1.Introduction
Bacillus anthracis is the etiological agent of anthrax; a common disease of livestock and, occasionally, of humans. It can be grown ordinary nutrient medium. Genus bacillus have only Bacillus anthracis as a obligate pathogen, Bacillus anthracis is a Gram-Positive bacteria, and it is also an endospore forming bacteria. It is road-shaped with a width of 1.0-1.2µm and a length of 3-5µm. (R.C. et al, 2003).
Fig: 1| Gram strain of the bacterium Bacillus anthracis, the cause of the anthrax disease.
1.1. Historical Background:
French physician Casimir Davaine demonstrated the symptoms of anthrax were invariably accompanied by the microbe Bacillus anthracis. Bacillus anthracis derives its name from Greek word anthrax meaning coal, due to its ability to cause black, coal-like Cutaneous eschars and referring to the most common form of the disease. Bacillus anthracis was the first bacterium conclusively demonstrated to cause disease, by Robert Koch in 1876. On 5th October 200 a 63 year old white man in Florida died of inhalation anthrax, the first person to develop this disease in USA for 25 years. Bioterrorism, in the form of letters containing the Ames strain of Bacillus anthracis spores, had also arrived. After the sending of probably six such letters, as of 26th January 2002, a total of 11 cases of inhalation anthrax (five of these patients died) and 11 (seven confirmed and four suspected) cases of Cutaneous anthrax have been identified. Long considered a potential biological warfare agent (Spencer et al.1993; Ingleby T.V. et al, 1999; R.C. et al, 2001). Bacillus anthracis had been investigated and weaponised for potential use against animals in World War I (Germany) (Redmond C. et al,1998; Wheelis M. 1999) and both animals and man in World War II.
Fig: 2| Structure of the Bacillus anthracis.
1.2. Bacteriology:
It is one of few bacteria known to synthesize a protein capsule (poly-D-gamma glutamic acid).Like Bordetella pertussis; it forms a calmodulin-dependent adenylate cyclase exotoxin known as Anthrax edema factor, along with anthrax lethal factor. It bears close genotypical and phenotypical resemblance to Bacillus cereus and Bacillus thuringiensis. All three species share cellular dimensions and morphology. All form oval spores located centrally in an unswollen sporangium. Bacillus anthracis endospores, in particular, are highly resilient, surviving extremes of temperature, low-nutrient environments, and harsh chemicals treatment over decades or centuries.
The endospore is a dehydrated cell with thick walls and additional layers that forms inside the cell membrane. It can remain inactive for many years, but if it comes into a favorable environment, it begins to grow again. It initially develops inside the rod-shaped form. Features such as the location within the rod, the size and the shape of endospore, and whether or not it causes the wall of the rod to bulge out are characteristic of particular species of Bacillus. Depending upon the species, the endospores are round, oval, or occasionally cylindrical. They are highly refractile and contain dipicolinic acid. Electron micrography sections show they have a thin outer endospore coat, a thick spore cortex, and an inner spore membrane surrounding the endospore contents. The endospores resist heat drying and many disinfectants (including 95% ethanol). Because of these attributes, Bacillus anthracis endospores are extraordinarily well-suited to use (in powdered and aerosol from) as biological weapons.
1.3. Genomic structure of Bacillus anthracis:
Bacillus anthracis has s single chromosome which is a circular, 5,227,293bp DNA molecule. It also has two circular, extrachromosomal double stranded DNA plasmids. Virulence of most Bacillus anthracis strain is associated with two megaplasmids pXO1 and pXO2; and strains lacking any plasmid are either avirulent or significantly attenuated.
• Fig:3 | Plasmid gene expression profile (Bourgogne et al. 2003).
