703 Matching Annotations
- Jun 2019
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shodhganga.inflibnet.ac.in shodhganga.inflibnet.ac.in
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GFP-β-catenin-C-N (151-423 a.a) (ARM-N)
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GFP-β-catenin-C (ARM)
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GFP-β-catenin- C (634 -781 a.a)
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GFP-β-catenin- a.a
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GFP-β-catenin T41A mutant
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Constructs made in the study
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GFP-β-catenin-FL
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shodhganga.inflibnet.ac.in shodhganga.inflibnet.ac.in
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Chemicals/inhibitors
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Antibodies
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Instruments
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Plasmid
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Bacterial Strain
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Mammalian cell lines
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Commonly used Buffers/Reagents
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IL-2 Bioassay
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IL-2 Bioassay
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Statistical Ana(vsis
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Western Blot Ana(v.\'is
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IL-2 Bioassay
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Assay of IFN-yand IL-10 using sandwich ELISA
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IL-2 Bioassay
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LympllOkine Assays
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T cell hybridoma stimulation as.".ays
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T cell proliferation assay
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Anti-Stm and anti-E coli /gG subclass assay
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Total Anti-Stm and anti-E coli antibody assay
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Enzyme linked Immunosorbant Assays (ELISA .... )
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Injections/or determining MHC-peptide ligand densities
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Delayed type hypersensitivi(V (DTH) response
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Injections/or determining MHC-peptide ligand densities
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Immunisation regimen
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Bleeding of mice and Collectiml of Sera
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Kinetics of bacterial clearance
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Expression of foreign protein in bacteria
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Transformation ofStm754 with Ea-cmyc-CiST plasmid
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Protein estimation of sonicate
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Sonication of bacteria
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Irradiation of bacteria
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Bacterialstrain
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Cellline
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Reagents
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Statistical Analysis
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Columns for Affinity Chromatography
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SOS PAGE
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2 Bioassay
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Proliferation Assay
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Target binding assay
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High Performance Liquid Chromatography
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Ion Exchange Column Chromatography
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IFN Biossay
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Cytotoxicity assay
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Sephadex G-100 Column Chromatography
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Preparation of Mouse MHC-AF
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sg.inflibnet.ac.in sg.inflibnet.ac.in
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ACK lysis Buffer
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ELISA dilution buffer
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ELISA blocking buffer
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ELISA washing buffer
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ELISA binding Buffer
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FACS buffer
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Youman's modified medium
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Phosphate buffered saline (PBS), 0.15M, pH 7.2
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Reagents Used
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Statistics
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Histopathological studies
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Bacterial load in the spleens studied by cfu plating
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Protection studies
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Expression of costimulatory molecules on splenocytes harvested from immunized and control animals
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Expression of costimulatory molecules on the vaccines
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Phenotypic analysis by Flow cytometry
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lgG-isotyping in the sera of the immunized animals
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Lymphokines Assay
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Lymphoproliferation
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Immunization of the animals
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Preparation of the vaccines
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Cell lines
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Bacterial Cultures
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Animals
Tags
- mt-11-mt-2
- mt-11-mt-3
- mt-3
- mt-12-mt-1
- mt-12-mt-6
- mt-9-mt-1
- mt-5
- mt-12-mt-4
- mt-4
- mt-12
- mt-1
- mt-6
- mt-11-mt-1
- mt-9
- mt-12-mt-7
- mt-2
- mt-12-mt-5
- mt-12-mt-2
- mt-10
- mt-8
- mt-12-mt-8
- mt-12-mt-3
- mt-11
- mt-7
Annotators
URL
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shodhganga.inflibnet.ac.in shodhganga.inflibnet.ac.in
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Microscopy
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Drugs
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Lucifearase assay
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Cytotoxicity assay
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Immunoblotting
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Dot-Blotting
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Transblotting
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SDS-Poly acrylamide gel electrophoresis (PAGE) of proteins
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Protein extraction and quantification
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RNA extraction/c-DNA synthesis
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Lipofectamine method
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CaPO4 method
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Phosphate buffered saline
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Dulbecco’s Modified Eagle Medium
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Agarose gel electrophoresis
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Transformation
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Polymerase Chain Reaction (PCR)
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Genomic DNA extraction
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Large scale plasmid extraction
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Resuspension solution
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Transformation buffer
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Ultra-competent cells
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X-gal
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Antibiotics
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Luria-Bertani (LB) medium
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T-Cell Receptor Mutation Assay
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RS-1 in Cancer samples
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RS-1 Assay
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Flow Cytometric analysis of variant erythrocytes
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Monoclonal Antibodies against the Human Glycophorin
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MN Blood group typing
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shodhganga.inflibnet.ac.in shodhganga.inflibnet.ac.in
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Instruments used for the study
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MATERIALS
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Chemicals
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Single Cell Test
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Electrochemical Measuremen
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Physical Characterization
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Catalyst Preparation
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Materials
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Single Cell Test
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Electrochemical Measuremen
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Physical Characterization
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Catalyst Preparation
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Material
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Plant material
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Isolation of inclusion bodies from E. coli cells
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Overnight grown primary culture of E. coli cells (1 % v/v final concentration) was inoculated into 1 litre of LB media containing antibiotics. Culture was incubated at 37 oc at 200 rpm. Growth was monitored by measuring absorbance of E. coli broth at 600 nm. Culture was induced by adding 1 mM IPTG at an OD of 0.6 and was harvested after 4 hrs of induction. Samples were taken on an hourly basis after induction to check the kinetics of protein expression. Un-induced and induced E. coli cells were analyzed by SDS-PAGE to check the expression of recombinant protein.
