Gel electrophoresis pMB40 positive control. Transformation assay 3Recipient strains Serotype Transformants/ml Transformants/ml (kanamycin (chloramphenicol resistant) resistant ) liu 1235 a liu1195 b liu1196 c smooth rough 103 e liu1193. Gel electrophoresis pjak 17 positive control. Transformation assay 4 With liu 1212 twist spent medium Recipient strain Transformants/ml Transformants/ml (kanamycin resistant) (chloramphenicol resistant ) liu with liu 1223 spent medium Recipient strain Transformants/ml Transformants/ml (kanamycin resistant) (spectinomycin resistant) liu. Gel electrophoresis of genomic dna transformants pMB 79 positive control li genomic dna transformants. Gel electrophoresis of plasmid dna transformants with uss pMB 40 positive control li plasmid dna transformants. Gel electrophoresis of plasmid dna transformants without uss pjak 17 positive control li plasmid dna transformants. Transformation assay.
Km(40) Cm(2) Km(40) Sp(20). Transformation Assay : 3 Transformation of li liu 4 Nal(20) Nal(20 Nal(20) Nal(20) Km(50) Cm(50) Km(50) Sp(50). Transformation assay 4 Transformation with heated growth medium. Transformation assay 5 Transformation with frozen supernatant. Catalase test Individual colonies of transformant were picked with tooth pick without touching the plate surface and dipped into eppendorf tube containing 1 ml of 3 hydrogen peroxide. Results Transformation assay 1 and 2 Number of transformants Fresh donor growth medium Frozen/thawed donor growth medium Growth medium volume Growth medium volume.22.214.171.124 mlRecipient Serotype Incubation time Incubation time Incubation time Incubation time 2h 3h 5h. Gel electrophoresis: pmb 78 positive control. Transformation assay 2 Transformants/ml write Transformants/ml (spectinomycin Recipient strains Serotype (kanamycin resistant) resistant) liu 1235 a liu1195 b liu1196 c smooth rough 176. E liu1193.
Pjak 17 is a derivative of the broad host range plasmid rsf1010. PMB40 contains a two copies of uss cloned into pJAK13, a similar derivative of rsf1010. Li strain used: liu 4 (MV10Nal). Transformation with genomic donor dna containing kanamycin resistance marker present in vesicles in the growth medium of donor stains Set 1: 24 hours culture tubes Incubation for different length of time. Set 2: recipient cells were resuspended.5 ml of donor dna instead of 100. Transformation Assay : 2 Transformation with plasmid dna with and without uss present in growth medium of donor strains liu 12 respectively. 4 steps were as above.
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Cell surface proteins or transporters that bind the uss have not yet been identified uss are often found in inverted-repeat pairs downstream from coding region. Need for natural transformation The lack of summary genetic tools Scarcity of standard and biological techniques which work well. Preference to take dna containing sequences which are present frequently in its own genome. Lack of treatments which permanently cure lap. Aim of the study: The objective of this study is to demonstrate a novel mechanism of natural transformation. With genomic and plasmid dna present in micro vesicles secreted by donor cells in growth medium. To demonstrate that.
Can be naturally transformed by this method, both in the presence or absence of Uptake signal sequences (USS) or Tfox gene. Materials and MethodsRecipient strains. Formal name of liu names serotype reference/Source strains Strainsused: liu 1235 DF2200Nal a david Figurski, columbia recepient strains. Actinomycetemcomitans University nj1000Nal liu 1195 b dan Fine, umdnj nj 2700Nal liu 1196 c dan Fine, umdnj idh781Nal liu 1231 d dan Fine, umdnj nj9500Nal liu 1201 for e dan Fine, umdnj nj9100Nal liu 1193 f dan Fine, umdnj cu1000Nal liu 1188 f david Figurski, columbia. Donor strains. Al.19 Y4NalTn:katA(pjak 17) pjak 17 mobilized from li into liu11 Y4nalTn:Kat(pMB 40) pMB40 mobilized from li into liu 1121 All donor strains were catalase negative.
