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Summer Research Fellowship Programme of India's Science Academies

Over-expression and purification of Escherichia coli RNA polymerase

R Baskaran Janaani Sri

Integrated M.Tech, Department of Biotechnology and Genetic Engineering, Bharathidasan University, Tiruchirapalli, 620024, Tamil Nadu

Dr. B. Anand

Associate Professor, Department of Bioscience and Bioengineering, Indian Institute of Technology, Guwahati, Assam 781039

Abstract

Escherichia coli is a gram negative, facultative anaerobic, non-sporulating and rod–shaped bacterium. E. coli is the most widely studied and best understood prokaryotic model organism. Due to its rapid growth rate and ability to express proteins at very high levels, E. coli is used as a workhorse of recombinant protein production. Purification of bacterial RNA Polymerase (RNAP) was carried out as it is useful for structural and biochemical studies. E. coli RNA Polymerase is a complex enzyme made up of multiple subunits such as α, β, σ, ω & β’. RNAP is the key enzyme which is responsible for transcription in prokaryotes. The planned work involves over-expressing and purifying the bacterial RNAP using E. coli BL21(DE3) cells. In this study Plasmid A encodes for RNAP core subunit and has ampicilin resistance. The transcriptional machinery gets activated only when the σ factor binds to the promoter region. So a second expression vector Plasmid B encoding for the σ70 subunit is used to express it and has kanamycin resistance. The recombinant proteins were produced from vector A and B and were purified using an affinity tag (Histidine-tag) at the C-terminus and N-terminus, respectively. The study involves over-expression of RNAP core complex and σ70 subunit and purifying them using Immobilized Metal Ion affinity chromatography. Upon purification to homogeneity, an in vitro transcription system to synthesize RNA was also designed to check the activity of the purified RNAP enzyme.

Keywords: expression, RNA polymerase, recombinant protein, σ70 subunit, In vitro transcription, affinity chromatography.

Abbreviations

Abbrevations
RNA Ribonucleic acid
RNAP RNA Polymerase
DNA Deoxyribonucleic acid
EDTA Ethylenediaminetetraacetic acid
SDS Sodium dodecyl sulfate
ssDNA Single-stranded DNA
dsDNA Double-stranded DNA
LB Luria Bertani
TAE buffer Tris- acetate- EDTA buffer
Kb kilobase
IPTG Isopropyl β-D-1-thiogalactopyranoside
OD Optical density
β-ME B- mercaptoethanol
DTT Dithiothreitol
TEMED Tetramethylethylenediaminne
APS Ammonium persulfate
TG buffer Tris-glycine
KDa Kilodalton
IDA Iminodiacetic acid
IMAC Immobilized Metal Affinity Chromatography
FPLC Fast protein liquid chromatography
BSA Bovine serum albumin
NTP Nucleoside triphosphate
ATP Adenosine triphoshate
GTP Guanosine triphosphate
UTP Uridine triphosphate
CTP Cytidine triphosphate
PAGE Polyacrylamide gel electrophoresis
HAC Heparin affinity chromatography
IEC Ion- exchange chromatography
NaCl Sodium chloride
NaOH Sodium hydroxide
CaCl2 Calcium chloride
MgCl2 Magnesium chloride
HCl Hydrochloric acid
PMSF Phenylmethylsulfonyl fluoride.
KCl Potassium chloride
UV Ultraviolet
CV Column volume
EtBr Ethidium bromide
MPa Megapascal Pressure
mA milliampere
μg microgram
mg milligram
ml milliliter
min minutes
ºC Degree centigrade
PCR Polymerase chain reaction
V Volt
M Molar
mM millimolar
nm Nanogram
et.al., Co-workers

INTRODUCTION

Transcription is the fundamental step in gene expression. It is the process of synthesis of mRNA from DNA. The process of transcription is mediated by the enzyme RNAP in both prokaryotes and eukaryotes. In bacteria, the multi-subunit RNAP transcribes all genes. The bacterial RNAP core enzyme is capable of catalyzing the polymerization of NTPs into RNA but it does not bind to specific sequence of DNA. It requires a σ- subunit which recognizes promoters and initiate transcription. Escherichia coli is the most extensively used organism by researchers in the field of molecular biology to investigate different biological processes. E.coli RNAP is a hetero multimeric enzyme and has two α -subunit, one β, β’, and ω- subunits. It has a molecular weight of about 440kDa. In this work, we have used plasmids one of which contain genes that encodes core complex and other contains genes that encodes σ70 subunit.

