APPENDICES Figure A.1 Map of pGEM®-T Easy Vector 181 Figure A.2 Production of recombinant proteins. (a) The expression vector contains the lac promoter which drives the expression of lacZ gene encoding for βgalactosidase. Lactose or its analog IPTG will stimulate the expression of βgalactosidase. (b) If lacZ is replaced by the gene encoding the protein of interest, lactose or IPTG will stimulate the expression of desired proteins. Many proteins of interest are expressed in low concentration in endogenous system. Through recombinant DNA technology as above, large quantities of recombinant proteins can be produced using an in vitro expression system. (Modified from http://www.web-books.com/MoBio/Free/Ch9H.htm) Note Blue white selection: Many vectors (such as pUC series) carry coding information for the first 146 amino acids of the β-galactosidase gene. Embedded in this coding region is the multiple cloning site (does not disrupt the reading frame of the gene) into which the insert DNA is cloned. After IPTG induction, this 146 amino acid fragment of βgalactosidase protein is expressed but incapable of acting on the chromogenic substrate (X-gal). It is only when transformed in an appropriate host cells which expressed the other half of the carboxyl terminal fragment of the β-galactosidase protein, these two protein fragments can then associate to form an enzymatically active protein to cleave the chromogenic substrates. This process is called as α-complementation. Properly expressed β-galactosidase protein (after α-complementation) will turn cells into blue colonies when plated on plates containing X-gal. If the insert DNA is cloned in the multiple cloning sites and disrupted the reading frame, amino terminal fragment of β-galactosidase protein is not produced. Hence, colonies will remain white. However, if the inserts not disrupt the reading frame of LacZ gene, there is still a possibility to have blue colonies harboring the desired inserts. 182 Table A.1 Reagents and chemicals used for molecular biology experiments. Name Components Amount in l TAE buffer (10X) Tris-base EDTA 0.40 M 0.01 M Æ Prepare from the stock solution of the chemicals, adjust to pH 7.8 using glacial acetic acid and autoclave Æ Dilute to 1X before use SOB medium (for preparation of competent cells) Tryptone Yeast extract NaCl KCl MgCl2.6H2O MgSO4.7H2O 20.00 g 5.00 g 0.58 g 0.19 g 2.03 g 2.46 g Æ Prepare without Mg2+, adjust to pH 7.0 and autoclave Æ A M stock of Mg2+ (1 M MgCl2 and MgSO4, filter stelize) is used to make the medium 20 mM in Mg2+ TB buffer (for preparation of competent cells) Pipes MnCl2 CaCl2 KCl 10 mM 55 mM 15 mM 25 mM Æ Prepare from the stock solution of the chemicals, adjust to pH 6.7 and filter sterilize IPTG stock (20 mg/ml) IPTG 2.00 g Æ Dissolve in dH2O, filter sterilize and store at -20 oC Æ IPTG induces synthesis of a β-galactosidase. It is used to detect lacZ gene expression in cloning experiment Æ Use for blue-white colonies selection X-gal stock (50 mg/ml) X-gal 5.00 g Note Æ Dissolve in dimethyl formamide, filter sterilize, wrap containers with aluminium foil and store at -20 oC Æ Use as chromogenic substrates in blue-white colonies selection 183 Table A.2 Antibiotics, reagents and chemicals used for growing bacterial culture and for recombinant protein expression. Name Components Amount in l Note LB Medium Tryptone Yeast extract NaCl 10.00 g 5.00 g 10.00 g Æ Adjust to pH 7.5, sterilize by autoclaving Æ To make the agar medium, add 15 g agar to l liquid medium before autoclaving Ampicillin (Amp) (100 mg/ml) Ampicillin 100.00 g Æ Dissolve in dH2O, filter sterilize and store at -20 oC Æ Use for inhibition of cell-wall synthesis by interfering with peptidoglycan cross-linking IPTG stock (1 M) IPTG 238.30 g Æ Dissolve in dH2O, filter sterilize and store at -20 oC Æ Use to induce expression of plasmid-based genes for the production of recombinant proteins under control of the lac promoter Lysozyme (100 mg/ml) Lysozyme 100.00 g Æ Dissolve in dH2O, filter sterilize and store at -20 oC Æ Use for hydrolyzing peptidoglycan. Osmotic lysis will occur as a result of destruction of the bacterial cell wall. 184 Table A.3 Solutions for preparing 15 % resolving and % stacking gels for SDSglycine Polyacrylamide Gel Electrophoresis (SDS-PAGE). Solutions components Component volumes/ gels (0.75 mm thickness) Note Resolving H2O 30 % Acrylamide mix (37.5:1) 1.5 M Tris-base (pH 8.8) 10 % SDS 10 % Ammonium persulfate TEMED 4.70 ml 10.00 ml 5.00 ml 200.00 µl 100.00 µl 10.00 µl Æ Mix the components in the order shown, swirl the mixture rapidly and pour the resolving mixture into the gap between glass plate Stacking H2O 30 % Acrylamide mix (37.5:1) 0.5 M Tris-base (pH 6.8) 10 % SDS 10 % Ammonium persulfate TEMED 6.00 ml 1.32 ml 2.52 ml 100.00 µl 50.00 µl 10.00 µl Æ Mix the components in the order shown, swirl the mixture rapidly and pour the stacking mixture onto the surface of the polymerized resolving gel Æ Insert a clean Teflon comb into the stacking solution 185 Table A.4 Reagents and chemicals used for SDS-PAGE. Name Components SDS-glycine buffer (5X) Tris-base Glycine SDS Staining solution Coomassie Brilliant Blue R-250 Ethanol Acetic acid 300.00 ml 100.00 ml Methanol Acetic acid 400.00 ml 100.00 ml Æ Mix 500 ml dH2O with methanol and acetic acid Gel drying solution Methanol Acetic acid Glycerol 400.00 ml 100.00 ml 100.00 ml Æ Mix 400 ml dH2O with methanol, acetic acid and glycerol Destaining solution Amount in l 15.10 g 72.00 g 5.00 g 1.00 g Note Æ Dissolve Tris and Glycine, adjust pH to 8.3 Æ Add SDS and adjust the volume to 1000 ml Æ Dilute to 1X before use Æ Dissolve Coomassie Brilliant Blue R-250 in 600 ml dH2O, ethanol and acetic acid Table A.5 Preparation of SDS gel-loading buffer. Name Components Amount Note SDS gelloading buffer (4X) Bromophenol blue Glycerol Upper Tris (pH 6.8) 20 % SDS 0.0185 g ml 10 ml ml Æ 4X SDS gel-loading buffer without the β-mercaptoethanol can be store at room temperature Æ Add 28 µl of β-mercaptoethanol to 190 µl of loading buffer before use, store the remaining loading buffer with β-mercaptoethanol at 20 oC after use 186 Table A.6 Preparation of DNA ladders for molecular biology experiments. Components Note DNA ladders stock (1 kb plus, 100 bp, 50 bp) (Working stock: µg/ µl) Æ Mix 10 µl of stock DNA ladders with 90 µl dH2O and 20 µl loading dye, store at -20 oC Æ Load µl each time 187 . fragments can then associate to form an enzymatically active protein to cleave the chromogenic substrates. This process is called as α-complementation. Properly expressed β-galactosidase protein (after. substrate (X-gal). It is only when transformed in an appropriate host cells which expressed the other half of the carboxyl terminal fragment of the β-galactosidase protein, these two protein fragments. 184 Table A. 2 Antibiotics, reagents and chemicals used for growing bacterial culture and for recombinant protein expression. Name Components Amount in 1 l Note LB Medium Tryptone Yeast