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Growth and expression of recombinant proteins in E. coli cells
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Primary culture of E. coli was grown in LB medium containing either ampicillin (Amp) and/or kanamycin (Kan) to final concentration of 100 j..tg/ml and 25 J..tg/ml respectively. Depending on the vector construct, antibiotics were used for expression of different proteins as described in Table 3.1. Medium was inoculated with 1 ml glycerol stock of E. coli and incubated overnight at 37 oc at 200 rpm.
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Preparation of primary culture of E. coli cells
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sg.inflibnet.ac.in sg.inflibnet.ac.in
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Fmoc protected amino acids and other chemicals used in peptide synthesis were obtained from Novabiochem (Switzerland). V8 protease and TF A were procured from Pierce Chemical Company (USA), while }-propanol, PMB," Hemin, Dithiothreitol, EDT A were obtained from Sigma Chemical Company (USA). DE52 and CM52 ion exchange resins were purchased from Whatman (UK). Sodium diothinite was procured from Fluka (Switzerland) and Catalase from Boehringer Mannheim (Germany). Carboxypeptidase was obtained from Worthington Biochemical Corporation (USA).
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Materials
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- May 2019
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shodhganga.inflibnet.ac.in shodhganga.inflibnet.ac.in
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250 mM KCl 55 mM MnCl2.4H20 50 x TAE (1 litre): 242 g of tris base 57.1 ml of glacial acetic acid 100 ml of 0.5 M EDTA Alkaline Lysis Solution 1: 50mM tris-HCl (pH 8.0) 10.0 mM EDTA 50 mM glucose Alkaline Lysis Solution 2: 0.2M NaOH 1% SDS Alkaline Lysis Solution 3: 3.0M Potassium acetate
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2.7 mM KCl 10 mM Na2HPO42 mM KH2PO4Tris Buffer Saline (TBS) (10X): 12.1gm Trizma Base 40.0gm NaCl Adjust PH to 7.6; make up the volume to 1 lit with milli Q water. HEPES Buffer Saline: 20 mM HEPES (pH 7.5) 150 mM NaCl Blocking Buffer: 5% fat free milk or 2% BSA in PBST or TBST. Stripping Buffer: 100 mM β-mercaptoethanol 2% (w/v) SDS 62.5 mM Tris-HCl (pH6.7) Luria Broth: 10g tryptone 10g NaCl 5g yeast extract, make up the volume to 1 lit with water. TB buffer for preparation of competent cells: 10 mM PIPES (free acid) 15 mM CaCl2.2H20
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2.5 ml of 1.5 M Tris-Cl (pH 8.8) 4.0 ml of 30% acrylamide; bisacrylamide (29:1) mix 50.0 μl of 20% SDS 3.35 ml of milli-Q water 100 μl of 10% APS 10.0 μl of TEMED. 2X SDS loading Buffer: 130 mM Tris-Cl (pH 8.0) 20% (v/v) glycerol 4.6% (w/v) SDS 0.02% bromophenol blue 2% DTT SDS PAGE Running Buffer: 25mM Tris base, 0.2M glycine 1% SDS Western Blot: 1 x Blotting Buffer (2Litres): 25mM tris base, 0.2M glycine 20% methanol Phosphate Buffer saline (PBS): 137 mM NaCl
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Whole cell lysis buffer: 20mM Tris (PH 7.5) 150mM NaCl 1mM EDTA 1mM EGTA 1 % triton X 100 2.5mM sodium pyrophosphate 1mM β-glycerophosphate 1mM Na3VO41μg/ml aprotinin, 1μg/ml leupeptin and 1μ.ml pepstatin SDS-PAGE: Stacking Gel Mix (4ml, 5%): 380μl of 1M Tris-Cl (pH 6.8) 500μl of 30% acrylamide ; bisacrylamide (29:1) Mix 15 μl of 20% SDS 2.1 ml of milli-Q water 30 μl of 10% APS 5 μl of TEMED. 12% Resolving Gel Mix (10ml):
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Buffers and Media
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Human lung epithelial type II cells (HPLD) were a kind gift from Dr. T Takahashi, Japan. All the cell lines were maintained in 5% CO2 with the recommended media containing 10% FBS (3% FBS for HPLD) and following the standard guidelines.