At present, researchers utilize T7 RNA Polymerase for synthesis of RNA by in vitro transcription and this RNA transcript is used for structural and translational studies. T7 RNA Polymerase is a single-subunit and very active enzyme. It can synthesize RNA much faster than E.coli RNAP does. It performs all the functions required for transcription without the need of auxiliary protein factors. However, there are some demerits which limit its usage. Due to its rapid polymerization activity, T7 RNA Polymerase lowers RNA folding during mRNA in vitro transcription. Unlike T7 RNA Polymerase, E. coli RNAP is a less active enzyme which in turn allows proper RNA folding. Hence, E. coli RNAP is more suitable for structural characterization of RNA.

Our intension here is to purify the E. coli RNAP core complex by performing various chromatographic techniques. Upon purification, we have planned to check the RNA activity by in vitro transcription.

Strains and Plasmid

E. coli BL21(DE3)

It is one of the most popularly used expression strain as it is deficient in Lon protease (cytoplasm) and ompT protease (outer membrane protease). This reduces proteolysis of expressed protein upon induction by IPTG. It contains λ DE3 lysogen that carries gene for T7 RNAP under the control of lacUV5 promoter.

Plasmid A

Plasmid A is a polycistronic vector which is used for expression of E. coli RNAP core. The vector consist of rpoA, rpoB, rpoC and rpoZ that codes for the subunits: α, β, β’, and ω respectively ( Fig 1).All rpo genes are expressed under T7 promoter with pET vector as its backbone. The affinity tag i.e., His-tag is attached to the C-terminal of the rpoC gene. The vector has Ampicillin reistance.

P A_1.png
    Fig.1 Schematic representation of Plasmid A

    Plasmid B

    Plasmid B is a monocistronic vector that contains the rpoD gene which codes for the initiation factor σ70 under T7 promoter. The N-terminal of rpoD gene is His6 – tagged ( Fig 2). It has pET28b as its vector backbone and has Kanamycin resistant.

    p B_1.png
      Fig.2 Schematic representation of Plasmid B

      E. coli RNAP subunits

      Table 1. Properties of the subunits of E. coli RNAP
      SUBUNIT GENE ENCODED MOLECULAR WEIGHT(KDa)
      α rpoA 36.5
      β rpoB 151
      β’ rpoC 155
      ω rpoZ 10.1
      σ rpoD 70.2

      OBJECTIVES

      • To over-express Escherichia coli RNAP
      • To acquire highly purified fractions of E. coli RNAP for further structural and functional studies.
      • To examine RNA Polymerase activity by in vitro transcription.

      Scope

      •   Mechanistic analysis of transcriptional machinery.
      •   Structural analysis of bacterial transcription.
      • Development of antibiotics to treat infections and particularly to treat infections caused by Gram-negative pathogens.
      •  In vitro translational studies.

      LITERATURE REVIEW

      The present review incorporates a concise report on the research work carried out earlier on the expression and purification of E. coli RNAP and RNA synthesis by in vitro transcription.

      The E.coli DNA-dependent RNAP core enzyme is capable of transcription, elongation and termination (Maxim V. sukhodolets and Susan Garges,2003). The core RNAP of E. coli initiates transcription when it interacts with the σ factor which contains amino acid residues recognizing 10 to -35 promoter elements (Dombroski.et.al.,). Initially, Thermus aquaticus RNAP became structural model of choice after the discovery of its structure by Darst lab (1999). However, the regulatory stratergies and auxiliary factors most probably binds to distinct sites and lacks many accessory proteins which are characterized in E. coli (Artsimovitch.et.al.,). By obtaining the structure of E. coli RNAP, both functional and structural studies are carried out on same model system (Murkami.et.al, Steitz.et.al, Darst.et.al) (Fig.3).

      This became possible by purifying a recombinant RNAP core in vivo from co-expressed subunits. The multicistronic vector contained rpoA, rpoB, rpoC genes expressed from T7promoter and his tag placed at C-terminal of β’ subunit (Artsimovitch I, Svetlov V, Murakami KS et.al.) for purification. Later it became clear that ω is an important factor for RNAP regulation by ppGpp (Ross W et.al). So, the rpoZ gene was added to the rpoABC cassette, the construct used by Steitz.et.al, Darst.et.al. In our work, over-expression and purification of recombinant RNAP was carried out using the similar to construct.

