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  1. May 2019
    1. Flies were maintained at 18°C or 25°C as appropriate. Through out this thesis, flies defined as wild-type were yellow white of the genotype: y67c23w118. BEAF32 null lines BEAF32AB-KO/CyOGFP, kindly provided by Craig Hart, University of Illinois (Roy et al., 2007a). Homozygous BEAF32AB-KOlines were obtained by selection against the CyOGFPmarker at the 3rdinstar larvae stage, using a Leica M165 FC with a GFP filter. Lethality of the BEAF32AB-KOallele was assessed against the dppHr27hypersensitive allele (genotype: dppHr27,cn1,bw1/CyO P{dpp-P23}). For this embryos were collected from the following crosses as set up by Catherine Sutcliffe:BEAF32AB-KO/+ ×dppHr27,cn1,bw1/CyO P{dpp-P23}and+/+ ×dppHr27,cn1,bw1/CyO P{dpp-P23}
    2. Fly Stocks and Crosses
    1. eppendorf tube had DNA of interest. The purified DNA fragments were checked on an agarose gel, with an appropriate marker, before setting up the ligation reaction
    2. The plasmid DNAs for cloning, were digested with the respective enzymes, checked on an appropriate percentage of agarose gel along with 100 bp ladder or .A Hind ill marker (Promega, USA) and the required fragments were eluted from the gel using the Qiagel Gel Extraction kit (Qiagen, U.K.). According to the manufacturer's directions, the area of the gel containing the DNA fragment was excised using a clean and sharp blade, minimizing the amount of surrounding agarose excised with the fragment. The gel slice was weighed and placed in a microfuge tube. Three volumes of Gel Solubilization Buffer (QG) was added for every one volume of gel. The gel piece was then vortexed and incubated at 500C for 10 min. The contents were mixed in between, by inverting the tube few times, to ensure gel dissolution. It was then centrifuged at 13,000 rpm for 1min. The flow through in the discard column was carefully removed. Then 500 p.l of buffer PB was added to remove the traces of gel and the tube was centrifuged at 13,000 rpm for 1 min. The flow through in the discard column was removed and 750 p.l of Wash Buffer (PE) (containing ethanol) was added and the tube was centrifuged at 13,000 rpm for 1 min. The flow through was discarded and another spin at maximum for 2 min was given to remove the traces of wash buffer. The column was then put on a fresh tube and finally, to elute the DNA, 40 p.l of Tris-EDTA buffer (TE) or RNase-DNase-free water was added and then centrifuged at 13,000 rpm for 1 min. The flow through in the
    3. Gel elution of DNA fragments:
    1. Western blotting by adding 2X sample buffer as described in 3.2.B.19 and 3.2.B.20 respectively
    2. Triton X-114 has a property of a low cloud point (23°C). At temperatures above the cloud point, detergent solutions separate into aqueous and detergent-enriched phases thus separating membrane proteins (detergent phase) from cytosolic ones (aqueous phase). Promastigotes in the logarithmic phase of growth were harvested and dead cells pelleted at 129 x g. 109 live promastigotes were resuspended into 10mL of 0.5% vI v Triton X-114 containing protease inhibitor cocktail. The cells were homogenized using a 30-40 strokes of dounce homogenizer. The homogenate was incubated on ice for 90 min with intermittent stirring. Following this insoluble material was pelleted by centrifugation at 3700 x g for 35 min at 4°C. The supernatant was incubated at 37°C for 2 hr., till layers separated well. The top aqueous layer was separated from the bottom detergent layer. Samples of both these were prepared for SD
    3. Triton X-114 extraction of membrane proteins
    4. First strand synthesis of mRNA into e-DNA was performed using First strand e-DNA synthesis kit from Invitrogen (Carlsbad, CA) following manufacturer's protocol. Briefly, 4 !lg of total RNA was denatured at 65°C for 5 min in the presence of Oligo dT12-18 and dNTPs and then cooled on ice for 1 min. DTT, MgCb and RNaseOUT in lOX reverse transcriptase buffer added to the above mixture and incubated at 42°C for 2 min. 1!!L/reaction of the Superscript Reverse Transcriptase enzyme was added to the denatured RNA and incubated at 42°C for 50 min. The enzyme was denatured by heating at 70°C for 15 min. The reaction was completed with degradation of the complementary RNA strand by incubating with RNase H for 20 min at 37°C. The DNA preparation was stored at -20°C.
