2018

GEBZE TECHNICAL
UNIVERSITY
MOLECULAR GENETICS LABORATORY
TURKEY

EBRU AKHARMAN
142204026
17.05.2018
 RECOMBINANT GENE EXPRESSION AND SDS-PAGE
AIM

Cloning vectors provide a backbone for the DNA insert to be reproduced and propagated in bacteria;
however, these vectors are only useful for storing a genetic sequence. By themselves, they are incapable of
allowing for transcription and translation of the gene into a functional protein product. Therefore, genes will
be stimulated with IPTG to produce functional protein. The presence of functional protein will be detected by
SDS-PAGE. SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) is commonly used in the
lab for the separation of proteins based on their molecular weight.

INTRODUCTION
Many proteins are expressed at low levels in vivo. To produce high levels of a protein, it is often useful to clone the
gene downstream of a well-characterized, regulated promoter. Inducing transcription from the regulated promoter
thus results in elevated expression of the downstream gene product. Isopropyl β-D-1-thiogalactopyranoside,
abbreviated IPTG, is a molecular biology reagent. This compound is used as a molecular mimic of allolactose, a
lactose metabolite that triggers transcription of the lac operon. Unlike allolactose, the sulfur (S) atom creates
a chemical bond which is non-hydrolyzable by the cell, preventing the cell from “eating up” or degrading the
inductant; therefore the IPTG concentration remains constant. IPTG binds to the lac repressor and releases
the tetrameric repressor from the lac operator in an allosteric manner, thereby allowing the transcription of
genes in the lac operon, such as the gene coding for beta-galactosidase, a hydrolase enzyme that catalyzes the
hydrolysis of β-galactosides into monosaccharides. One advantage of IPTG for in vivo studies is that since it
cannot be metabolized by E. coli its concentration remains constant and the rate of expression of lac p/o-
controlled genes, is not a variable in the experiment. IPTG intake is independent on the action of lactose
permease, since other transport pathways are also involved.

Image 1: Induction of the Lac Operon

In the previous lab sessions, a bacterial enzyme gene was cloned under the control of lac promoter and
operator in pUC19 plasmid vector, and the clones carrying the desired gene fragment were selected by using
blue-white colony screening technique. Next step is the expression of the gene which will result in a functional
protein product. So, in this experiment, the gene will be expressed by means of IPTG induction. Then, the
expression level will be observed by means of SDS-PAGE analysis and enzymatic activity assays.

The separation of macromolecules in an electric field is called electrophoresis. A very common method for
separating proteins by electrophoresis uses a discontinuous polyacrylamide gel as a support medium and
sodium dodecyl sulfate (SDS) to denature the proteins. The method is called sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE). SDS (also called lauryl sulfate) is an anionic detergent,
meaning that when dissolved its molecules have a net negative charge within a wide pH range. A polypeptide
chain binds amounts of SDS in proportion to its relative molecuar mass.

The negative charges on SDS destroy most of the complex structure of proteins, and are strongly attracted
toward an anode (positively-charged electrode) in an electric field. Polyacrylamide gels restrain larger
molecules from migrating as fast as smaller molecules. Because the charge-to-mass ratio is nearly the same
among SDS-denatured polypeptides, the final separation of proteins is dependent almost entirely on the
differences in relative molecular mass of polypeptides. In a gel of uniform density the relative migration
distance of a protein (Rf, the f as a subscript) is negatively proportional to the log of its mass. If proteins of
known mass are run simultaneously with the unknowns, the relationship between Rf and mass can be plotted,
and the masses of unknown proteins estimated.

Protein separation by SDS-PAGE can be used to estimate relative molecular mass, to determine the relative
abundance of major proteins in a sample, and to determine the distribution of proteins among fractions. The
purity of protein samples can be assessed and the progress of a fractionation or purification procedure can be
followed. Different staining methods can be used to detect rare proteins and to learn something about their
biochemical properties. Specialized techniques such as Western blotting, two-dimensional electrophoresis,
and peptide mapping can be used to detect extremely scarce gene products, to find similarities among them,
and to detect and separate isoenzymes of proteins.