      The recombinant protein expression at low temperatures results in several advantages such as, increase in solubility of aggregation-prone recombinant protein and decrease in degradation by heat shock proteases (JESS A. VASINA et.al). Hence we optimized the temperature conditions to over-express the recombinant RNAP. The purified E. coli RNAP core was obtained by performing Ni-NTA affinity chromatography, Heparin affinity chromatography, Ion Exchange Chromatography techniques (Vladimir Svetlov and Irina Artsimovitch). For obtaining high level of purity, we also tried to purify it using Gel-filtration chromatography. The purified E. coli RNAP was used for in vitro transcription to check RNA synthesis activity by following protocols suggested by Ju-Sim Kim.et.al, Anna Maciag.et.al.

      ecoli_1.jpg
        Fig.3 Three- dimensional  structure of  E. coli RNAP σ70 holoenzyme(Murakami.et.al.,2013)

        PLASMID ISOLATION

        Principle

        Plasmid is a double stranded circular DNA molecules. The size of plasmid ranges from 1 kbp to 1000kbp. The term “Plasmid” was coined by a molecular biologist Joshua Lederberg in 1592. Plasmid isolation and purification is an essential step in cloning, protein expression, DNA sequencing, etc.

        Alkaline lysis method is used to isolate plasmid DNA from bacterial cell suspension by rupturing the cell wall. First the bacteria containing plasmid of interest is resuspended in a resuspension buffer that contains EDTA and RNase A. EDTA helps in chelating divalent cations (Mg2+,Ca2+) in the solution preventing DNases from damaging plasmid and RNase A degrades the cellular RNA. It is followed by lysis of cells by adding an alkaline lysis buffer that consist of SDS and sodium hydroxide. SDS is an anionic detergent that solubilize the cell membrane and the alkali denatures the dsDNA to ssDNA. Then the neutralization buffer is added which contains potassium acetate that neutralizes the pH. The plasmid DNA being smaller in size renatures back dsDNA form bus genomic DNA because of its complexity cannot renature back to its original form, getting aggregated and forming insoluble fractions. Now, the precipitated protein, genomic DNA, cell debris are pelleted by centrifugation and the supernatant is loaded onto a column. Contaminants are removed by washing with ethanol and the pure plasmid DNA is finally eluted with the help of elution buffer.

        Materials Required

        Sterile micropipettes, eppendorf tubes, centrifuge, cells containing plamids (plasmid A and plasmid B in E. coli DH5alpha), silica beads, vortex, tips.

        BUFFERS:

        Resuspension buffer (P1 buffer): 50mM Tris-Hcl(pH 8.0), 10mM EDTA, 100μg/ml RNase.

        Lysis buffer (P2 buffer): 200mM NaOH, 1% SDS.

        Neutralizing buffer (P3 buffer): 4.2 M Guanidium Hydrochloride, 0.9 M Potassium acetate (PH 4.8)

        Wash buffer: 10mMTris-HCl (pH 7.5), 80% ethanol

        Procedure       

        •  DH5alpha cells containing Plasmids A and B were procured from Addgene.
        • LB broth was prepared by mixing 25g of LB powder in 1000ml of Milli-Q water
        • Two test tubes were taken and named as A and B respectively. In each tube 5ml of LB broth was taken.
        • 5 μl of Ampicillin was added to test tube A and 2.5 μl of kanamycin was added to test tube B.
        • Cells containing plasmid A and plasmid B were inoculated in LB media that contains Ampicillin and Kanamycin respectively.
        • Cells were incubated overnight at 180rpm in 37ºC.
        • 150 μl of primary inoculum (1%) was inoculated in 15ml LB agar media containing the respective plasmids.
        • Cells were inoculated at 37ºC4-5 hours till OD reaches 0.6.
        • Cell culture was centrifuged at 13000 rpm for 2 min in 25ºC.
        • The pellet was resuspended by adding 250 μl of ice cold resuspension buffer.
        • 250 μl of lysis buffer was added and swirled.
        • Gently,350 μl of neutralizing buffer was added and mixed well.
        • The solution was centrifuged at 13000rpm for 10 min in 25ºC.
        • Then 750μl of wash buffer was added.
        • The column was centrifuged at 13000rpm for 2 min in 25ºC.
        • Silica column was transferred to 1.5ml vial and 60μl of elution buffer was added.
        • It was kept for 1 min and the column was centrifuged at 13000rpm for 2 min in 25ºC.
        • The isolated plasmid were then stored in -20ºC.

        Result

        Plasmid A and Plasmid B were successfully isolated using alkaline lysis method which was confirmed by agarose gel electrophoresis.

        PLASMID QUALITY CHECK BY AGAROSE GEL ELECTROPHORESIS

        Principle

        Agarose gel electrophoresis is a widely used laboratory technique to separate nucleic acids based on their size under the influence of an applied electric field. The negatively charged DNA molecules migrates towards the positively charged anode.