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A549 (human lung adenocarcinoma epithelial) and murine fibroblast cell line NIH3T3 was purchased from American Type Culture Collection. Murine lung epithelial cell line E-9 and E-10 were a kind gift from Dr. L.M.Anderson, (NCI-FCRDC, Frederick, Maryland)
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Cell Lines
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Monoclonal antibody against KRAS were purchased from Merck Research Laboratories, phospho p44/42 (ERK1/2)and total p44/42 (ERK1/2)antibodies were purchased from Cell Signaling Technologies. Anti tubulin antibody was obtained Sgima-Aldrich Chemicals. HRP conjugated anti-mouse and anti-rabbit secondary antibodies were purchased from Bangalore Genei Pvt. Ltd.
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Antibodies
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All the chemicals used for routine molecular biology work were procured from Sigma-Aldrich Chemicals (St Louis, MO, USA) unless otherwise mentioned. Taq polymerase for PCR and standard DNA markers and protein markers were purchased from MBI Fermentas. Tissue Culture materials like DMEM medium (for A549), Ham’s F-12 medium (for HPLD), Opti-MEM medium, 0.5% trypsin-EDTA, 100X antibiotic-antimycotic, freezing medium, fungizone, 200mM L-glutamine, fetal bovine serum (FBS), Lipofectamin-2000 and TRIzol were obtained from GIBCO BRL (Gaithersburg, Maryland, USA). CMRL medium was purchased from ICN laboratories. M-MLV reverse transcriptase, RNase inhibitor, dNTPs and MgCl2 were obtained from Invitrogen Corporation (Carlsbad, CA). ECL western detection kit and HybondTM- P were purchased from Amersham biosciences (GE Healthcare, UK).
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Chemicals and Reagents
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shodhganga.inflibnet.ac.in shodhganga.inflibnet.ac.in
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Most chemicals were obtained from commercial sources. The sources for some of the fine chemicals used in this study are given below. Most of the chemicals such as amino acids, antibiotics, sugars, IPTG, ONPG and X-gal were obtained from Sigma Chemical Co. The media components for the growth of bacteria were routinely from Himedia. The materials used in the recombinant DNA experiments such as restriction endonucleases, T4 DNA ligase, DNA polymerases for PCR amplification and DNA size markers were obtained from companies including New England Biolabs and Fermentas. Quiagen or HiPura Kits used for plasmid isolation, purification of DNA fragments. The oligonucleotide primers used in this study were mainly synthesized on order by Ocimum Biosolutions or MWG Biotech Pvt. Ltd
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Chemicals
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PonceauS stain Instant Blue (Biorad)
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Protein loading dye (6X) Tris-Cl (pH 6.8) 300 mM SDS 12% (w/v) Bromophenol blue 0.6% (w/v) Glycerol 60% (v/v) 600 mM β-mercaptoethanol
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Stains and Dyes
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Antibiotics were used at the following final concentrations (μg/ml): Rich media Minimal media Ampicillin (for plasmids) 100 50 Ampicillin (chromosome) 30 30 Chloramphenicol (for plasmids) 50 25 Chloramphenicol (chromosome) 25 25 Kanamycin 50 25 Nalidixic acid 50 - Rifampicin 100 - Streptomycin 50 100 Streptomycin 100 200 Spectinomycin 50 100 Tetracycline 15 8 Trimethoprim (for plasmids) 60 30 Chloramphenicol 0.1mg/ml The 10 mg/ml chloramphenicol stock in ethanol was used to make 0.1mg/ml solution in water
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Antibiotics
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NP-40 1% Tris 50 mM Sodiun deoxycholate 0.5% SDS 0.1% pH adjusted to 8.0 Running buffer for Western blotting Glycine 14.4g/l Tris base 3.05g/l SDS 1.0g/l Transfer buffer for western blotting Glycine 14.4g/l Tris base 3.03g/l The above salts were dissolved in 800ml of miliQ water and 200ml of methanol was then added. The buffer was chilled before use. PBST for Western blot 10X PBS (1000 ml) Sodium chloride 80 g Potassium chloride 2 g Disodium hydrogen phosphate 14.1 g (Na2HPO4) Potassium dihydrogen phosphate 2.49 g (KH2PO4) 1 l of 1X PBS + 1 ml of Tween-20
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TBF-I buffer (200ml) Potassium acetate 0.588 g Calcium chloride 0.249 g Manganese chloride 1.98 g Rubidium chloride 2.418 g 15% Glycerol 30 ml pH adjusted to 5.8 with 1M acetic acid TBF-II buffer (100 ml) MOPS 0.209 g Calcium chloride 1.102 g Rubidium chloride 0.120 g 15% Glycerol 15 ml pH adjusted to 6.5 with 1M potassium hydroxide Acrylamide solution (30%) Acrylamide 29 g Bis-acrylamide 1 g H2O 100 ml Non denaturing polyacrylamide gel (12%) 30% acrylamide 38.6 ml H2O 40.6 ml TBE 20 ml 10% APS 0.7 ml RIPA buffer (Radio Immuno Precipitation Assay buffer): RIPA buffer for bacterial cell lysis Sodium chloride 150 mM
