Bacterial Gene Expression

GeNeiTM

Bacterial Gene Expression

GeNeiTM

GeNeiTM Bacterial Gene Expression Teaching Kit Manual

Cat No. KT69 KT69A

Bangalore Genei, 2007 Bangalore Genei, 2007

New Cat No. 106334 106335

Revision No.: 00061204

Bacterial Gene Expression

GeNeiTM

Bacterial Gene Expression

GeNeiTM

CONTENTS
Page No.

Objective Principle Kit Description Materials Provided Procedure Observation & Interpretation Appendix

3 3 5 6 7 9 10

SDS-PAGE

Principle Procedure

13 16
19

ORDERING INFORMATION

Bangalore Genei, 2007

Bangalore Genei, 2007

Bacterial Gene Expression

GeNeiTM

Bacterial Gene Expression

GeNeiTM

Objective:
To learn the concept of bacterial gene expression.

Principle:
Molecular cloning or gene cloning involves insertion of a DNA segment of interest into an autonomously replicating DNA molecule, i.e., a cloning vector. Transforming the vector into a suitable host organism results in production of large amounts of the inserted DNA fragment. For expression of genes, the insert DNA should be flanked by correctly oriented control sequences for RNA and protein synthesis. Hence, one uses an expression vector, such that the host produces large quantities of RNA and subsequently the protein which can be isolated and purified. Five major expression systems have been developed: Bacterial expression system Yeast expression system Bacillus expression system Baculovirus expression systems and Mammalian expression systems.

Bacterial expression vectors contain necessary elements for bacterial transcription and translation, including a strong bacterial promoter containing appropriate recognition sequences for RNA polymerase. A suitable ribosome binding site (Shine-Dalgarno sequence) is required for efficient translation initiation. These sequences are placed in the plasmid at an appropriate distance upstream from inserted DNA fragment. It is important that the gene is inserted into the plasmid in a proper reading frame to ensure expression of the right protein.

Bangalore Genei, 2007

Bangalore Genei, 2007

Bacterial Gene Expression

GeNeiTM

Bacterial Gene Expression

GeNeiTM

An efficient expression vector system can convert a bacterial cell into a protein manufacturing machine with yields as high as 10-20% of total cellular protein. Since, foreign proteins can be toxic to host cells, genes must be under very strict control. These vectors generally contain inducible promoters that can be controlled by presence/absence of an inducing agent. For example, Lac operon is induced by addition of IPTG. In the absence of lactose in the growth medium, the E.coli lac promoter is repressed, i.e., turned off, by the lac repressor protein that prevents the lac operon from being transcribed. Induction or turning on of the lac promoter is achieved by the addition of either lactose or IPTG (Isopropyl--D-thiogalactopyranoside), a gratuitous inducer, to the medium. Either of these substances prevent the lac repressor from binding to the lac operator, thereby enabling transcription to occur. Factors that influence the choice of a system for expression of a particular protein in E.coli are: Size of protein Amount of protein required and Whether active protein is required.

Kit Description:
The kit supplies an E.coli strain having a recombinant expression vector. Glutathione-S-transferase (GST) gene is cloned into the expression vector having tac (lac + trp) promoter and lac I operator elements. Students will grow recombinant cells to mid-log phase and induce it with IPTG to allow expression of GST protein. Cells will then be lysed and the GST protein (29 kDa) analysed by SDS-PAGE. The expression will be verified by comparing samples before and after induction for the amount of GST protein produced. KT69 : The kit is designed to carry out 5 experiments, each involving induction of the strain with IPTG for expression of GST protein and analysis of protein by SDS-PAGE. The kit also includes PAGE equipment with accessories (ETS-4) required for polyacrylamide gel electrophoresis. The kit is designed to carry out 5 experiments, each involving induction of the strain with IPTG for expression of GST protein and analysis of protein by SDS-PAGE. PAGE equipment with accessories is required for KT69A.

