin and neomycin-SO4 can efficaciously extinguish all contaminations to bring forth axenic civilization of Chlorella vulgaris ( Jones et al. , 1973 ) . Hence, it is extremely suggested to reiterate this experiment by utilizing the antibiotics combination used by Jones.
Agrobacterium tumefaciens GV3101 ( pMP90RK ) has successfully been transformed with pVT200 by electroporation. All Agrobacterium tumefaciens GV3101 ( pMP90RK ) transformants was able to turn on LA medium with the presence of Kanamycin. However, farther analysis such as digestion of the extracted plasmid from Agrobacterium tumefaciens GV3101 ( pMP90RK ) should be done to corroborate this consequence.
Cultivation of Chlorella sp on Walne ‘s medium was successful. However, the civilization was to a great extent contaminated with unknown micro-organisms. It was propose that there was an obligatory relationship between algae and bacteriums which seem to forestall from obtaining an axenic civilization. Future survey should be directed at detecting the length of this obligatory relationship.
An axenic civilization of Chlorella sp failed to accomplish due to the trouble to extinguish all contaminations by utilizing antibiotics intervention. A new set of antibiotics combination consist of Benzyl-penicillin-SO4, Tetracycline, Chloramphenicol, Aureomycin, Ceporin and neomycin-SO4 should be usage in future effort to obtain an axenic civilization of Chlorella sp.
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