MATERIALS
Required Materials For Recombinant Gene Expressions

 LB medium
 Ampicillin ( 10 mg/ml )
 IPTG ( 100 mM )

Preparing the IPTG (100 mM) Solutions:

Dissolve 0,238 g of IPTG in 8 ml of dH2O. Filter sterilize with a 0.2 μm syringe filter. Store in 1 ml aliquots at -
20 °C.

Required Materials For SDS - Page

 Separating and stacking gel solutions

 5X SDS/PAGE Electrophoresis Buffer
 Cracking Buffer ( Loading Dye )
 Coomasie Blue R 250 Staining Solution
 Destaining Solution
 Protein Molecular Weight Standard
 70 % alcohol
 Isopropyl alcohol
Prepairing the Solutions:

Separating Gel

 750 μl distilled water

 3750 μl 0.75 M Tris – HCl, pH 8.8 / 0.2 % SDS
 3 ml Acrylamide / Bisacrylamide ( 30:1 )
 90 μl 10 % APS
 6 μl TEMED

The percentage of acrylamide in the separating gel depends on the molecular size of the protein
being separated. As a guideline, use 5 % gels for SDS-denaturated proteins of 60 to 200 kDa, 10 % gels for SDS-
denaturated proteins of 16 to 70 kDa, and 15 % gels for SDS-denaturated proteins of 12 to 45 kDa.

Stacking Gel

 1050 μl distilled water

 1500 μl 0.25 M Tris – HCl, pH 6.8 / 0.2 % SDS (SDS is a detergent with a strong protein-denaturing effect
and binds to the protein backbone at a constant molar ratio. In the presence of SDS and a reducing agent that
cleaves disulfide bonds critical for proper folding, proteins unfold into linear chains with negative charge
proportional to the polypeptide chain length.)
 390 μl Acrylamide / Bisacrylamide ( 30:1 )( Acrylamide and Bisacrylamide, both are very important for
proper polymerization. Acrylamide forms linear polymers whereas Bisacrylamide cross links these linear
polymer. More numbers of cross links means smaller pore size. So, ratio of Acrylamide and Bisacrylamide
determines pore size)
 60 μl 10 % APS
(Ammonium Persulfate (APS) and TEMED catalyze the polymerization of acrylamide solutions into gel
matrices. These gels are then used to separate a variety of macromolecules by size in the presence of an
electric field. Optimal polymerization requires the daily preparation of fresh APS stock solution since APS is
unstable in aqueous solution)
 3 μl TEMED

Preparing the 5X SDS / Page Electrophoresis Buffer:

Dissolve 15g Tris base, 72 g glycine, 5g SDS in distilled water and adjust final volume to 1 l.

Cracking Buffer

 50 mM Tris – HCl, pH 6.8, 1 % SDS

 2 mM EDTA
 1 % β-mercaptoethanol (Beta-mercaptoethanol is a reducing agent that will irreversibly denature RNases by
reducing disulfide bonds and destroying the native conformation required for enzyme functionality.)
 10 % glycerol (Glycerol is added so the sample you load is more viscous and won't diffuse out of the well in
the time it takes to load the other samples before the current is turned on.)
 0.002 % Bromophenol Blue (Bromophenol blue is used as a migration front indicator during running.)

Coomassie Blue R Staining Solution

 100 ml 10 % acetic acid

 100 ml 0.25 % Coomasie Blue R250 ( dissolved in 95 % ethanol )( Coomassie is used for protein staining
after SDS/PAGE running)

