Tn 4351 was originally isolated from bacteroides fragilis [30] . The transposon was successfully introduced into Cytophaga succinicans, Flavobacterium meningosepticum, Flexibacter canadiansis, Flexibacter strain SFI and Sporocytophaga myxococcoides by conjugation [25]. Tn 4351carries two antibiotic resistance gene. One of the codes for resistance to erythromycin and clindamycin which is expressed in bactroides but not in E.Coli. The other gene codes for resistance in tetracycline and is expressed in aerobically grpwn E. coli, but not in anaerobically grpwn E. coli or in bacteroides. The transposon of Tn4351 was originally detected in E. coli which carried an unstable chimeric plasmid, pSS-2. The mobilization of pSS-2 from onestrain of E. coli …show more content…
chinesis. A construct of R751::Tn4351 (the physical map of R751::Tn4351 and restriction sites are shown in fig. 7) was selected for introduction into F. chinesis to discover if the introduction and insertion of the vector R751 and the transposition of T4351 into the F. chinesis chromosome by a triparental mating occurred. One parent was E. coli GJ342 which carried a helper plasmid, the second parent was E. coli HB101 which contained R751::Tn4351 and the third parent was the F. chinesis target strain. 189 colonies were isolated on LB agar plates which in passage in fresh media were able to grow in 200µgml-1 erythromycin. The erythromycin resistance gene is carried by Tn4351. erythromycin resistance colonies were transfer to LB agar containing 200µgml-1 thrimethoprim. Non of the colonies could grow in this medium and no free vector (R751) was obtained in plasmid miniprep. This indicates that no replication of R751 occurred. Colony blot hybridization was done separately to discover if Tn4351 and/or R751 had inserted into the chromosome of F. chinesis. Duplicated blots were probed separately with radiolabled pVOHI (for the detection of the Tn4351) and R751 (for the detection of the transposon delivery vector). pVOHI is a derivated of pBR328 that carries Tn4351 but there is no common sequence with R751. Approximately half of the colonies selected from the first screen were positive in the second screenfor the detection of Tn4351 (Fig. 8.a). A few colonies were positive for the detection of R751 (Fig. 8.b). A few mutant defective in spreading were isolated (Fig. 8.c) and some auxotroph mutants were also
The Mut section is expected to have no growth as mutants require the amino acids leucine and valine to grow which is not provided in the minimal medium. Results Figure 2. Testing of mutant mixed with DNA, mutant bacteria and DNA on LB medium Growth was observed on the Transformed (Trsf) section and the Mutant (Mut) section but not on the DNA section. Due to human errors, the photo of our experiment was lost, but we have obtained similar results as from group1.1 and their photo is presented.
By what mechanism do the authors propose that the mcr-1 gene confers colistin resistance, and what evidence do they use to support this assertion? The protein sequence of mcr-1 showed its similarity to the polymyxin-producing bacterium, Paenibacillus spp., which showed the possibility of gene transfer occurring. The mcr-1 gene enables protection from polymyxin. The mechanism that the authors proposed on how the mcr-1 gene confers colistin resistance is that mcr-1 causes a modification in lipid A, present in the lipopolysaccharides of most bacteria, which leads to lessened polymyxin affinity.
Human beings are hosts for many bacterial species that colonize our skin as their natural flora. The skin acts as a superior barrier and first line of defense against bacterial infections. When they do occur, these infections are mild and easily treatable; however some can become very serious and even life-threatening. Staphylococcus aureus and Streptococcus pyogenes are uncommon bacteria, but they are responsible for a wide variety of bacterial pyodermas [1]. In some cases, the host for bacterial infections can become contagious to others.
Typical sample dimensions 9.51 × 4.83 mm2in surface area and1.58 mm in thickness were coated with conductive silver paint formetallic contacts. The dielectric constant of the sample was mea-sured for the applied frequency that varies from 100 Hz to 1 MHz atdifferent temperatures (40◦C, 60◦C, 80◦C). The observations weremade while cooling the sample. The dielectric constant εrwas cal-culated using the relation, εr =
Abstract The transformation principle suggests that bacteria use DNA as their genetic material and are able to exchange their genetic material via a process of transformation. Griffith had theorised the concept of the transformation principle using two strains of bacteria and studied their ability to recombine. Avery and MacLeod followed his studies and suggested DNA was sensitive to DNase, and that the enzyme would destroy the bacteria's ability to exchange genetic material and transform into a new strain. This was then tested in the labs at Wits by second year students where they studied the transformation of ampicillin sensitive E. coli to ampicillin resistant E. coli.