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Water to 1000 ml MacConkey lactose agar: MacConkey Agar Base (Difco) 51.5 g Lactose 1% Water to 1000 ml Maloy agar: Tryptone 5 g Yeast extract 5 g NaCl 10 g NaH2PO4 10 g Chlorotetracycline (12.5 mg/ml) 4 ml Water 1000 ml Bacto-agar 15 g After autoclaving, the following solutions were added, ZnCl2 (20 mM) 5 ml Quinaldic acid (10 mg/ml) 10 ml Citrate buffer: (0.1 M; pH 5.5) Citric acid (0.1 M) 4.7 volumes Sodium citrate (0.1 M) 15.4 volumes TBE and TAE buffers: TBE: 90 mM Tris-borate, 2 mM EDTA (pH 8.0) and TAE: 40 mM Tris-acetate, 2 mM EDTA (pH 8.0) were used as standard electrophoresis buffers. TBE and TAE were prepared as 10X and 50X concentrated stock solutions, respectively, and used at 1X concentration
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cto-agar 15 g LBON agar: LBON medium1000 mlBacto-agar 15 gLB soft agar: LB medium 100 ml Bacto-agar 0.6 gK-Medium: KH2PO4 1.0 mM FeSO4 0.5 mg/l (NH4)2SO4 1.5 mM MgCl2 0.08 mM Casamino acids 5 g/l Thiamine 2 mg/l pH was adjusted to 7.0 with Tris free base. K-medium is low osmolarity (70 mOsm) medium (Kennedy, 1982). Z broth: LB medium 100 ml CaCl2 (0.5 M) 0.5 ml MacConkey agar: MacConkey Agar (Difco) 51.5 g Water to 1000 ml MacConkey galactose agar: MacConkey Agar Base (Difco) 51.5 g Galactose 1%
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Glucose/Glycerol-minimal A 19 amino acid medium: This medium is essentially the same as glucose/glycerol-minimal A medium described above except that all the 19 amino acids (except tryptophan) were added after autoclaving in a final concentration of 40 μg/ml from autoclaved 4mg/ml amino acid stock solutions. Minimal agar: Contained 1.5% Bacto-agar (Difco) in minimal A Medium. The plates were poured after mixing double strength minimal A medium with 4% agar (in water) that had been autoclaved separately. Wherever required, to test polaity relif at lacZ(am) or trpE(fs), meliobose (0.2%) was replaced for glucose and anthranilate at 100 μg/ml (4 mg/ml stock prepared in DMF) was replaced for tryptophan respectively. LB medium: Tryptone 10 g Yeast extract 5 g NaCl 10 g Water to 1000 ml pH adjusted to 7.0 - 7.2 with 1 N NaOH. LBON medium: Tryptone 10 g Yeast extract 5 g Water to 1000 ml pH adjusted to 7.0 - 7.2 with 1 N NaOH LB agar: LB medium 1000 ml
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All the media and buffers were sterilized by autoclaving for 15 minutes at 121°C. Media and buffers used in this study are described below. Glucose/Glycerol-minimal A medium: K2HPO4 10.5 g KH2PO4 4.5 g (NH4)2SO4 1 g Sodium citrate, 2H2O 0.5 g Water to 1000 ml After autoclaving the following solutions were added. MgSO4 (1 M) 1 ml Glucose (20%) 10 ml Or Glycerol (80%) 5ml Vitamin B1 (1%) 0.1 ml Amino acids and bases, when required, were added to a final concentration of 40 μg/ml. When growth on other carbon sources was to be tested, glucose was substituted by the appropriate sugar at 0.2%; when used as carbon source, the final concentration of Casamino acids was 0.5%
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Media and buffers
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Primers used in this study
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aStrain DH5α, MC4100 and MG1655 was from our laboratory stock collection. Strains described earlier include GJ3107, GJ3110, GJ3161, GJ3168, GJ3171 (Harinarayanan and Gowrishankar, 2003), and RS353 and RS445 (Chalissery et al., 2007). Strain GJ5147 is an Ilv+ derivative of GJ3073 (Chalissery et al., 2007). Strains GJ6504, GJ6509, GJ6511, GJ6516, GJ6520 and GJ6524 were constructed by S. Aisha (unpublished). Strains GJ5108, GJ5146, GJ5153 were constructed by K. Anupama (unpublished). b Genotype designations are as described in Berlyn (1998). cK7906 strain is described in Zheng and Friedman (1994). d MDS42 strain is as described in Posfai et al. (2006)
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Table 2.1 : List of E. coli K-12 strains
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Bacterial strains
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Plasmids constructed in this study
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genome cloned in a ColEI-based replicon, and obtained from Dr. Manjula Reddy. pHYD2556 is spectinomycin resistant and carries the minimal nusA+ open-reading frame with its native ribosome-binding site between genomic nucleotide co-ordinates 3314061and 3315548 cloned downstream of the ara regulatory region in a pSC101-based replicon, and obtained from Dr. Ranjan Sen. pHYD2557 is chloramphenicol resistant and carries a 2.3-kb PCR-amplified region between genomic nucleotide co-ordinates 3314061 and 3316393 (containing yhbC nusA region with its own promoter) cloned in a pSC101-based Ts replicon, and obtained from Dr. Ranjan.Plasmid DNA preparations were routinely prepared from recA strains such as DH5αand were stored in 10mM Tris-Cl (pH 8.0) plus 1mM EDTA at ─20 ̊C
