11. ANALYSIS OF RECOMBINANT CLONES

Recombinant clones are analyzed using restriction enzyme (RE) digestion. RE is selected based on the recognition sequences present in insert and vector. Select such a REs which has recognition sequence in insert and the vector, digestion with enzyme releases a fragment which confirms the presence of insert in the clone. Orientation of the gene with respect to promoter is determined using two methods RE digestion and PCR. As mentioned earlier select two unique -Res present in insert and vector, digest and subsequent analysis of product size on Agarose gel would give the orientation of the insert in the vector. For expression of the insert 5` side should be towards the promoters 3` side. Once the orientation of the gene is confirmed the plasmid carrying the insert is transformed into an E. coli strain (For Example if pET vector are used the host should be BL21(DE3).) Single colony is picked from the transformation plate and checked for the expression. In most of the E.coli expression vectors the gene is cloned downstream to T7 promoter.

Experiments:

1.      Restriction digestion.

2.      Agarose gel electrophoresis

3.      Culture of Bacterial cells for expression

4.      Polyacrylamide gel electrophoresis for detection of protein expression.

 

I.                   Restriction digestion:

      Restriction endonucleases bind specifically to and cleave double-stranded DNA at specific sites within or adjacent to a particular sequence known as the recognition sequence. These enzymes have been classified into three groups. Type I and type III enzymes carry modification  (methylation) and ATP-dependent restriction (cleavage) activities in the same protein. Type III enzymes cut the DNA at the recognition site and then dissociate from the substrate. However, type I enzymes bind to the recognition sequence but cleave at random sites when the DNA loops back to the bound enzyme. Neither type I nor type III restriction enzymes are widely used in molecular cloning.  Type II restriction endonuclease cleaves at a specific sequence of nucleotides and a separate methylase that modifies the same recognition sequence. The widely used type II restriction enzymes recognize specific sequences that are four, five, or six nucleotides in length and display palindrome sequence (eg. EcoRI: 5’-GAA TTC-3’). Two types of cuts are produced by restriction endonucleases.  They are the blunt ends (both strands are cut at the same location) and sticky ends which have single stranded projections at both ends.

Materials:

1.      Restriction enzyme

2.      Plasmid DNA

3.      Buffer (10X Buffer supplied with the Enzyme) 

4.      Distilled water

5.      Water bath

Protocol for Restriction digestion:

1. Prepare the reaction for restriction digestion by adding the following reagents in the order listed to a microcentrifuge tube:

sterile ddH20          q.s (where "q.s." means quantity sufficient)

   10X assay buffer           one-tenth volume

   DNA                             x ul

   restriction enzyme*        y ul (1-10 units per ug DNA)

   Total volume                 z ul

*If desired, more than one enzyme can be included in the digest if both enzymes are active in the same buffer and the same incubation temperature.

Note: The volume of the reaction depends on the amount and size of the DNA being digested. Larger DNAs should be digested in larger total volumes (between 50-100 ul), as should greater amounts of DNA.

Refer to the vendor's catalogue for the chart of enzyme activity in a range of salt concentrations to choose the appropriate assay buffer (10X High, 10X Medium, or 10X Low Salt Buffers, or 10X SmaI Buffer for SmaI digestions). Restriction enzymes are purchased from Bethesda Research Laboratories, New England Biolabs, or United States Biochemicals.

2. Gently mix by pipetting and incubate the reaction at the appropriate temperature (typically 37°C) for 1-3 hours.

3. Inactivate the enzyme(s) by heating at 70-100ºC for 10 minutes or by phenol extraction (see the vendor's catalog to determine the degree of heat inactivation for a given enzyme). Prior to use in further protocols such as dephosphorylation or ligation, an aliquot of the digestion should be assayed by agarose gel electrophoresis versus non-digested DNA and a size marker, if necessary.

II.  Agarose gel electrophoresis

Agarose gel electrophoresis is employed to check the progression of a restriction enzyme digestion, to quickly determine the yield and purity of a DNA isolation or PCR reaction, and to size fractionate DNA molecules, which then could be eluted from the gel.

1.      Prior to gel casting, dried agarose is dissolved in buffer by heating and the warm gel solution then is poured into a mold  made by wrapping clear tape around and extending above which is fitted with a well-forming comb.

2.      The percentage of agarose in the gel varied. Although 0.7% agarose gels typically are used, in cases where the accurate size fractionation of DNA molecules smaller than 1 kb is required, a 1, 1.5, or 2% agarose gel is prepared, depending on the expected size(s) of the fragment(s).