1.3.1. pXO1 plasmid:
The pXO1 plasmid is of 110 MDa (184 kb). Bacillus anthracis require pXO1 plasmid for synthesis of toxin protein of Bacillus anthracis. Toxin proteins like edema factor, lethal factor, and protective antigen. It harbors the structural genes that encode for the anthrax toxin components: pag (protective antigen, PA), lef (Lethal factor, LF), and cya (Edema factor, EF). These factors are contained within a 44.8kb pathogenicity island (PAI). The protein for lethal toxin and edema toxin; lethal factor and edema factor respectively, bind with protective antigen in binary combination. The lethal toxin is a combination of protective antigen (PA) with lethal factor (LF) and the edema toxin is a combination of protective antigen (PA) with edema toxin (EF). The pathogenicity island (PAI) also contains trans-acting regulatory genes which encode a transcriptional activator atxA and the repressor pagR, both of which regulate the expression of the anthrax toxin genes. The pXO1 plasmid also contain a gene topA; encoding a type I topoisomerase. It also contains an operon harboring three genes whose functions appear to affect germination (Luna et al. 2006) (Liange et al. 2016).
1.3.2. pXO2 plasmid:
The pXO2 plasmid is of 60 MDa (97 kb). Bacillus anthracis containing pXO2 harbors three genes required for capsule synthesis; capB, capC and capA. It also contain a gene which associated with capsule degradation dep, And acpA a trans-acting regulatory gene. The AcpA expression is controlled by AtxA from pXO1. Plasmid pXO2 carries gene which required for the synthesis of an antiphagocytic poly-γ-D-glutamic acid (polyglutamate) capsule. These genes located in the pXO2 pathogenicity island (PAI) (35kb) (Green et al. 1985) (Koehler et al. 2002).
1.4. Pathogenesis of Bacillus anthracis:
This zoonitic pathogen is mainly lives in soil, animal and water. Spores produced are stable and resistant to harsh external conditions like heat, cold, pH, desiccation, and chemicals. They can germinate when exposed to a nutrient-rich environment, such as the tissues or blood of an animal or human host. Bacillus anthracis possess an anti-phagocytic capsule essential for complete virulence. The organism also produces three plasmid-coded exotoxins: edema factor, a calmodulin-dependent adenylate cyclase, causes elevation of intracellular cAMP, and is responsible for the severe edema usually seen in Bacillus anthracis infection, lethal toxin responsible for tissue necrosis by releasing tumor necrosis factor (TNF) and Interleukin-1 (IL-1) which are responsible for rapid health deterioration during the inflammatory process; from macrophages. Protective antigen mediates cell entry of edema factor and lethal factor. The edema toxin causes the formation of edema in tissue as a result of water and Cl− ions loss from cells and may inhibit neutrophil phagocytic activity and oxidative burst. Anthrax can also result in necrosis, septicemia, organ failure, and death (Banerjee et al. 2017).
1.5. Manifestations in human disease:
The symptoms is anthrax depend on the type of infection and can take anywhere from 1 day to more than 2 months to appear. All types of anthrax have the potential, if untreated, to spread throughout the body and cause severe illness and even death. Four forms of human anthrax disease are recognized based on their portal or entry.
Cutaneous anthrax follows entry of the infection through the skin. It the most common form (95%). The face, neck, hands, arms and back are the usual sites. It start lesion as a papule 1-3 days after infection and becomes vesicular, containing fluid (which may be clear or bloodstained). This whole papule area is congested and edematous, and several satellite lesion filled with serum or yellow fluid. And finally causes a localized, inflammatory, black, necrotic lesion (Eschar). The lesion is called a malignant pustule. The disease is common in dock workers carrying loads of hides and skins on their bare backs and hence was known as the hide porter’s disease. If untreated develop fatal septicemia or meningitis.
Pulmonary anthrax follows entry of infection through the inhalation of dust from infected wool. Inhalation is characterized by flu like symptoms, chest discomfort, diaphoresis, and body aches. This pulmonary anthrax is also called wool sorter’s disease due to it is more common in workers in wool factories. This is a hemorrhagic pneumonia with a high fatality rate. As a complication Hemorrhagic meningitis may occur. It is a rare but highly fatal form.