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pWSK30 an Ampicillin resistant vector with pSC101 origin of replication and blue-white screening facility (Wang and Kushner, 1991). pHYD272 is a derivative of pMU575, an IncW-based single copy vector with Trimethoprim resistance marker carrying lacZYA reporter genes under proU promoter (Dattananda et al., 1991). pHYD751 a ColE1 replicon plasmid with ampicillin resistance marker and 2.1kb EcoRI-SalI fragment carrying nusG+cloned into EcoRI-SalI sites of pAM34 vector. The plasmid exhibits IPTG dependent replication (Harinarayanan and Gowrishankar, 2003). pHYD763 is a Ts (maintained at 30 ̊C but not at 37 ̊ or 39 ̊C), CmR, pSC101 derivative carrying 3.8 kb BamHI-SacI fragment of nusG+ cloned into BamHI-SacI sites of pMAK705 (Harinarayanan and Gowrishankar, 2003). pHYD1201 a ColE1 replicon plasmid with ampicillin resistance marker and 3.3kb HindIII-SalI fragment carrying rho+cloned into HindIII-SalI sites of pAM34 vector. The plasmid exhibits IPTG dependent replication (Harinarayanan and Gowrishankar, 2003). pHYD1622 is the derivative of pHYD1201 where the Ampicillin resistance marker has been replaced with Chloramphenicol using Wanner method of gene replacement. Cm gene was amplified from pKD3 plasmid (K. Anupama, unpublished). pHYD1623 is the derivative of pHYD751 where the Ampicillin resistance marker has been replaced with Chloramphenicol using Wanner method of gene replacement. Cm gene was amplified from pKD3 plasmid (K. Anupama, unpublished). pHYD2368 is a derivative of pBAD18 (AmpR) with 1.7 kb fragment encompassing RBS and coding region of uvsW from phage T4gt7 into SacI site of pBAD18 (K. Leela, unpublished). pHYD2554 is a derivative of pMBL18 with ampicillin resistance, carrying the 10-kb EcoRI-HindIII fragment between kilobase co-ordinates 3310.06 and 3320.08 of the E. coli
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to CCT mutation leading to a Glutamic acid to Glycine change at the 53rd amino acid and a Threonine to Proline change at the 55th amino acid in the H-NS protein (Willams et al., 1996). pLG-H-NS-I119T is a derivative of pLG-H-NS plasmid with ATC to ACC mutation leading to a Isoleucine to Threonine change at the 119th amino acid in the H-NS protein (Willams et al., 1996). pLG-H-NS-P116S is a derivative of pLG-H-NS plasmid with CCA to TCA mutation leading to a Proline to Serine change at the 116th amino acid in the H-NS protein (Willams et al., 1996). pLG-H-NS-Y97C is a derivative of pLG-H-NS plasmid with TAT to TGT mutation leading to a Tyrosine to Cysteine change at the 97th amino acid in the H-NS protein (Willams et al., 1996). pPMrhoCam is a Ts (maintained at 30 ̊C but not at 37 ̊ or 39 ̊C), CmR, pSC101 derivative carrying PuvII-HindIII fragment containing trxArho+ cloned into PuvII-HindIII sites of pPM103 (Martinez et al., 1996). pTrc99A an expression vector with ColE1 origin of replication and ampicillin resistance marker. Provides IPTG dependent induction of the insert (Amann et al., 1988). pUC19 is a high-copy-number ColE1 based E.coli cloning vector (500-700 copies/cell) with an Ampr selectable marker. It is one of a series of related plasmids constructed by Messing and co-workers and contains portions of pBR322 and M13mp19 (Yanisch-Perron et al., 1985). It carries a multiple-cloning site (MCS) region in the lacZα fragment, and therefore allows for blue-white screening of recombinant clones
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pAM34 is a pBR322-derived cloning vector with Ampr and Specr selectable markers. The replication of this plasmid is dependent on the presence of IPTG, the gratuitous inducer of the lac operon (Gil and Bouche, 1991). pBAD18 is an expression vector with a pBR322 derived origin of replication and allows for tightly regulated expression of the genes cloned under the PBAD promoter of the araBADoperon (Guzman et al., 1995). The vector also carries the araC gene, encoding the positive and negative regulator of this promoter. pBluescript II KS (pBKS) is also a high-copy-number ColE1 based cloning vector with Ampr selectable marker and blue-white screening facility (obtained from Stratagene). pCL1920 is a low-copy-number vector with pSC101 replicon (~ 5 copies/cell), that carries streptomycin (Str)/spectinomycin (Spec)-resistance marker (encoded by aadA) and also carries a MCS region within the lacZα that allows blue-white screening to detect recombinants (Lerner and Inouye, 1990). pCP20 pSC101-based Ts replicon, CmR AmpR, for in vivo expression of Flp recombinase (Datsenko and Wanner, 2000). pLG339 is a low-copy-number cloning vector with pSC101 replicon that has a Kanrselectable marker (Stoker et al., 1982). pLG-H-NS is a pLG339 derivative where the hns ORF had been cloned into the EcoRI-SalIsites of pLG339 vector (KanR, pSC101) (Willams et al., 1996). pLG-H-NSΔ64 is a derivative of pLG-H-NS plasmid with AT base pair deletion after codon 63 in the hns gene resulting in a frameshift (Willams et al., 1996). pLG-H-NS-L26P is a derivative of pLG-H-NS plasmid with CTG to CCG mutation leading to a Leucine to Proline change at the 26th amino acid in the H-NS protein (Willams et al., 1996). pLG-H-NS-E53G/T55P is a derivative of pLG-H-NS plasmid with GAG to GGG and ACT
