a The expression vector contains the lac promoter which drives the expression of lacZ gene encoding for β-galactosidase.. Lactose or its analog IPTG will stimulate the expression of β-g
Trang 1APPENDICES
Figure A.1 Map of pGEM®-T Easy Vector
Trang 2Figure A.2 Production of recombinant proteins (a) The expression vector contains the
lac promoter which drives the expression of lacZ gene encoding for
β-galactosidase Lactose or its analog IPTG will stimulate the expression of
β-galactosidase (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 β-galactosidase 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
Trang 3Table A.1 Reagents and chemicals used for molecular biology experiments
Name Components Amount in 1 l Note
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 2 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 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
Trang 4Table A.2 Antibiotics, reagents and chemicals used for growing bacterial culture and
for recombinant protein expression
Name Components Amount in 1 l Note
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 1 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
Trang 5Table A.3 Solutions for preparing 15 % resolving and 4 % stacking gels for SDS-
glycine Polyacrylamide Gel Electrophoresis (SDS-PAGE)
Solutions components Component volumes/
4 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
Trang 6Table A.4 Reagents and chemicals used for SDS-PAGE
SDS-glycine
buffer (5X)
Tris-base Glycine SDS
15.10 g 72.00 g 5.00 g
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
Staining solution Coomassie
Brilliant Blue R-250
Ethanol Acetic acid
1.00 g
300.00 ml 100.00 ml
Æ Dissolve Coomassie Brilliant Blue R-250 in 600 ml dH2O, ethanol and acetic acid
Destaining
solution
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
Table A.5 Preparation of SDS gel-loading buffer
SDS
gel-loading
buffer (4X)
Bromophenol blue Glycerol
Upper Tris (pH 6.8)
20 % SDS
0.0185 g
8 ml
10 ml
4 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
Trang 7Table A.6 Preparation of DNA ladders for molecular biology experiments
Components Note
DNA ladders stock
(1 kb plus, 100 bp, 50 bp)
(Working stock: 1 µg/ µl)
Æ Mix 10 µl of stock DNA ladders with 90 µl
dH2O and 20 µl loading dye, store at -20 o
C
Æ Load 6 µl each time