Fig: 4 | The cycle of infection in anthrax (WHO 1998).
Intestinal anthrax is a rare but also fatal (causes death to 25%) type, results from ingestion of spores. It mainly occurs in primitive communities who eat the carcasses of animal’s dying of anthrax. Symptoms include: violent enteritis with bloody diarrhea (with high case fatality), fever and chills, swelling of neck, painful swallowing, hoarseness, nausea and vomiting (especially blood vomiting), flushing and red eyes, and swelling of abdomen.
Injection, symptoms are similar to those of harder to recognize and treat compared to cutaneous anthrax.
Human anthrax may be industrial or non-industrial (agricultural). The industrial anthrax found in industries like meat packing or wool factories, whereas non-industrial anthrax is an occupational disease in those who associate frequently with animals, such as veterinarians, butchers and farmers (Ananthnarayan et al. 2000).
1.6. Laboratory research
Bacillus anthracis and its toxin activity are inhibit by polyphenols; Component of tea The addition of milk to the tea completely inhibits its antibacterial activity against anthrax. Activity against the Bacillus anthracis in the laboratory does not prove that drinking tea affect the course of an infection, since it is unknown how these polyphenols are absorbed and distributed within the body.
1.7. Recent research:
Advances in genotyping methods have led to improved genetic analysis for variation and relatedness. These methods include multiple-locus variable-number tandem repeat analysis (MLVA) and typing systems using canonical single-nucleotide polymorphisms. The Ames ancestor chromosome was sequenced in 2003 and contributes to the identification of genes involved in the virulence of Bacillus anthracis. Recently, Bacillus anthracis isolate H9401 was isolated from Korean patient suffering from gastrointestinal anthrax. The goal of the Republic of Korea is to use this strain as a challenge strain to develop a recombinant vaccine against anthrax.
The H9401 strain isolated in the Republic of Korea was sequenced using 454 GS-FLX technology and analyzed using several bioinformatics tools to align, annotate, and compare H9401 to other Bacillus anthracis strain. The sequencing coverage level suggests a molecular ratio of pXO1: pXO2: chromosome as 3:2:1 which is identical to the Ames Florida and Ames Ancestor strains. H9401 has 99.679% sequence homology with Ames Ancestor with an amino acid sequence homology of 99.870%. H9401 has a circular chromosome (5,218,947bp with 5,480 predicted ORFs), the pXO1 plasmid (181,700bp with 202 predicted ORFs), and the pXO2 plasmid (94,824bp with 110 predicted ORFs). As compared to the Ames Ancestor chromosome above, the H9401 chromosome is about 8.5kb smaller. Due to the high pathogenicity and sequence similarity to the Ames Ancestor, H9401 will be used as a reference for testing the efficacy of candidate anthrax vaccines by the Ames Ancestor; H9401 will be used as a reference for testing the efficacy of candidate anthrax vaccines by the Republic of Korea.
1.8. Type IV secretion systems (T4SSs OR TFSSs):
The type IV secretion system (T4SSs) is one of several types of secretion systems, which microorganisms use for the transport of macromolecules such as proteins and DNA across the cell envelop. It is the most versatile family of secretion systems, mediating transport of monomeric proteins as well as multi-subunit protein toxins and nucleoprotein complexes, and has been found in both Gram-negative bacteria as well as in some archea. Type IV secretion system translocate DNA and protein substrates across the bacterial cell envelope. These effector molecules are toxic to host cell which was discovered in many microorganism Agrobacterium tumefaciens, Helicobacter pylori, and Bordetella pertusis (Christie et al. 2004).
These systems are classified on the basis of an ancestral relatedness to bacterial conjugation machines. There are tree functional types of T4SSs.