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pACYC184 is a medium-copy-number cloning vector (~ 20 copies/cell) with Cmr and Tetrselectable markers. It carries the origin of replication from plasmid p15A (Chang and Cohen, 1978), which is related to and yet is compatible with that of ColE1. This property enables pACYC184 to co-exist in cells with ColE1 plasmid vectors, including all the ones mentioned above
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Plasmids that have been described elsewhere
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Plasmids
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The bacteriophage P1kc was from our laboratory collection and is referred to as P1 throughout this thesis. Phage λcI857 was also from our laboratory collection. Other bacteriophages that were used in this study included the following: (i) λNK1098 carries a Tn10 transposon with a tertracycline (Tet) ressistance marker. (ii) λNK1324 carries a mini-Tn10 transposon Tn10dCm with a chloramphenicol (Cm)-resistance marker, Cmr. The lambda phage vectors above (Kleckner et al., 1991) were used to make random transposon insertions in the chromosome either for the purpose of insertional mutagenesis or for tagging antibiotic resistance markers to point mutations
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Bacteriophages
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Bacterial strains
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All the bacterial strains that were used in this study are derivatives of Escherichia coliand their genotypes have been listed in Table 2.1 Bacterial strains were routinely stored on solid agar plates at 4°C and also as thick suspensions in 40% glycerol either at −20°C or at −70°C. Plasmid harboring strains, were reconstructed whenever necessary by fresh transformations
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shodhganga.inflibnet.ac.in shodhganga.inflibnet.ac.in
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purification of DNA fragments werefrom Qiagen or HiMedia. The oligonucleotide primers used in this study were mainly synthesised by Ocimum Biosolutions or MWG Biotech. The radioactive chemicals were procured from BRIT Mumbai
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Chemicals were obtained from commercial sources. Most of the chemicals such as amino acids, antibiotics, sugars, IPTG, ONPG and X-gal were obtained from Sigma Chemical Co. The media components for the growth of bacteria were mostly from HiMedia laboratories. The materials used in the recombinant DNA experiments such as restriction endonucleases, T4-DNA ligase, DNA-polymerases and DNA size markers were obtained from companies including New England Biolabs, MBI Fermentas and Stratagene.RNA isolation chemicals like Reverse transcriptase, trizol, RNA loading buffers and dyes and RNA size markers were obtained from Invitrogen and Sigma. Protein markers were obtained from MBI Fermentas. Kits for plasmid isolation,
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Chemicals
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Antibiotics were used at the following final concentrations in various media as given inTable 2.4.Table 2.4Concentrations of antibiotics (μg/ml)
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Antibiotics
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Waterto 3 mlTEMED 10 μlDenaturing (urea) sequencing gel (6%) composition10 X TBE 50 ml40% acrylamide 75 mlUrea 210 gm (7 M)Waterto 500 mlThis was filtered through a 0.45/0.22 μ milipore filter.For casting the gel 35 ml of the sequencing gel mixure was mixed with 150 μl10% APS and 25 μlTEMED
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Formaldehyde agarose gel(For 50 ml)DEPC treated water 43 mlMOPS buffer 5.3 mlAgarose0.63 gmFormaldehyde2.6 mlThe above mix was boiled without formaldehyde to dissolve agarose and then at around 50ºC formaldehyde was added just before casting the gel.40% Acrylamide solutionAcrylamide39 gmBis-acrylamide 1 gmWater to 100 mlNon denaturing gel composition (50 ml)40% acrylamide solution 5 ml10 X TBE 5 mlH2O 40 ml10% APS 250 μlTEMED 30 μlSDS PAGE gel (12%)For resolving Gel (15 ml):30% Acrylamide solution 6 ml1.5 M Tris-Cl (pH 8.8)3.8 ml10% SDS150 μl10% APS 150μlWaterto 15 mlTEMED 10 μlFor stacking gel (3 ml):30% Acrylamide solution 500 μl1 M Tris Cl (pH 6.8) 380 μl10% SDS 30 μl10% APS 30 μl