Destaining Solution

 5 % acetic acid
 50 % ethanol
 METHOD
For Recombinant Gene Expression:
1. As a starter culture, inoculate 2 ml of sterile LB containing ampicillin with a colony of the selected clone
from the master plate.
2. Incubate at 37 °C with shaking at 200 rpm overnight to obtain a saturated culture.
3. Inoculate 30 ml of the LB medium with 300 μl of this starter culture and incubate the culture under the
same conditions until 𝑂𝐷600 value reaches 0,6-0,8; this usually takes 2,5-3 hours. Check the OD frequently
when it gets beyond 0,4 to avoid overgrowth.
4. When 𝑂𝐷600 value of the culture reaches 0,6, take 1 ml culture as “pre-induction” sample and kept on ice
until SDS-PAGE sampling mentioned at step 7.
5. Add IPTG (use 100 mM stock solution) to make a final concentration of 200 μM and immediately put the
cultures back to incubation at 37 °C with shaking at 200 rpm. (It should be noted that, IPTG amount calculation
should be done considering the retaining culture volume by excluding the amount of samples taken during 𝑶𝑫𝟔𝟎𝟎
measurements and pre-induction SDS-PAGE sampling. )
6. Take another sample after 3 hours of induction described as step 4 and kept on ice. Check 𝑂𝐷600 value of
the culture and note it down.
7. SDS-PAGE samples should be prepared by considering the 𝑂𝐷600 readings. The collected samples should
be kept at +4 °C and prepared SDS-PAGE samples at -20 °C. (For SDS-PAGE sampling, use an inverse proportion of
growth and sample amount. For example when 𝑶𝑫𝟔𝟎𝟎 value of the culture is 0,3, you should take 500 μl of the
culture as SDS-PAGE sample. While 𝑶𝑫𝟔𝟎𝟎 value of the culture is increasing, the amount of SDS-PAGE sample
decreases. When you calculate the required amount for SDS-PAGE analysis, pipet this amount of sample from the
cultures which you obtained at step 4 and 6. Then centrifuge at 10.000 rpm for 10 min at 4 °C. Remove the
supernatant and keep the pellet at -20 °C. )

For SDS - PAGE:

1. Clean glass plates first in detergent and distilled water then in 70 % ethanol.
2. Assemble the glass-plate sandwich using two clean glass plates and two spacers.
3. Lock the sandwich to the casting stand.
4. Prepare the separating gel solution.
5. Using a pipette, apply the 3.2 ml separating gel solution to the sandwich along an edge of one of the
spacers. (Use the solution immediately; otherwise it will polymerize in the flask.)
6. Using a syringe, slowly cover the top of the gel with a layer of isopropyl alcohol. (Be careful not to disturb the
gel surface.)
7. Allow the gel to polymerize 20-40 mins at RT. (A sharp optical discontinuity at the overlay/gel interface will be
visible upon polymerization. Failure to form a firm gel usually indicates a problem with the ammonium persulfate
(APS), TEMED (N,N,N’,N’-tetramethylethylenediamine), or both. APS should be made fresh before use. APS should
“crackle” when added to the water. If not fresh APS should be purchased. Purchase TEMED in small bottles so, if
necessary, a new previously unopened source can be tried.)
8. Pour off the layer of isopropyl alcohol and rinse with distilled water. (Residual isopropyl alcohol can reduce
resolution of the protein bands; therefore, it must be completely removed. The isopropylalcohol should not be left on
the gel for >2 hr.)
9. After rinse, take the remained water with 3 MM Whatman paperPrepare the stacking gel solution and using
a pipette, slowly allow the stacking gel solution to trickle into the center of the sandwich along an edge of one
of the spacers. (Use the solution immediately; otherwise it will polymerize in the flask. Be careful not to introduce
air bubbles into the stacking gel.)
10. Insert a comb into the layer of stacking gel solution. If necessary, add additional stacking gel to completely
fill the spaces in the comb. (Again, be careful not to trap air bubbles in the tooth edges of the comb as they will
cause small circular depressions in the well upon polymerization that will lead to distortion in the protein bands
during separation.)
11. Allow the stacking gel solution to polymerize 30-45 mins at RT.
12. Dissolve the protein sample with 40 μl cracking buffer and mix gently. Prepare the protein marker. Use 3
μl protein molecular weight standard. Add 7 μl 1x SDS/PAGE electrophoresis buffer and 10 μl cracking buffer.
(To achieve the highest resolution possible, the following precautions are recommended. Prior to adding the sample
buffer, keep samples at 0 °C. Add the SDS/sample buffer (RT) directly to the 0 °C sample (stil on ice) in a
microcentrifuge tube. Cap the tube to prevent evaporation, vortex, and transfer directly to 100 °C water bath for 5
mins. DO NOT leave sample in SDS/electrophoresis buffer at RT without first heating to 100 °C to inactivate
proteases.)
13. After vortex, heat 5 mins at 100 °C in a boling water bath.
14. Spin the samples shortly.
15. Carefully remove the comb without tearing the edges of the polyacrylamide wells. After the comb
removed, rinse wells with 1x SDS/electrophoresis buffer to remove unpolymerized monomer; otherwise, the
monomer will continue to polymerize after the comb is removed, creating uneven wells that will interfere
with sample loading and subsequent separation. (If well walls are not upright, they can be manipulated with a
flat-tipped needle attached to a syringe.)
16. Using a pipette load the protein samples into wells by carefully applying the sample as a thin layer at the
bottom of the wells. Add 1x SDS/PAGE electrophoresis buffer onto sample wells to prevent spreading of
adjoining lanes. (Prepare the samples at approximately the same concentration, and load an equal volume to each
well. This will ensure that all the lanes are the same width and that the proteins run evenly. If unequal sample buffer
volumes are added to wells, the lane with the larger volume will spread during electrophoresis and constrict the
adjacent lanes, causing distortions. The samples will layer on the bottom of the wells because the glycerol added to
the sample buffer makes the solution density greater than the electrophoresis buffer.)
17. Attach the gel sandwich to upper buffer chamber into lower buffer chamber.
18. Partially fill the upper bufer chamber with 1x SDS/PAGE Electrophoresis buffer so that the sample wells of
the stacking gel are filled with buffer.
19. Fill the remainder of the upper bufer chamber with additional 1x SDS/PAGE Electrophoresis buffer so that
the upper platinium electrode is completely covered. Do this slowly so that samples are not swept into
adjacent wells.
20. Connect the power supply to the cell and run at 100 V of constant current until the bromophenol blue
tracking dye enters the separating gel. Then increase the current to 200 V.
21. After the bromophenol blue tracking dye has reached the bottom of the separating gel, disconnect the
power supply.
22. Discard electrode buffer and remove the upper buffer chamber and the attached sandwich.
23. Orient the gel so that the order of the sample wells is known, remove the sandwich from the upper buffer
chamber, and lay the sandwich on a sheet of absorbent paper towels.
24. Carefully slide one of the spacers halfway from the edge of the sandwich along its entire lenght. Use the
exposed spacer as a lever to pry open the glass plate, exposing the gel.
25. Carefully remove the gel from the lower plate. Cut a small triangle off one corner of the gel so the lane
orientation is not lost during staining and drying. The gel can be stained with coomassie blue or silver,
electroeluted, electroblotted onto PVDF membrane for subsequent protein sequence analysis, or transferred
to a membrane fitler for western blotting.
26. Place the polyacrylamide gel in a plastic or glass container. Cover the gel with coomassie blue staining
solution and shake gently until desired intensity is reached, 2 hr to overnight at RT.
27. Pour out staining solution and cover the gel with 5 % acetic acid + 50 % ethanol to destain, shaking gently
2 hr at RT until a clear background is obtained.
28. If necessary, pour out destaining solution and add more. Continue destaining until clear background is
obtained. Store gel in destaining solution or water, or in plastic wrap at 4 °C. (It is usually unnecessary to add
additional destaining solution.)
29. If desired, photograph or dry the gel.
 RESULTS