KT69A :

Note

Bangalore Genei, 2007

Fig 1: Schematic representation of an expression vector 4

Duration of experiment: Experiment is carried out over a span of 3 days, approximate time taken on each day is indicated below: Day 1: 2 hours (Preparation of media and Revival of strain) Day 2: 10-15 minutes (Inoculation of media) Day 3: 6-7 hours (Induction and analysis of protein by SDS-PAGE) 5 Bangalore Genei, 2007

Bacterial Gene Expression

GeNeiTM

Bacterial Gene Expression

GeNeiTM

Materials Provided:
The list below provides information about the materials supplied in the kit. The products should be stored as suggested. Use the kit within 6 months of arrival.
Materials E.coli strain (Lyophilized) Ampicillin 0.1 M IPTG Control GST protein Cell lysis buffer SDS-separating gel mix SDS-stacking gel mix Sample loading buffer Protein marker Ammonium persulphate 10X Reservoir buffer Ezee blue LB Broth Agar 1.5 ml vials Quantity KT69/69A (5 expts.) 1 vial 100 mg 5 x 1 ml 0.125 ml 1.5 ml 25 ml 10 ml 0.5 ml 0.125 ml 50 mg 100 ml 100 ml 15 g 1g 1 x 10 & 1 x 25 Nos. Store 4C 4C 4C 4C 4C 4C 4C 4C 4C RT RT RT RT RT RT

Note:
Read the entire procedure before starting the experiment. All microbiological procedures should be done strictly under aseptic conditions. Revive the strain as soon as the lyophilized vial is opened. Induce the bacterial culture at mid-log phase with exact quantity of IPTG. Ensure complete lysis of the cell pellet. For preparation of SDS-PAGE, staining, refer SDS-PAGE electrophoresis. Ensure that all components are ready prior to starting the experiment. For preparation of media, antibiotic, refer appendix.

Procedure:
Day 1: Revival of Host 1. Break open the lyophilized vial, add 0.1 ml of LB medium. 2. Streak a loopful (each) of this suspension onto two LB plates containing ampicillin at a concentration of 100 g/ml. 3. Incubate the plates at 37C, overnight. Note: Store the revived plates at 4 C, use within 2 weeks to carry out 5 experiments. Day 2: 4. Pick a single colony from the LB plate and inoculate into 5 ml LB broth containing ampicillin (100 g/ml). 5. Incubate at 37C in a shaker, overnight.
Bangalore Genei, 2007

Materials Required:
Equipment : Incubator, Microcentrifuge, 37C shaker. Glassware : Conical flask, Petri plates, Staining tray, Measuring cylinder, Test tubes. Reagent : Distilled water. Other Requirements : Micropipette, Tips, Water bath.
Bangalore Genei, 2007

Bacterial Gene Expression

GeNeiTM

Bacterial Gene Expression

GeNeiTM

Day 3: Induction and analysis of protein by SDS-PAGE 6. Inoculate 1ml of overnight culture into 100 ml of LB broth (in a 500 ml conical flask) containing ampicillin at a concentration of 100 g/ml . 7. Incubate the culture flask at 37C shaker, until cells reach mid-log phase of growth i.e., A 600 is 0.5. (Takes approximately 2-3 hours). 8. Transfer 5 ml of the uninduced culture from step 7 into a sterile test tube and label it as before induction (BI) sample. 9. Induce the remaining culture by adding 1.0 ml of IPTG, continue incubating the culture on a shaker at 37C for 2 hours. 10. Transfer 5 ml of induced culture into a sterile test tube and label it as after induction sample (AI). 11. Spin down 3.0 ml (1.5 ml at a time) of BI and AI samples in sterile 1.5 ml vials at 8000 rpm for 10 minutes. Discard the supernatant each time. 12. Resuspend the cell pellets in 100 l of cell lysis buffer. 13. Add 25 l of the sample loading buffer to BI and AI cell suspensions and mix gently. 14. Add 10 l of sample loading buffer to 25 l of control protein and 25 l of protein marker each. 15. Boil the BI, AI, protein marker and control protein samples in a water bath for 20 minutes. 16. Spin the BI and AI samples at 6000 to 8000 rpm for 10 minutes. 17. Load 35 l each of protein marker, control protein sample and supernatants of BI and AI sample onto SDS-PAGE. Note down the order in which the samples are loaded. 18. Run the SDS-PAGE at 50 volts until bromophenol blue reaches the bottom of the resolving gel. 19. Stain the gel with Ezee blue.
Bangalore Genei, 2007

Observation:
Compare the GST protein band (29 kDa) in the before induction sample with that of after induction sample.