The first type is similar to conjugation mechanism. The first type is present in some archaea and in gram positive and gram negative bacteria. It is used for transferring DNA from one cell to other cell to the other in a cell-to-cell contact dependent manner. It increases genomic plasticity, help bacteria to adapt them self in changing environment. This system is most important in transferring antibiotic resistance genes in pathogenic bacteria. Some self transmiable plasmids also encode these conjugative T4SSs with genes that provide selective advantage for cell such as antibiotic resistance, some metabolic function which enhance cell survival and virulence. They are also integrated in chromosome like a part of transposons (E.Cascales et al, 2003).
Agrobacterium tumefaciens have another T4SS transferring DNA (oncogenic nucleoprotein complexes) into plant cells. It is a causal agent of crown gall disease (the formation of tumours) (Smith et al, 1907), by transferring small segment of T-DNA from Ti plasmid, into the host plant cell. Bordetella pertusis secretes pertusis toxin by type IV secretion system, the causative agent of whooping cough (Cascales et al, 2003). Helicobacter pylori deliver CagA into gastric epithelial cells by using T4SS, which is responsible for gastric carcinogenesis (Hatakeyama et al, 2005).
Fig: 5| Schematic representation of Type 4 secretion system found in bacteria (Alvarez et al. 2009).
The second type is used for DNA uptake and release from the extracellular milieu. The Neisseria gonorrhoeae gonococcal genetic island (GGI) secretes DNA to the extracellular milieu and Helicobacter pyloriComB system uptake DNA from extracellular milieu (Walden et al. 2010).
The transfer of protein is facilitated by the third type of T4SS. These systems are mostly found in pathogenic bacteria like Helicobacter pylori, Bordetella pertusis, Legionella pneumophila, Brucella spp. and Bartonella spp. rely on T4SSs for their pathogenicity.
The type IV secretion system form by several subunits. The in silico analysis of Bacillus anthracis genome shows few genes have similarity with known type IV secretion system subunits (Grynberg M. et al, 2007). This in sillico clue prompted us to explore possibility of existence of T4SS in Bacillus anthracis using molecular techniques and also to explore the possibility of its role in toxin secretion.
1.9. Cloning vector:
Clone the plasmid gene by the cloning vector. pXO2-22 gene clone with the E.coli strain vector like pET and pGEX. The pET vector with 6-Histidine tag (His-tag), is usually the first choice to get recombinant protein because His-tag is a smaller affinity tag with high levels of expression due to the presence of a strong promoter and the availability of robust purification system like Ni-NTA. The pET vector is provided by ‘Novagen’. Consecutive 6-Histidine can possibly decrease the solubility of a fused protein. The pET vector is commercially provided by ‘Clontech’ , and encoded a novel polyhistidine epitope tag. This tag is 19 amino acids long and has a non adjacent 6-Histidine.
When a histidine tagged gene does not express the protein or recombinant protein forms inclusion bodies, the gene of interest should be cloned in pGEX system of ‘GE helthcare’ or GST tag. It has been well established that maltose binding protein (MBP tag) and GST tag increases solubility of fused proteins and expression of genes. The drawback of pGEX vector is their large sizes of fusion tag (MBP:44kDa; GST:25kDa). The large size of fusion tags may interfere with the activity of recombinant proteins if the tag is not cleaved properly. Different enzyme sites have been incorporated in this vector so that cleavage would be precise and no extra amino acid remain fused with recombinant protein. Tags may Interfere with the structure and function of the target protein, therefore provision tags must be removed after the expression and purification of the protein. Multiple cleavage sites can be engineered into the expression construct to remove tags.