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Storage buffer for proteinTris-Cl (pH 8.0) 20 mMNaCl 300 mMDTT10 mMGlycerol 40 % Hybridization bufferTris-Cl (pH 8.0) 9 mMEDTA 0.35 mMSample buffer (for SDS-PAGE)Tris-Cl (pH 6.8) 150 mMSDS (20%) 6% v/vGlycerol 30% v/vβ-mercaptoethanol (5%) 15%Bromophenol blue 0.6% (w/v)EMSA binding bufferTris-Cl (pH 7.5) 10 mMNaCl 50 mMEDTA1 mMGlycerol 5 %DTT 5 mMDenaturing gel loading buffer with dyeFormamide 95%EDTA 20 mMXylene Cyanol 0.05 gmBromophenol blue0.05 gmNon denaturing gel loading buffer with dyeTris-Cl (pH 7.5) 250 mMBromophenol blue 0.02%Glycerol 20%
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MOPS bufferMOPS 4.16 gm0.5 M EDTA 1.0 mlSodium acetate 0.68 gmWater (nuclease free) to 500 mlIt was filter sterilized and stored in an amber colored bottle. This was prepared as 10 Xstock solution and used at 1 X concentration.INOUE (PIPES) bufferPIPES (free acid) 10 mMCaCl2.2H2O15 mMKCl 250 mMMnCl2.4H2O 55 mMpH was adjusted to 6.7 with 1 N KOH.PIPES gets into solution when the pH is greater than 6.7. MnCl2was dissolvedseparately and added drop by drop with stirring. The pH was adjusted to 6.7 and filtersterilized and stored at –20ºC.Z buffer (for β-Galactosidase assay)Na2HPO416.1 gmNaH2PO45.5 gmKCl0.75 gmMgSO4.7H2O 0.246 gmβ-mercaptoethanol 2.7 mlWaterto 1000 mlpH was adjusted to 7.0 and stored at 4ºC.SDS running bufferTris-base 30.3 gmGlycine144 gmSDS 10 gmWaterto 1000 mlIt was prepared in 10 X concentration and diluted to 1 X for running
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Citrate bufferCitric acid (0.1 M)4.7 volumeSodium citrate (0.1 M) 15.4 volumeTE bufferTris-Cl (pH 8.0) 10 mMEDTA 1 mMTBE bufferTris-Borate 90 mMTris-Borate 90 mMEDTA (pH 8.0) 2 mMThis was prepared as 10 X stock solution and used at 1 X concentration.TAE bufferTris-acetate 40 mMEDTA (pH 8.0) 2 mMThis was prepared at 50 X concentrated stock solution. Both TBE and TAE were usedas standard electrophoresis buffers
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Buffers and solutions
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LB agarLB medium 1000 mlBacto-agar 15 gmZ broth (for P1 transduction)LB medium100 mlCaCl2(0.5 M) 0.5 mlZ agar (for P1 transduction)Z broth 100 mlBacto-agar0.75 gm
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Amino acids when required, were added to a final concentration of 40 μg/ml. Whengrowth on other carbon sources was to be tested, glucose was substituted withappropriate sugar at 0.2%.Glucose-minimal A medium, pH 7.4This medium was same as Glucose-minimal A medium described above except for the difference in K2HPO4and KH2PO4which were as mentioned below:K2HPO414.0 gmKH2PO42.7 gmGlucose-minimal A medium, pH 5.8This medium was same as Glucose-minimal A medium described above except for the difference in K2HPO4andKH2PO4which wereas mentioned below:K2HPO41.5 gmKH2PO412.4 gmGlucose /Glycerol-minimal A 19 (18 or 17) amino acidmediumThis medium is essentially the same as glucose/glycerol-minimal A medium described above except that all 19 or 18 or 17 otherthan either Lys or Lys and Arg or Lys and Arg and His amino acids were added after autoclaving at a final concentration of 40μg/ml from autoclaved 4 mg/ml stock solutions.Minimal A agarIt contains 1.5% bacto-agar (Difco) in minimal A medium. The plates were pouredafter mixing double strength minimal A with 3% agarthat had been autoclaved separately.LB mediumTryptone 10.0 gmYeast Extract5.0 gmNaCl 10.0 gmWaterto1000 mlpH adjusted to 7.0 to 7.2 with 1 N NaOH
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All media and buffers were sterilised by autoclaving at 121ºC for 15 mins. Mediaand buffers used in this study are given below:Glucose /Glycerol-minimal A mediumK2HPO410.5 gmKH2PO44.5 gm(NH4)2SO41.0 gmSodium citrate, 2H200.5 gmWater to 1000mlAfter autoclaving the following solutions were addedMgSO4(1M) 1 mlGlucose (20%) 10 mlOr Glycerol (80%)5 mlVitamin B1 (1%) 0.1 ml
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Media
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The primers used in this study are listed in Table 2.3.Table 2.3 Oligonucleotide primersa
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Primers
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bindingsite lie upstream of the MCS to ensure the high level expression of any genecloned in MCS. A stretch of hexa-histidine (His6)-encoding codons followed by stopcodon is incorporated downstream of MCS to give a C-terminally His6-taggedrecombinant protein (EMD Biosciences).6. pBAD18:It is an expression vector with a pMB9derived origin of replication and allows for tightly regulated expression of genes cloned under the PBADpromoter of the araBADoperon (Guzman et al.,1995). The vector also carries thearaCgene, encoding the positive and negative regulator of this promoter.7. pCP20: pSC101-based Ts replicon, chloramphenicol resistant, ampicillin resistant, for in vivoexpression of Flp recombinase (Datsenko and Wanner, 2000)Plasmid DNA preparations were routinely made from recAstrainDH5αandwerestored in 10 mM Tris-Cl (pH-8.0) with 1 mM EDTA at –20ºC. The plasmid constructsused in this study are given in Table 2.2.Table 2.2Plasmid constructs