Image 2: SDS Page Image 3: Protein Marker

The all protein profiles of cells can be detected with results of Sds Page. The holes of 1-2 show before
induction of pUC19. The holes of 3-4 show induction of pUC19. The holes of 5-6 show before induction of
clon. The holes of 7-8 show induction of clon. The nineth hole shows pure enzyme(this enzyme is penicillin
acylase) and tenth hole shows protein marker. The pure enzyme was used for control and has 60 % purity
ratio. Thus, there is not only one enzyme in this enzyme. The enzyme has an amount of other cellular enzyme.
The enzyme can be seen clearly in two bants of SDS Page. The size of forward band is 64 kDa and size of last
band is 24 kDa. If cloning is correct, cloned bacterial cells must use penicillin acylase enzyme and produce.The
insert DNA provides to produce penicillin acylase. Thus, this method shows correctness of cloning. The wanted
proteins must be found in level of these bands. The protein marker is used for controlling size of protein
because of the protein marker has several weight level. 64 kDa'daki bantlar, 1, 2, 3, 4, 5, 6, 7, 8'lik delikler
görülebilir, ancak sadece pUC19'un indüksiyonundan önce 24 kDa'da bantlar vardır.
 DISCUSSION

Cloning vectors provide a backbone for the DNA insert to be reproduced and propagated in bacteria;
however, these vectors are only useful for storing a genetic sequence. By themselves, they are incapable of
allowing for transcription and translation of the gene into a functional protein product. Therefore, genes will
be stimulated with IPTG to produce functional protein.
PUC19 cloning vector provides Plac promoter, which could be induced by a highly stable synthetic lactose
analog called IPTG (isopropyl-β-D-thiogalactoside). Plac promoter is under control of a repressor protein,
which is encoded by the lacI gene. This lac repressor binds to a site (lac operator sequence) on the vector DNA
where transcription of the lac operon is initiated, thereby blocking transcription. When IPTG is present in the
cell, it binds to the lac repressor, decreasing its affinity for the repressor-binding site on the DNA. As a result,
the repressor falls off the DNA and transcription is initiated, leading to synthesis of β-galactosidase. The
presence of functional protein will be detected by SDS-PAGE. SDS-PAGE (sodium dodecyl sulphate-
polyacrylamide gel electrophoresis) is commonly used in the lab for the separation of proteins based on their
molecular weight. According to results of SDS Page, there are bands in same level with 60 % pure enzyme but
the bands are observed in pUC proteins. The pUCs do not have to gene of this enzyme. There are some bands
in holes of 5, 6, 7, 8 but these bands are not clear. The thin bands must be waited in before induction of clon
and the large bands must be waited in induction of clon. There are thin bands in before and after induction of
clon. The reason of this results, bacterial cells can have to inhibitor of insert gene.
The protein try to express may be degraded immediately after it has been synthesized. Some times, under
selection with continues antibiotics media, mutations could occur, the non transfected cells could have
become resistant and overtake the rest of cells. While plasmids are integrated into genome, they get
linearized (if they are circular), and this is done at a random position on the plasmid sequence by the cellular
machinery.
Therefore, some percentage of your stable cells will have the vector with in their genome with the intact
antibiotic resistance casette, but with the interrupted GOI ( gene of interest )casette (due to the linearization
at the promoter site or the coding sequence), which will eventually give a population of cells resistant to
antibiotic but lacking GOI expression. Thus, we say that, wrong cells can be used in the holes of 1, 2, 3, 4 and
there is not enough concentration of protein in the holes of 5, 6, 7, 8 so the bands can not be clear. The some
problems occured in prepairing of SDS-Page. The buffer was fulled excess and holes remained into buffer.
Thus, the samples do not filled enough in holes for this reason the samples do not run in enough
concentration. According to SDS Page, there are not some protein bands of pUC in clon. The reason of this
result can be cutting region of pUC for producing clon. The cutting region can produce different some proteins
from clon.

RESOURCES

https://www.biologicscorp.com/blog/iptg-induction-protein-expression/#.WvmNrGjRDDc
http://agscientific.com/blog/2013/01/iptg-triggers-the-transcription-of-the-lac-operon/
http://www.ruf.rice.edu/~bioslabs/studies/sds-page/gellab2.html
https://bitesizebio.com/580/how-sds-page-works/
https://www1.udel.edu/biology/fschmieg/411acrylamide.htm
http://ruo.mbl.co.jp/bio/e/support/method/sds-page.html