1.10. Expression vector:
Recently, Many biotech companies provide different types of genetically altered E.coli strain as per suitable expression of foreign genes. Genes cloned with ‘tac’ promoter (pGEX system) can be expressed in the cloning host itself but the BL21 strain is the preffered choice for expression because of absence of two main proteases genes cloned in pET system with t7 promoter should be expresses in BL21 (De3). E.coli strain BL21 (De3) has a t7 polymerases encoding gene introduced in its genome as well as Lon and OmpT protease deleted. Lon and OmpT protease deficient strain of E.coli are unable to degrade foreign protein. Leaky expression of the desired gene in BL21 (De3) cell is possible. To minimize leaky expression of toxic gene, the BL21 host strain was improved. The improved strain is BL21 (De3)pLys S abd BL21 (De3)pLys E. Both the strain have lysozyme coding plasmid. Lysozyme is inhibitor of T7 polymerases which inhibits residual T7 polymerase and thus prevents leaky expression. Leaky expression of toxic gene is detrimental to the host cell. Host cell will not survive or it will change its mechanism so that even in presence of inducer it will not allow production of toxic protein. Rosetta host strains are BL21 (De3) derivatives designed to enhance the expression of heterologous proteins by containing codons rarely used in E.coli.
OBJECTIVE
On the basis of these background and importance of secretory system, I would like to carry out following study in this proposal.
1. Cloning of pXO1-85
2. Expression of gene in E.coli
3. Purification and characterization of protein
2. Materials and Method
2.1 Materials
2.1.1. Strains:
E.coli: Dh5α
BL21 (De3)
2.1.2. Vectors:
pUC18
pET28
pET84
pET92
pGEX64
2.1.3. Protein size marker for SDS:
Pierce unstained protein MW marker,
Pierce prestained protein MW marker
2.1.4. Chemicals:
Tris-base, SDS, Acrylamide, Bis-acrylamide, 2-Mercaptoethanol, TEMED, IPTG, Ammonium persulfate, Coomassie Brilliant Blue, Glycine, Saturated Phenol, Calcium Chloride Dehydrate, Triton X-100, Ammonium sulphate, Ethidium Bromide, Bromophenol Blue, EDTA, Tris-HCl, PCI, Glcial acetic acid, Ethanol, Glycerol, Hydrochloric acid, Methanol, Acetone, Acetic acid, Dextrose, Sodium chloride, Sodium hydroxide, Sodium acetate, Potassium acetate, Potassium chloride.
2.1.5. Bacterial cell culture media:
LB Agar, LB Broth
2.1.6. Antibiotics:
Ampicillin (Stock: 100mg/ml, Working: 100µg/ml)
Kanamycin (Stock: 50mg/ml, Working: 50µg/ml)
2.1.7. Solution for Plasmid Isolation:
Solution I: 25mM TrisHCl (pH 8.0), 50mM Glucose and 10mM EDTA
Solution II: 0.2 N NaOH (Freshly diluted from 10N stock) and 1% SDS
Solution III: 60ml 5M Potassium Acetate, 11.5ml Glacial Acetic Acid per 100ml solution . In H2O
TE: 10mM TrisHCl(pH 8.0) and 1mM EDTA
2.1.8. DNA Gel Loading Dye (6X):
0.25% Bromophenol Blue in 30% Glycerol in H2O
Ethidium Bromide: 10mg/ml H2
2.1.9. 50X stock TAE buffer:
Tris base: 242g
Glacial Acetic Acid: 57.1ml
0.5M EDTA: 100ml
2.1.10. SDS PAGE Stacking Gel Buffer:
0.062 Tris HCL, pH 6.8 from 1M stock
2.1.11. SDS PAGE Resolving Gel Buffer:
0.375M Tris HCL, pH 8.8 from 1.5M stock
2.1.12. SDS PAGE Electrophoresis Buffer:
25mM Tris, 191mM Glycine and 0.1%SDS (pH 8.8)
2.1.13. SDS PAGE Gel Loading Buffer:
50Mm TrisHCl (pH 6.8), 5%β-Mercaptoethanol (V/V), 1mM EDTA, 0.1% Bromophenol Blue, 2% SDS and 10% Glycerol.
2.2Method
2.2.1 Bacterial strains and growth condition
E.coli strains DH5α and Bl-21(DE3) were grown in LB broth or on LB agar
Plates supplemented with ampicillin (100µg/ml), Kanamycin (50µg/ml).
2.2.2 Isolation of plasmid DNA
Plasmid DNA was isolated by alkaline Lysis method (Birnboim HC et al 1979).