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The plasmid vectors used in this study were as follows:1.pCU22: It is a derivative of pUC19 used to prepare supercoiled DNA for in vitrotranscription where two strong phage fdtranscription terminators flank MCS. This ensures that the transcripts originated from vector based promoters will not interferewith the transcription from the cloned promoter and that the transcript originated fromthe cloned promoter will be terminated after the MCS (Ueguchi and Mizuno,1993).2.pMU575: It is an IncW-based, single-copy, trimethoprim resistance bearingpromoter probe vector. It carries its MCS upstream of a promoterless galK’-lacZfusion. This fusion has the first 58 codons of galKfused to the 8th codon oflacZ, andthe resultant hybrid polypeptide possesses functional β-Galactosidase activity(afterassembly as a tetramer). Translation of the hybrid gene is controlled by the ribosomebinding site of galK. There are stop codons in all the three reading frames between MCS and initiation codon of galKso that there is no interference caused bytranslational read-through from inserts cloned into MCS region. A strong pheRterminator located upstream of the MCS prevents read through from any vector-basedpromoter into the lacZgene (Andrews et al.,1991).3. pTrc99A:It is an expression vector with ColE1 origin of replication and ampicillin resistance marker. It provides IPTG dependent induction of the cloned gene (Amann et al., 1988)4. pCL1920: It is a pSC101-based, low-copy-number vector with spectinomycin and streptomycin resistance marker carrying the MCS in lacZαregion and henceprovides the advantage of screening the insertions using α-complementation (Lernerand Inouye,1990).5. pET21b: It is a ColE1-based, high-copy-number expression vector bearing ampicillinresistance marker. A strong T7 RNAP-recognised promoter and an efficient ribosome
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Plasmids
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TheE. coli strains used in this study with their genotypes are shown in Table 2.1. All strains other than BL21 (DE3) employed in protein overexpression experiments are derivatives of E. coli K12. Bacterial strains were routinely stored on solid agar plates at 4ºC and also as thick suspensions in 40% glycerol at –70ºC. Plasmid harboring strains were freshly prepared by transformation of the required plasmid. The bacteriophage P1kc from the laboratory collectionwas used for routine transduction tomove a locus from one strain to anotherand is referred to as P1 throughout this thesis.Table 2.1 E. coli strains used in this study
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Strains and bacteriophages
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sg.inflibnet.ac.in sg.inflibnet.ac.in
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Oligonucleotides/primers used in this study were designed using either free online-tool Primer3 (http://frodo.wi.mit.edu/) or Gene Runner software (http://www.generunner.net/). Oligonucleotides used in this study were commercially
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synthesized from MWG Biotech Pvt. Ltd., Bangalore. All primers used in this study are listed in Table 2.3.Table 2.3: List of primers used in this study
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Oligonucleotides
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10mM EDTA0.1% SDS 1 M ureaToluidine blue staining solution:0.05% Toluidine blue20% Methanol2% GlycerolSolution was prepared in H2O.Destaining solution for polyphosphate gels:20% Methanol2% GlycerolSolution was prepared in H2O.Spheroplast buffer:50 mM Potassium phosphate (pH 7.5)0.6M Sorbitol0.2 X YPD mediumPS(PIPES-Sorbitol)buffer:10 mM PIPES-KOH (pH 6.8)200mM Sorbitol1 X protease inhibitor cocktail (Roche Cat # 04693159001)**To be added fresh before use
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Citric-Phosphate buffer:0.5 M citric acid0.5 M dibasic sodium phosphatepH was adjusted to 5.0 with phosphoric acid and filter-sterilized.MES/TEA buffer:1 mM MES(2-(N-morpholino)ethanesulfonic acid)pH was adjusted to pH 5.0 with TEA(triethanolamine).Plasma membrane suspension buffer:50 mM Tris-HCl(pH 7.5)0.1mM EDTA0.1 mM Dithiothreitol 20% GlycerolPolyphosphate extraction buffer:50 mM HEPES (pH 7.2)
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Genomic DNAisolation buffersBuffer A:50 mM Tris-HCl10mM EDTA150 mM NaCl 1% Triton-X 1% SDSBuffer B:50 mM Tris-HCl (pH 7.5)10 mM EDTA1.1 M Sorbitol50 mM β-mercaptoethanol(To be added just before use
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Other buffers
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