Interpretation:
Due to induced expression of GST-gene, one observes GST protein (29 kDa) as a very intense band in the after induction sample which is otherwise barely visible in the before induction sample, indicating increased level of GST protein production. (Refer fig 2).

1 kDa 66 43 29

4
Lane 1: Protein Marker Lane 2: Control GST Lane 3: BI Sample Lane 4: AI Sample

GST Protein

14
Fig 2 : GST Protein analyzed on SDS-PAGE after IPTG induction.

Bangalore Genei, 2007

Bacterial Gene Expression

GeNeiTM

Bacterial Gene Expression

GeNeiTM

Appendix:
Preparation of LB Agar/broth (1 litre): Dissolve 25 g of media in 800 ml of distilled water. Adjust the pH to 7.0 with 5N NaOH (if necessary) and make up the volume to 1000 ml. Sterilize by autoclaving. For LB agar, add 1.5% agar and autoclave. Ampicillin Preparation: Dissolve 100 mg of the antibiotic in 1 ml sterile water to get a stock concentration of 100 mg/ml. Store at 4C for 2 weeks. Use the antibiotic within this period. For Ampicillin LB media: Add ampicillin to LB broth or agar at a final concentration of 100 g/ml, when the temperature of the media is around 40-45C. Following aliquots of media are required for each experiment (Excludes preparation of media required for revival of strain): LB Broth 1 x 5 ml; 1 x 100 ml

SDS-PAGE

Note: Prepare 100 ml of LB broth in a 500 ml conical flask.

Bangalore Genei, 2007

10

Bangalore Genei, 2007

11

Bacterial Gene Expression

GeNeiTM

Bacterial Gene Expression

GeNeiTM

Principle:
SDS-PAGE is the most widely used method for qualitatively analyzing any protein mixture, monitoring protein purity and to determine their molecular weight. It is based on the separation of proteins according to their size and then locating them by binding to a dye. SDS or sodium dodecyl sulphate is an anionic detergent that binds strongly to proteins, causing their denaturation. In the presence of excess SDS, about 1.4 g of the detergent binds to each gram of protein, giving the protein a constant negative charge per unit mass. As a result, proteinSDS complexes move towards the anode during electrophoresis and owing to molecular sieving properties of the polyacrylamide gel, get separated based on their molecular weights. Since, the principle of this technique is separation of proteins based on size differences, by running standard proteins of known molecular weight on the same gel as unknown protein, molecular weight of the unknown protein can be determined. Mobility of protein in SDS gel electrophoresis is expressed as relative mobility (Rf) with respect to the tracking dye, bromophenol blue.

Bangalore Genei, 2007

12

Bangalore Genei, 2007

13

Bacterial Gene Expression

GeNeiTM

Bacterial Gene Expression

GeNeiTM

Rf =

distance migrated by protein____ distance migrated by tracking dye

Rf values of protein standards of known size is used to generate a standard curve by plotting the molecular weight against the Rf value on a semi-log graph. The molecular weight of an unknown protein can then be extrapolated from its Rf value. The technique consists of three basic steps: Step I: Preparation of Polyacrylamide Gel Crosslinked polyacrylamide gels are formed by co-polymerization of acrylamide monomer and a cross-linking agent N, N- Methylene bisacrylamide. This reaction is catalyzed by N, N, N, N-Tetramethylenediamine (TEMED) and initiated by ammonium persulphate (APS). Porosity of the gel is determined by the amount of acrylamide and bis-acrylamide mix used. Lower percentage gels have larger pore sizes, thereby offering less resistance to passage of larger molecules, while higher percentage gels favour separation of smaller molecules. Most protein separations are carried out using gels ranging from 5-15% acrylamide. Step II: Electrophoresis The polyacrylamide gel slab is prepared and fixed to a vertical electrophoresis apparatus. Protein samples are usually denatured by boiling in sample loading buffer containing Tris (pH 6.8), SDS, -Mercaptoethanol (to reduce disulphide bonds), sucrose/glycerol (to increase the density), and bromophenol blue (as a tracking dye) and loaded onto wells in the gel.