3.0 ml of overnight grown E.coli cells harboring desired plasmid was collected in a micro centrifuged tube by centrifugation. Cell pellet was resuspended in 200µl of solution I (25mM TrisHCl pH 8, 50mM Glucose and 10mM EDTA), Lysed by addition of 400µl of solution II (0.2 NaOH in 1% SDS). Chilled in ice, neutralized by addition of ice –cold
300µl solution III (3M potassium acetate in a glacial acetic acid) and chilled further in ice for 5 minute. In the subsequent step, bacterial lysate was centrifuged at 10K for 10 minute at 4°C, supernatant was collected and nucleic acids were precipitated by the addition of 0.6 volume of isopropanol at room temperature. Precipitated nucleic acids were collected by centrifugation at room temperature, dissolved in 5µl TE buffer, treated with 20µg/ml of RNase at 37°C, extracted twice with phenol/chloroform/isoamyl alcoholmixture and plasmid DNA was then precipitated from the aqueous layer by addition of two volume of ethanol in presence of 0.3M sodium acetate. The precipitate was collected by centrifugation at 10K for 10 min at 4°C. Resultant precipitate was washed twice with 70% ethanol, dissolved in 50µl TE and stored at -20°C.
2.2.3. Preparation of Competent E.coli cell
Competent cell of E.coli strain [DH5α and BL21 (De3)] were prepared by CaCl2 method (Sambrook J., et al.1989).
Briefly, E.coli cells were streaked on LB agar plate to obtain single colonies. LB medium was inoculated with a single colony and grown overnight at 37°C with moderate shaking. Next day 100ml of LB medium was inoculated with 0.1% starter culture and grown at 37°C with shaking, until the OD600 reached between 0.3-0.4. The culture was chilled over ice and transferred to a chilled 50ml Oakridge tube and centrifuge at 5K for 5 minute at 4°C. The supernatant was discarded and the pellet was gently resuspended in 50ml of freshly prepared ice-cold 100mM CaCl2 solution (filter sterilized)and incubated on ice for 30 minutes. Cells were centrifuged at 5K for 5minute at 4°C and pellet was resuspended in 10ml ice-cold 100mM CaCl2. Equal volume of sterile ice-cold 30% glycerol was added to the cell suspension and mixed gentle swirling. Aliquots of 100µl cell suspension were transferred into sterile pre-chilled tubes and stored at -70°C.
2.2.4. Transformation of competent E.coli:
Plasmid DNA (5-10ng) was added to an aliquot of 100µl of competent cells, mixed and kept on ice for 30minute. Following incubation, cell suspension was subjected to heat shock treatment at 37ºC for 2min followed by chilling on ice for 2-3minute. In the next step, 1ml LB was added to the cell suspension and incubated further at 37ºC for 1hour. Finally cells were spreaded on LB plates containing selective antibiotics as desired and incubated over night at 37ºC (Chung et al. 1989).
2.2.5. Protein expression:
Induction of protein using IPTG:
Transferred E.coli BL21 (De3) colonies were incubated into 5ml of LB broath containing appropriate antibiotic and grown overnight at 37ºC for expression of recombinant protein. This primary culture was then used to incubate 100ml of fresh LB broath containing the same antibiotic. The culture was allowed to grow at 37ºC till OD reaches 0.6-0.8 at Abs600nm. At this stage, culture was induced with 1mM isopropyl-β-D-thiogalactoside (IPTG) for 3 hour. Further, Protein expression was checked by SDS-PAGE ((Lee et al. 1994).
SDS-PAGE:
Preparation of sample, Setting up SDS-PAGE assembly, Preparation of resolving gel, Preparation of stacking gel, SDS-PAGE electrophoresis, Staining and distaining, Observation under illuminator (Schagger et al. 1987).
Preparation of sample:
Take 2ml of culture whose protein analysis to be carried out. Centrifuge at 50K for 5minute. Take pellet and discard supernatant. Dissolve the pellet in 180µl of sample buffer and 20µl of β-mercaptoethanol. Now heat the sample till it gets less viscous approx at 60ºC for 10 minute.