    5. First strand synthesis by reverse transcription
    6. Murine macrophage cell line J774A.1 (ATCC no. TIB-67) was maintained in phenol red free DMEM supplemented with 10% heat inactivated (45 min at 65°C) foetal bovine serum at 37°C in 5% C02 and 95% air. The cultures were sub-cultured every three days or at the attainment of 80 % confluency.
    7. In vitro J774A.l murine macrophage cultur
    1. For bacterial isolates, a single colony from a nutrient agar slant was inoculated into 50 ml of nutrient broth in a 250 ml Erlenmeyer flask. These flasks were incubated at 37±1°C in a incubator shaker till an optical density of 0.6 at 660nm. Now these cultures were used to inoculate 50 ml of the tannase production medium in 250 ml Erlenmeyer flasks using 2% v/v inoculum. These flasks were incubated at 37±1°C in an incubator shaker (Multitron AG-27; Switzerland) at 200 rpm for 72h. The experiments were carried out in triplicates. Samples (2.0 ml for bacteria and same for fungi) were withdrawn at regular intervals of 12h upto 72 h. The samples thus obtained were centrifuged at 10,000 rpm in a refrigerated centrifuge (SIGMA 4K15 Germany) for 10 min at 4°C. The supernatant/s were analyzed for tannase activity
    2. Microorganisms were isolated from the above mentioned sources using direct plating method. Serial dilution of the different soil samples with normal saline was carried out and the different dilutions were spread plated on to potato dextrose agar (PDA) for isolation of fungi and on to nutrient agar (NA) for the isolation of bacteria. The plates were incubated at either 30 or 37±1°C in a bacteriological incubator so that the different organisms could grow and form visible colonies. The different fungal and bacterial colonies isolated by the procedure mentioned above were purified by subculturing on respective media, and subsequently screened for tannase production. The new isolates, alongwith different cultures obtained from laboratory stock culture collection, were revived on potato dextrose agar (PDA) slants. These cultures were regularly subcultured and stored at 8±1°C in a BOD incubator. Their purity was periodically checked by microscopic examination.
    3. Isolation of bacteria and fungi from the samples
    1. The slides for microscopy were prepared as described in Dajkovic et al.,(2008)with slight modifications. After wiping the glass slide with ethanol, 200μL of 1% molten agarose was layered on it between two strips of tape and clean cover-slip placed on it to obtain levelled surface. The agarose was allowed to solidify and the cover-slip was carefully removed and5μlof sample was put on top of the agarose and carefully covered with a cover-slip
    2. Preparation of microscopic slides
    3. Semi-dry transfer apparatus (Bio-Rad trans blot semi dry transfer cell)was used for the transfer of RNA from the gel to the membrane. The Hybond-N+ membrane from Amersham biosciences was used which was cut as per dimensions of the gel containing the RNA samples. For each transfer 6 pieces of Whatman3mmsheets of the size of the membrane were used. The membrane was soaked for 30-60 minutes in 0.5XTBE before transfer. The transferapparatus was set up as describedby the manufacturer. Transfer was done in 0.5XTBE buffer at 20V, 400mA and 100W for 1.15 hours
    4. Transfer of RNA to the membrane
    5. Total RNA extraction from E. colicells was doneusing Qiagen RNeasy minikit. Cells were grown to an A600of 0.6 and harvested(amaximumof107cells)at 6000rpm for 5min at room temperature to prevent cells for encountering any stress in cold. Rest of the steps were followed exactly as mentioned in the manufacturer’s protocol. The quality of RNA preparations was assessed following electrophoresison 1.4% agarose-formaldehyde-MOPS gels.Ingeneral,forawild-typestrainRNAyieldwouldbe~0.5-1μg
    6. Isolation of total cellular RNA