Resolution of the protein bands is greatly increased by applying the samples onto a short stacking gel on top of the separating gel. Differences in pH and composition between these two gels cause the samples to be concentrated into narrow bands by isotachophoresis. As the samples migrate through the separating gel, proteins get resolved depending on their molecular weight. Electrophoresis is stopped when the dye front reaches the bottom of the gel. Step III: Visualization of Proteins Generally, proteins are colourless and hence cannot be visualized directly. Suitable dyes are used to stain the samples, eg., Coomassie Brilliant Blue R-250

Bangalore Genei, 2007

14

Bangalore Genei, 2007

15

Bacterial Gene Expression

GeNeiTM

Bacterial Gene Expression

GeNeiTM

Note:
Read the entire procedure before starting the experiment. Bring the gel mix to room temperature before polymerization. Wear gloves while handling the gel mix and gel, as acrylamide is a neurotoxin. Plates should be clean and free of detergents. Comb should be cleaned with water and alcohol. Resuspend ammonium persulphate with 500 l of water. Store at 4C. Use within 3 months. Dilute the required amount of reservoir buffer to 1X with water.

Procedure:
1. Assemble the plates for casting gel as shown below: Glass Plates & Spacers Assembled for casting

Notched

Base Plate

Spacers 2. Clamp the assembly of plates. Ensure the assembly is leak proof by filling water between the plates. Silicon grease can be applied to spacers or 1% agarose can be used for sealing to make it leak proof. 3. Add 50 l of ammonium persulphate to 5 ml of separating gel mix and mix thoroughly.
Bangalore Genei, 2007

4. Pour the gel solution between the plates till the level is about 2 cm below the top edge of notched plate. 5. Add 200 to 250 l of water to make the surface even. 6. After the gel is set (approximately 20-30 minutes), wash the top of the separating gel with distilled water and drain off the water completely. 7. Add 20 l of APS solution to 2 ml of stacking gel mix, mix thoroughly and pour directly onto the polymerized separating gel. 8. Insert the comb into the gel solution carefully without trapping any air bubbles, approximately 1 cm above the separating gel. The stacking gel will set in about 10 minutes. 9. After the stacking gel has set, carefully remove the comb and the bottom spacer. Wash the wells immediately with distilled water to remove non-polymerized acrylamide. 10. Fill the bottom reservoir with 1X reservoir buffer. 11. Carefully fix the plate to the PAGE apparatus without trapping any air bubbles between the buffer and the bottom of the gel, with the notched plate facing the top reservoir. 12. Fill the top reservoir with 1X reservoir buffer. 13. Load samples into the wells, rinse the micropipette tip in the bottom reservoir buffer between each load. Note down the order in which the samples have been loaded. 14. Connect the cords to the power supply, according to the convention red : anode, black : cathode. 15. Set voltage at 50 - 100 V and switch on the power supply. 16. When the dye front reaches 0.5 cm above the bottom of the gel, turn off the power.

16

Bangalore Genei, 2007

17

Bacterial Gene Expression

GeNeiTM

Bacterial Gene Expression

GeNeiTM

17. Remove the gel plates and gently pry the plates apart. Use a spatula or similar tool to separate the plates (not at the notch). 18. Transfer the gel to a tray containing water, wash the gel for 5 minutes.Discard the water. 19. Add 20 ml of Ezee blue and stain the gel for 30-60 minutes. Continue staining the gel overnight if the bands appear light. 20. Destain the gel with water, if the background is not clear. (This is an optional step). Note: For uniform staining and washing, place the tray on a rocker or shake intermittently every 10 to 15 minutes.

Ordering Information
Product GeNeiTM Bacterial Gene Expression Teaching Kit (Consumables for 5 experiments & Elpho Kit (ETS 4)) GeNeiTM Bacterial Gene Expression Teaching Kit (Consumables for 5 experiments) Size 1 Pack Cat # KT69

1 Pack

KT69A

Email: Sales: geneisales@sanmargroup.com Customer Support: geneitechsupport@sanmargroup.com

Bangalore Genei, 2007

18

Bangalore Genei, 2007

19

Bacterial Gene Expression

GeNeiTM

Bacterial Gene Expression

GeNeiTM

Note:

Bangalore Genei, 2007

20

Bangalore Genei, 2007