Preparation of resolving gel (10%):
Prepare 10ml of resolving gel (Water: 4.00ml, 30% Acrylamide stock: 3.30ml, 1.5M TrisHCl pH 8.8: 2.50ml, 10%SDS: 0.10ml, 10% Ammonium persulfate: 0.10ml, TEMED: 0.004)
Preparation of stacking gel (5%):
Prepare 4ml of stacking gel (Water: 2.7ml, 30% Acrylamide stock: 0.67ml, 0.5M TrisHCl pH 8.8:0.50ml, 10%SDS: 0.04ml, 10% Ammonium persulphate: 0.04ml, TEMED: 0.004ml)
3. Result
3.1. Plasmid (pUC 18) isolation by alkaline lysis method:
Fig: 6 | a: standard DNA sample
b: pUC-18 Plasmid is successfully isolated
This arrow shows band of plasmid on 8% Agarose gel.
3.2. Plasmid (pET 92) isolation by alkaline lysis method:
a b b
Fig: 7 | a: standard DNA sample
b: pET-92 Plasmid is successfully isolated
This arrow shows band of plasmid on 8% Agarose gel.
3.3. Transformation of pUC 18:
Fig: 8 | Competent cells with plasmid pUC18 are successfully Transformed.
Colonies in LB+Amp (Ampicillin) plate are observed.
3.4. Protein expression of pET-92 in SDS-PAGE:
Fig: 9 | Commassie stained polyacrylamide gel showing induction of pET-92.
Total cell extract separated through 10% SDS-PAGE. Concentrations of IPTG were used 1mM.
3.5. Protein expression of pGEX-84 in SDS-PAGE:
Fig: 10 | Commassie stained polyacrylamide gel showing induction of pGEX-84. Total cell extract separated through 10% SDS-PAGE. Concentrations of IPTG were used 1mM.
4.Discussion
To fulfill our objective isolated plasmid was obtained by performing alkaline lysis method and purified plasmid was successfully isolated (as shown in fig: 6,7).To check the competency of Dh5α (cloning E.coli strain) Calcium chloride method was performed and subsequent transformation of pUC18 plasmid was carried out. By performing transformation protocol we found colonies on LB+Amp plates, which shows successfully transformation of pUC18 (as shown in fig: 8). For expression of the gene cloned we used BL21 (De3) (expression strain of E.coli) and performed transformation of expression strain with pET92 but transformed colonies were not found. This may be due to old reagents or it requires repeating procedure. Later we check induction of transformed pET92 wherein 1mM IPTG was used as inducer and total cell extract was separated through 10% SDS-PAGE TO check induction. Expression of pET92 was obtained successfully (as shown in fig: 9). Later we checked expression of pGEX84 whose clones are already available in laboratory. Expression of pGEX84 was obtained successfully (as shown in fig: 10). More experiments are required to get conclusive result. However, other researchers may get benefit from our established protocol.
5.Summary
The purpose of this project is to identify secretion system present in Bacillus anthracis (etiological agent for anthrax disease) depending upon the sequence homology of the subunits of known secretion system (type IV secretion system) like Agrobacterium tumefaciens, H.pylori. So in order to identify that whether proteins associated with secretion system are same or not to known proteins of type IV secretion system. For achieving this, cloning of gene possessing sequence homology (pXO2-22) is done. Then its expression is carried out in E.coli strain. Later isolation of protein is done and then bioassay to determine the type of protein is carried out. Hence if protein isolated is analog to protein constituting type IV secretion system it can be said that Bacillus anthracis might possess similar secretion system. By taking different gene and understanding which protein they make, type of secretion system in Bacillus anthracis can be identified. Henceforth we will be taking different gene which will eventually contribute to entire secretion system present. We have tried to express this gene in E.coli host to get good quality of recombinant purified protein. We got partial success.
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