3.      Ethidium bromide is included in the gel matrix to enable fluorescent visualization of the DNA fragments under UV light.

4.      Agarose gels are submerged in electrophoresis buffer in a horizontal electrophoresis apparatus. The DNA samples are mixed with gel tracking dye and loaded into the sample wells.

5.      Electrophoresis usually is at 100 V constant voltage till the bands get resolved completely at room temperature.

6.      When low-melting agarose is used for preparative agarose gels, electrophoresis is at 100-120 mA for 0.5-1 hour, again depending on the desired separation, and a fan is positioned such that the heat generated is rapidly dissipated.

7.      Markers (Ladders)  are co-electrophoresed with DNA samples, when appropriate for fragment size determination.

8.      After electrophoresis, the gel is placed on a UV light.

Materials:

1.      Agarose

2.      Ethidium Bromide (5mg/ml)

3.      50x TBE/TAE buffer

4.      Distilled water

5.      6x loading dye

6.      Submerged Gel electrophoresis system

7.      Power pack

8.      UV Trans-illuminator or a Gel Documentation system.

Protocol agarose gel electrophoresis:

Prepare an agarose gel, according to recipes listed below, by combining the agarose (low gel temperature agarose may also be used) and water in a 500 ml flask, and heating in a microwave for 2-4 minutes until the agarose is dissolved.

                                                  0.7%               1.0%             2.0%

          Agarose                          1.05 g              1.5 g            3.0 g

          50X TAE                           3 ml               3 ml             3 ml

          ddH2O                             147 ml             147 ml         147 ml

          EtBr (5 mg/ml)                25 ul                  25 ul            25 ul

          Total vol                       150 ml              150 ml    150 ml

 

1.      Add 50X TAE and ethidium bromide (EtBr), swirl to mix, and pour the gel onto a taped plate with casting combs in place. Allow 20-30 minutes for solidification.

          50x TAE (50ml) :

60.5 gm Tris base

14.25 ml Glacial acetic acid

25ml 0.5M EDTA

Distilled water to final volume of 50ml.

2.      Carefully remove the tape and the gel casting combs and place the gel in a horizontal electrophoresis apparatus. Add 1X TAE electrophoresis buffer to the reservoirs until the buffer just covers the agarose gel.

3.      Add at least one-sixth  volume of 6X DNA gel loading dye to each DNA sample, mix, and load into the wells.

4.       Set the voltage to 100V and start the run  until the required separation has been achieved.

5.      Visualize the DNA fragments on a long wave UV light box or a gel documentation system and take a photograph with a digital camera.

III.  Culture of Bacterial cells for expression

    Gene of interest is cloned under control of T7 polymerase promoter. Expression of the gene can be triggered by addition of optimal amount of IPTG(Generally 1mM final concentration). Induction time should also be optimized as some of the proteins would be toxic to the host cell and would kill the host upon over expression of the protein. Generally the culture is allowed to grow for 3hrs after induction. Sometimes the temperature of the culture after induction play an important role in stability of the expressed protein. Main problem of  inclusion bodies (partially folded) can be sometime be converted to soluble form by growing at culture at  lower temperature once the IPTG is added. As mention in this paragraph all the conditions are to standardized before setting up a large scale culture for expression and purification. 

General Protocol for expression of gene:

1.      1.Starting from glycerol stock, chip off a small piece from the frozen cells. Do not thaw the tube of bacteria. Streak out the cells from the ice chip on a plate with a loop. If necessary, the chip can be directly used to seed the starter culture.

2.      Pick and add a single colony of E. coli to 10 ml of LB media with the appropriate antibiotic ampicillin. 100µgm/ml final concentration

3.      Incubate at 37^oC overnight with shaking in the incubator.

4.      Add the 1% of overnight grown inoculum to 250  or 500 ml flask for up 50 100 ml cultures – of LB media with ampicillin

5.      Incubate at 37^oC in the shaker for 2 to 4 hours or until A550 ~      0.5-0.7

6.      Save 1.5 ml of cells as un-induced culture. Centrifuge for 10 min in a microfuge at max speed. Remove supernatant and add 75 µl of 5X SDS PAGE SDS-PAGE sample buffer and freeze until SDS-PAGE analysis.