Up to 1944, Avery et al demonstrated that deoxyribonucleic acid (DNA) was the transforming substance (Avery, 1944). In 1952, Hershey and Chase showed that DNA was the only material transferred during bacteriophage infection, which suggested that the DNA is the genetic material (Hershey, 1952). The basic technique for introducing DNA into E. coli have inspired procedures for the introduction of DNA into cells from a wide variety of organisms, including mammalian
coli (Table 1) by using the well agar diffusion method[26]. The multidrug resistant pathogens were initially propagated at 37 °C in nutrient broth. The overnight grown cultures were then again sub-cultured into nutrient broth media for 2 h till 0.01 OD. Subsequently, 100 μL of each culture was then spread uniformly onto nutrient agar plates. Wells of 6 mm diameter were made on agar plates using an agar well borer.
The sterile cotton swab was inserted in the S. epidermidis culture and twirled around to obtain a specimen. The entire plate was inoculate with the swab from top to bottom, to achieve a lawn of growth. The dry forceps was used to remove the antibiotic disk into the appropriate spot on the plate. This process was repeated for the all antibiotics with aseptic technique being used. The plate was incubate with lid up on the bookshelf at room temperature for 48 hours.
Then 1 ml from these bacterial solutions were added to 1 ml of MHII containing different concentration of ciprofloxacin 0.5x, 1x, 5x, 10x, 20x, 30x, 50x,75x and 100x MIC to each well in 12-well plate. Colony counts (CFU/ml) were determined at different time points (T0, T3, T24) by using appropriate dilutions of each culture. Using spot-plating method (32) 10µl was spotted on LB agar plates. However after 24 hour exposure the bacterial cell from dilutions of ≤ 1x102 were washed twice with sterile PBS prior to plating in triplicate. These were then incubated overnight at 37°C.
Tacrolimus is specific T-cell inhibitor and potent immunosuppressant agent [no]. Tacrolimus (FK506 or Fujimycin) is a 23-membered polyketide macrolide with immunosuppressant activity that was discovered in culture broth of the soil bacterium Streptomyces tsukubaensis in 1984 []. The immunosuppressant tacrolimus was initially discovered in fermentation broth of Streptomyces tsukubaensis, which was isolated from Japan soil of the Tsukuba region. Most of the initial studies performed with streptomyces tsukubaensis had been focused on strain improvement and development of fermentation procedures at industrial level []. In initial studies Fujisawa scientists isolate a tacrolimus producing strain of streptomyces tsukubeansis, no 9993 and established
Plasmid and PEI reagent were prepared both 1:3 and 1:5 ratios and incubated during 15 minutes at room temperature. Then, these plasmid-PEI mixtures were added to culture and incubate 24 hours and 48 hours for each ratios. The table 1 shows all prepared assay. Then, 10 µl MTT
This shows that there is no definite outcome of recombinational repair or mitotic crossing over caused by the mutagens, tartrazine and carmoisine. If tartrazine and carmoisine causes mitotic crossing over, then the homoallelic ade2 loci: ade2-40/ade2-40 would lead to an obstruction to the adenine pathway which then results in the formation of red colonies. However ade2-119/ade2-119 would result in the formation of pink colonies. The presence of pink-white and red-white colonies suggests that the crossing over between the ade2 loci and the centromere has taken place. However the exponential growth of the colonies and the presence of clumping were due to excess amounts of mixture added to the plates.
Materials and methods Gene cloning and protein over-expression in E.coli: The gene encoding Mbgl was amplified from the genome of Methylococcuscapsulatus(bath)with the following primers: forward primer- TGGTTGGTTCATATGAGCAGATACGAGTTTCCAGAGCGATTCCTCand reverse primer-TATATATTAAAGCTTATAGTCCCGATCCAGGACGGCACCGTTGGTC (restriction site NdeI and HindIII are in italics and underlined). Prime STAR HS DNA Polymerase premix (DSS Takara, New Delhi, India) and the set of above-mentioned primers were used in the program consist of the following polymerase chain reaction (PCR) steps: (1) 95°C for 5 minutes (1 cycle), (2) 98°C for 20 seconds, (3) 50°C for 20 seconds, and (4) 72°C for 2 minutes (steps 2 to 4 was repeated for 35 cycles). The obtained PCR product was digested with NdeI and HindIII restriction enzymes and
Proteins that play important role(s) in the structuring of DNA and having the potential to influence gene expression have been explored in all kingdoms of life. The organization of bacterial chromosome is influenced by several important factors. These factors include molecular crowding (de Vries, 2010), negative supercoiling of DNA (Postow et al., 2004), the influence of NAPs (nucleoid-associated proteins) and transcription (Dillon and Dorman, 2010; Dorman, 2009, 2013b;
Conjugative transfer which involves the acquisition of plasmid, and is mediated by cell to cell junctions and a pore through which DNA passes. This system has plasmid as the important component. The transfer of DNA in small units through the plasmid carriers is possible and is found to be preferred because, the transfer of whole chromosomes could take an hour, a duration too long to keep the interbacterial junction intact. Conjugation can mediate the transfer of genetic material between domains (for example, between bacteria and plants, and between bacteria and yeast; 29