Genetic and Phenotypic Characteristics of Transposon Mutant of Pseudomonas Aeruginosa PAO1 - Case Study Example

Materials and Methods: Strain and Growth Condition The strains and plasmids used in this study, Pseudomonas aeruginosa PAO1 wild type lausane and Escherichia coli Strains 517.1- λ pir and DH5α were cultured routinely at 37ºC in Luria Bertani (LB) broth (Pls include brand) or on LB agar plates supplemented with Gentamicin ( ) and Kenamycin (30 µg per ml). Table 1 summarizes the strains, plasmids, and primers used in the study. Strain Genotype /characterises References Escherichia Coli PLM1 Ctx :: lux Ctx::Lux reporter plasmid contains the Lux CDABE operon used as a donor strain Winzer et al (2001) Psudomonas aeruginosa PAO1 Wild type lausane Lausane PAO1 lec A:: lux Diggle et al (2003) Plasmid E.Coli 517.1- λ pir and DH5α Ctx lecA :: lux LecA:: Lux Plasmid insertion Primers Tn5-1 primer 100 PM Tn5-2 primer 100 PM Construction of Transposon library Construction transfer of pLM1 was done by growing the organism with E.Coli 517.1- λ pir. Both recipient strains were grown overnight in 50ml LB at 37 oC. The cultures were then transferred in 50 mL conical tubes and centrifuged for 10 minutes at 5000 rpm at 4 oC. The supernatant was removed and the remaining pellet was washed with 25 mL of cold PBS twice. Thereafter, 10 ml of PBS was added to the pellet and recentrifuged. Finally, the supernantant was removed and 1ml of PBS was loaded into the tube. Both strains were plated unto LB agar and incubated overnight at 37 oC. The following day, each droplet was plated unto LB agar supplemented with Gentamycin and incubated at 37 oC to make a transposon library using microplates 96 wells (960 strain). Screening of Transposons Library (phenotype assay) Replication using Genomic essay (do you mean ASSAY?) robot was done in different media such as Protease, swarming, Elastase,LB Kanamycin and LB agar for Lec expression Cloning and sequencing Isolation of Genomic DNA Isolation of genomic DNA was done using ____________ (the DNA kit you used and its brand) following the protocol of the manufacturer. Concentration and purity of the DNA were measured using Nanodrop Spectrophotometer (pls include brand of the equipment) DNA Digestion The digestion mixture (30 µl) was made by using 10µl of DNA ,3 µl of buffer,2µl of the restriction enzymes, Eco ˅ and Hin 111) and 15 µL H²O .The reaction mixture was placed in 37 oC overnight. Agarose Gel Electrophoresis (AGE) of dDNA Mixture 1% agarose gel was produced ,50 ml of TAE buffer and 0.50 g of agarose were added and heated in the microwave for 1 minute to dissolve the agarose, 2 µl of Ethidium bromide was added to the mixture was swirled. The gel was poured into a rack with a comb and left until it was loaded into the lane. The gel was run for around 45 minutes at 100 volt, the gel was taken and placed in the UV box ( ) program was used to visualize the gel with UV and a picture was taken. Friend, AGE is composed of stacking gel and loading gel, as far as I could remember. Can you give me more details on this part. thanks Ligation The ligation mixture (10µl) was made using 8µl of dDNA, 1µl ligaze buffer and 1 µl of T4 DNA ligaze . The reagents for the ligation were purchased from ..............The reaction was incubated at 4 oC overnight. Transformation Transformation was performed using DH5α E.coli competent cells. To optimize the conditions, one reaction was made with 50 µL of competent cells added with 2 µL of ligation mixture and another reaction was made with 50 µL of competent cell with 1µL of ligation mixture. The cells were thawed on ice and ligation mixture was added. The mixture was then loaded in the Gene pulser cuvette/E.coli pulser curvette. use pulser machine (electroperation)by using Eco ǁ,add 950 µl of LB,incubation 37C for 2 hours,transfere to clean appendofe tubes,centrifuge 5 minutes at 4000 rpm,remove the supernatant, plates 50µl from the pellet and incubation over night at 37C------ is this the procedure for the electroporation that you did? Sequencing The colonies that tested positive in the cloning steps had their DNA purified using.......prep miniprep spin kit from ( ) following the manufacturer’s protocol. DNA concentration of the plasmid and its purity were measured using Nanodrop spectrophotometer. Pyocynin Bacterial cultures were grown overnight in 10 mL _____(pls indicate the medium) without shaking. Afterwhich, the optical density of the bacterial culture at 600 nm was adjusted to 0.1 (is this correct? I also do this sometime, but I don’t know if this is what u meant?). Then, 1 mL of the adjusted culture was inoculated into a 100 mL ______ (what medium). To standardise the inocula, the OD was divided by the lowest OD and added with the relevant volume of medium. The cells were incubated for ____________ and their OD at 600nm was measured again. From this culture, 5 mL was transferred into a centrifuge tube and centrifuge for 10 mins at 10,000 rpm. Then, the supernatant was transferred into fresh tubes and added with 3 mL chloroform. The mixture was vortexed and 2 mL chloroform layer was transferred into a fresh tube, added with 2 mL of 0.2N HCl, and centrifuged. One millilitre of the top layer was removed and placed in an eppendorf tube. Amount of pyocianin was measured by taking the absorbance at 520 nm. (I think u don’t need to include the computation if this is a journal-formatted paper. Maybe you want to make an appendix for the detailed procedure. Swarming Growth medium for the swarming experiment was prepared by using the following components: 2 g Bacto Agar (Difco), 3.2 g Nutrient broth No 2 (Oxoid), 400 ml H²O). A filter-sterilized D-glucose was aseptically added to the medium to a final concentration of 0.5% and was poured in petri plates. Then, a 5 µl drop of overnight bacterial culture was inoculated in the center of the plates and was incubated at 37 oC (for how many hours) Protease The protease activity was done by spotting the bacteria onto 5 % skimmed milk agar. The plate was incubated at 37 oC. A clearing zone around the spot indicates a positive result. Elastase Amount of elastase produced was measured by reacting 1 mL of the reagents (100 mM Tris 1 mM CaCl², PH 7.5, elastin –Cango red 5 mg/ml buffer) with 100 uL of supernatant (is this bacterial supernatant?). The mixture was incubated at 37 oC with shaking for 2 hours, followed by addition of 100 uL of 120 mM EDTA. The mixture was centrifuged and the optical density of the supernant was read at 495 nm. Antibiotic susceptibility A 96-well microtiter plate was loaded with LB media supplemented with different concentration of Kanamycin( 10-30-50-70-100µg) and inoculated with different strain of mutants. TECAN (is this the equipment?) was used to measure the antibiotic susceptibility. 10. Determination of LecA:: lux gene experiments Determination of LecA:: lux gene by using two methods TECAN program and luminaneta Read More