7.      Add 1mM final conc. from stock 1M IPTG and continue to incubate at 37^oC for ~ 3-4 hours

8.      Save 1.0 ml of cells as induced culture. Centrifuge for 10 min in microfuge at max speed.

9.      Remove Supernatant and add 75 µl of 5X SDS PAGE sample buffer and freeze until SDS-PAGE analysis.

V.                 SDS-Polyacrylamide gel electrophoresis (SDS-PAGE) for detection of protein expression.

      Most of the analytical electrophoresis of the proteins is done in Polyacrylamide gels under condition that ensure the disassociation of proteins to monomeric forms and minimize aggregation. The commonly used anionic detergent SDS is used in combinatiion with a reducing agent and heat to disassociate the proteins before they are loaded on to gel. The denatured polypeptides bind SDS and become negatively charged. Because the amount of SDS bound is almost always proportional to the molecular weight of the polypeptide and is independent of its sequence, SDS-polypeptide complexes migrate through polyacryamide gels in accordance with the size of the polypeptide. Poly acryamide gels are composed of chains of polymerized acryamide that are crosss linked by a bifunctional agent such as N`N` methylenebis acrylamide. The effective range of separate on SDS-PAGE depends on the polyacryamide concentration used in gel preparation.

Choose a percentage acrylamide based on the molecular weight range of proteins you wish to separate:



Materials:

1.       30% acrylamide.

2.       Tris pH 8.8

3.       Tris pH 6.8

4.       Distill water

5.       10% SDS

6.       10% Ammonium sulphate.

7.       TEMED

8.       Vertical Gel Unit (Glass Plates, Comb and spacers)

9.       Casting stand

10.    Power Pack

 

Protocol for casting the gel:

Before pouring the gel assemble the glass plates, adjust the spacers (range from 0.5mm to 1.5mm thick Teflon strips) between the glass plates and get all the reagents listed to the work place. Get all the range of pipette, which will be useful in dispensing the required volumes of the reagents.

1.       Mix the ingredients needed for the chosen percentage and pour the solution quickly into your gel casting form. It is called the resolving gel. Leave sufficient space for the upper stacking gel usually 1cm.

2.        Look for air bubbles and remove them, add to the top of the gel water saturated butanol or, water. This will remove bubbles at the top of the gel and will ensure this part does not dry out.

3.       Wait for about 30 minutes for the gel to polymerize completely. (If you always use fresh ammonium persulfate, you're gel may polymerize more quickly and reliably.)

4.       Remove the overlayed butanol or water completely. Prepare the stacking gel by mixing the reagents as shown in the table.

5.       Mix in the polymerizing reagents and pour the stacking gel on top of the running gel. Insert Teflon combs trying not to get bubbles stuck underneath and allow another 30 min - 1 hour for complete polymerization. Teflon comb should be cleaned with water and dried with ethanol just before use.

6.       Be sure to leave a gap at the bottom of the comb for the stacking gel. You can do this by inserting the comb into the dry form, and marking a region below the comb for the height of the stacker you want.

Running gel:

1.       While stacking gel polymerizing, prepare the samples by heating them to 100ºC for 10 min in 1x SDS gel loading buffer to denature the proteins.

2.       Clamp in your gel and fill both buffer chambers top and bottom with gel running buffer (1X Tris-Glycine) according to the instructions for the specific apparatus. Remove any bubbles that become trapped at the bottom of gel between the glass plates using a bent needle.

3.       Load upto 15 ml of each of the sample in a predetermined order into the bottom of the well.

4.       Attach the electrophoresis apparatus to the powerpack initially at 25mA constant current till the dye reaches the resolving gel. Increase the current to 35mA till the end of the run.

5.       Stop the power supply when the dye reaches to the bottom of the gel.

6.       Remove the glass plates from the electrophoresis apparatus, using a spatula separate the plates. Mark the gel by cutting a corner from the bottom of the gel towards slot 1.

 

Staining after SDS PAGE with coomassie brilliant blue:

1.       Immerse the gel in at least 5 volumes of staining solution and place on the slowly rotating platform for a minimum of 4 hours at room temperature.

Staining solution:

0.25g coomassie brilliant blue in 90ml of methanol : water (1:1)

10ml of glacial acetic acid

Filter the solution through whatman no.1 to remove particulate matter.

2.       Destain the gel by soaking it in destaining solution for 4 to 8 hours.

Destaining solution:

90ml of methanol : water (1:1)

10ml of glacial acetic acid.