An unidentified supra antigen which cause t- cell apoptosis. Cytolethal distending toxin ( Cdt a, b, and C) causes arrest of cell growth in G2 phase. Natural Methods of gene transfer observed. Natural gene transfer or dna translocation occur during several important biological processes, such as - infection by bacteriophages, conjugative dna transfer of plasmids, t- dna transfer and natural genetic transformation. General mechanism of Natural transformation development of competence binding of dna to the cell surface processing and Uptake of dna into cell Incorporation of dna into host genome via homologous recombination. Natural transformation.
Appears to be strain specific and is of too low frequency to be useful in genetic studies. Requirements for natural transformation: Presence of 9-bp uptake signal sequences in incoming dna expression of tfox gene to activate genes of competence Induction of genes of competence similar features were observed in another gram negative bacteriaum fluezae. Development of Competence and role of Tfox gene Induction of competence. Requires the product of tfoX (sxy) gene Tfox protein itself does not bind to dna but interacts with crp(CAP) Turn on the genes of the competence regulon Thus expression of tfoX makes. Cell constitutively competent. Significance of uss in natural transformation usss are believed to be genomic identity tags. Seems to take up their own dna preferentially This specificity arises from presence of usss. Numerous copies of usss found in genome.
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This group of bacteria causes inflammation of heart valves. Studies show that infection by for these bacteria may impact the effectiveness of some medicines which are used to prevent heart attacks. Mechanism of pathogenicity:. Virulence factors. One of the best studied virulence factor. Is leukotoxin ( a 14 kda secreted lipoprotein homework that belongs to rtx family. Leukotoxin has been shown to kill polymorphonuclear leucocytes and macrophages. Other ill defined virulence proteins. Are Thioredoxins that inhibit lymphokine production.
Extra-oral pathologies includes, preterm low birth weight, atherosclerosis, plaque buildup in the arteries, which creates greater risk of stroke and heart attack. It also plays important role in accumulation of cholesterol in blood stream with a support of macrophage derived foam cells. Thus, contribute role in formation of atheroma. 0.6 of infective endocarditis are caused. Is a member of clinically important hacek group. ( haemophilus aphrophilus,. A., phd cardiobacterium ominis, eikenella corrodense, kingella kinge).
observed. Oral and Extra-oral Pathology:. S is one of the most powerful periodontal pathogen. It is naturally found in dental plaque, periodontal pockets and gingival sulcus. It is also present in periodontal pocket disease.
Apart from being a major periodontal pathogen, evidences of the presence of their genomic dna in lower respiratory tract, meninges of cns and urinary tract suggest that they are also non oral pathogens. Is predisposing factor for several other major diseases like atherosclerosis and related conditions of coronary artery disease, stroke, diabetes and pregnancy complications, abscesses, meningitis, pneumonia, septicemia, uti osteomyelitis. Distribution: In united states, periodontitis Affects 14 of adults writing aged 45-54, 23 of those aged 65-74. Mean prevalence was.53 among adolescents of all racial origins, and. Was isolated in 97 of those cases. Adolescents of African-American descent were found to have a 15- fold higher incidence of diseases than caucasian Americans. very few research that support genetic predisposition to localized aggressive periodontitis.
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Successfully reported this slideshow. Thesis Power point Presentation, upcoming SlideShare, loading. Show More, no downloads, no notes for slide. A thesis submitted by needed riddhika pandya graduate. Pharmacology toxicology guided. Index: genral introduction of actinobacillus actinomycetemcomitans aim of the study bacterial strains used and methods results discussion importance of project future applications. Actinobacillus Actinomycetemcomitans tinomycetemcomitans is gram negative facultative anaerobe, non-motile cocobacillus and a member of pasteurellacea. It is major causative agent of Localized aggressive periodontitis.