    7. Recombineering was performed as described in(Yuet al., 2000)for engineering the linear DNA on the chromosome. The oligonucleotide primers were designed to amplify the DNA cassette to be engineered. Oligonucleotidesused for recombination contained30–50nt homology at the 5ʹ endtothesequences at the target siteand 20nt homology tothe DNA cassette at the 3ʹ end. The DNA cassettefor recombinationwas generated by PCR and would contain30-50 bp homologiesto the target site. A strain with the target DNA and carrying a defective λ-prophage with gam,betaand exo genes (thatfacilitate homologous recombination)under the control of a temperature-sensitive λ cI-repressorwas grown at 30oC. At an A600of 0.4, the culture was shifted to 42oC for 15 minutes to express gam,betaand exo genes. Cells becomecapable ofrecombining linear DNA introduced into the cell by electroporation. 50-100ng ofamplified DNA cassettewas used for electroporation whichwas performed using theBio-Rad Gene Pulser set at 1.8 kV, 25 μF with Pulse controller of 200 ohms
    8. Recombineering
    9. Typically 400-500ng of DNA was used in each ligation reaction. The ratio of vectorto insert was maintained between 1:3 and 1:5 for cohesive end ligation. The reaction was generally performed in 15μl volume containing ligation buffer (provided by the manufacturer) and 0.075 Weiss unit of T4 DNA ligase at 16ºC overnight (14-16 hours)
    10. Ligation of DNA
    11. DNA fragments to be used for specific purposes like ligation or radioactive labellingwere eluted from the agarose gel after electrophoresis. The gel piece containing the desired band was sliced out from the gel and the DNA was purified using commercially available purification kit (Qiagen)for this purpose. The efficiency of elution was determined by checking a small aliquot of DNA sample on the gel
    12. Purification of DNA by gel elution
    13. Around 0.5-1μg DNA was regularly used for each restriction digestion. 2 to 5 units of restriction enzyme were used in the total reaction volume of 20μl containing 2μl of the corresponding buffer supplied at 10X concentration by the manufacturer. The reaction was incubated for 3hours at the temperaturerecommended by the manufacturer. The DNA fragments were visualized after electrophoresis on 0.8 to 1.5% agarose gels. Commercially available DNA size markers were run along with the digestion samples to compare with and to estimate the sizes of the restriction fragments
    14. Restriction enzyme digestion and analysis
    15. The DNA samples were mixed with appropriate volumes of 6X loading dye (0.25%bromophenol blue and 0.25% xylene cyanol and 30% glycerol in water) and subjected to electrophoresis through 0.8 to 1.5 % agarose gel in TAE buffer. The Goodview nucleic acid stain(supplied as 20000X; Beijing SBS Genetech Co. Ltd.) was added to the gel at the time of casting or 6X EZ-Vision One DNA dye(Amresco) was used as loading buffer, both being commercially available non-carcinogenic dyes to aid visualization of bands. The visualization was doneby fluorescence under UV light in a UV transilluminator
    16. Agarose gel electrophoresis
    17. following the manufacturer’s instructions. For genomic DNA, 1ml culture was used for DNA isolationusing Qiagen or Invitrogen kits. The quality of plasmid/genomic DNApreparations was assessed following electrophoresis on 0.8% agarose gels
    18. 3ml (for high copy number)or 10 ml (for low-copy number) of cells from an overnight culture were pelleted by centrifuging for 5 minutes at 6000rpm forthe plasmid isolation which was carried out with the commercially available kits (Qiagen or Invitrogen)
    19. Isolation of plasmid and chromosomal DNA
    20. Recombinant DNA techniques
    21. To 2 ml of fresh overnight culture of recipient strain, 108 pfu equivalent of phage lysate was added and incubated at 37ºC without shaking for 30 minutes to facilitate phage adsorption. The unadsorbed phage particles were removed by centrifugation at 6000 rpm for 5 minutes and the pellet ofbacterial cells was resuspended in 5 ml of LB broth containing 20 mM sodium citrate to prevent further phage adsorption. This was incubated for 25-60 minutes at desired temperaturewithout shaking to allow the phenotypic expression of the antibiotic resistance gene. The mixture was then centrifuged and the pellet was resuspended in 300 μl of 0.1M citrate buffer. 100 μl aliquots were spreadon appropriate antibiotic containing plates supplemented with 2.5 mM sodium citrate. A control tube without addition of P1 lysate was also processed in the same way. In the case of selection of nutritional requirement, the infection mixture was centrifuged, resuspended in 300 μl of 0.1M citrate buffer and plated without phenotypic expression
    22. Phage P1 transduction
  2. Nov 2017