        Agarose is a polysaccharide extracted from the red algae Gracilaria and Geledium and consist of repeated agarbiose (L- and D- galactose) subunits. It is only soluble in water on boiling. After cooling, it undergoes hydrogen bonding between adjacent molecules and creates a three-dimensional matrix with pores. The size of the pores varies as the concentration of agarose in the gel varies. The pore acts as a sieve through which the DNA migrates. The sieving properties of the gel influences the rate at which a molecule migrates. Smaller molecules move through the pores more easily than larger ones.

        The size of unknown DNA bands are determined by comparing them to a standard DNA Marker or ladder. Electrophoresis buffer facilitates the liquid medium for the migration of DNA into gel. The most commonly used buffer TAE buffer. It has better conductivity which helps the double-stranded DNA to migrate faster. Ethidium bromide is the most commonly used fluorescent dye to stain DNA. It stains the major grooves of DNA which imparts fluorescence in the presence of UV rays.

        Materials Required

        Agarose, TAE Buffer, Sample Loading Buffer, DNA ladder standard, Electrophoresis chamber, Power supply, Gel casting tray and combs, EtBr, Pipette and tips, casting tray

        TAE buffer: A 50X stock solution can be prepared by dissolving 242g Tris base in water, adding 57.1 mL glacial acetic acid, and 100mL of 500mM EDTA (pH 8.0) solution, and bringing the final volume up to 1 liter.

        Gel loading buffer: Sucrose & xylene cyanol / bromophenol blue (6x)

        Procedure

         Preparing the gel

        • O.4 g of agarose powder was weighed and mixed in 50 ml of dH2O (0.8% gel).
        • 1 ml of TAE buffer was added and the solution was heated in a microwave oven till the solution becomes clear.
        • Then the solution was allowed to cool down by swrilling the flask occasionally.
        • 2 μl of Ethidium bromide was added to the solution.
        • The combs were fixed in the casting tray.
        • Then the solution was poured into the casting tray and allowed to cool down until it solidifies.
        • They were then removed carefully and placed inside the chamber which contains 1% TAE buffer.

        Loading and running the gel

        • 2 μl of sample and 1μl of gel loading dye were mixed and loaded into each wells.
        • Then the electrodes were connected to the power supply and ran the gel at 80V.
        • When the blue dye approaches the end,the gel run is turned off and the gel is view in Gel Dock.

        Observation

        plsm check.png
          Fig.4 Plasmid quality check for Plasmid A and Plasmid B

          Result

          Plasmid quality was checked in agarose gel electrophoresis. The bands were compared with the DNA ladder. Plasmid A had a molecular weight of about 15.2Kb and Plasmid B had a molecular weight of about 7.1Kb respectively.

          COMPETENT CELL PREPARATION AND TRANSFORMATION USING BL21 (DE3) CELLS

          Principle

          Competence is the property of the cell to take up exogenous DNA. Most types of cells cannot take up DNA efficiently unless they have been exposed to physical or chemical treatments to make them competent. Once the bacterial cell becomes competent, the foreign genetic material is transferred into them by a process known as ‘Transformation’. Electroporation and CaCl2 methods are commonly employed for competent cell preparation. Here, we followed CaCl2 method in which bacterial cells are treated with divalent cations like Ca2+ and Mg2+. It causes negativity charged DNA to bind to the cell wall and Heat shock results in the development of transient pores on the surface through which DNA can enter which closes soon on cooling in ice.

          Materials Required

          LB broth, test tubes, ice cold solution I (0.08M Mgcl2 + 0.02M CaCl2), ice cold solution II (0.1M CaCl2), antibiotics (Ampicillin, Kanamycin), LB agar-Ampicillin and Kanamycin plates, centrifuge, water bath, falcon tube, micro pipettes and tips, microcentrifuge tubes, shaking incubator, culture plates.

          Procedure

          • 150μl of E. coli BL21(DE3) cells were inoculated into 15ml LB broth.
          • The cells were incubated at 37ºC for 45 min till OD600 reaches 0.3.
          • The media was transferred into a falcon tube taken in ice.
          • The solution was centrifuged at 2710g for 10 min in 4ºC.
          • The supernatant was discarded and 9 ml of ice cold solution I was added.
          • The pellet was resuspended by swirling in ice.
          • Again the solution was centrifuged at 2710g for 10 min in 4ºC.
          • The supernatant was discarded and 750μl of ice cold solution II was added.
          • Then the pellet was resuspended by swirling in ice.
          • 250μl of the competent cells was taken in 1.5 ml vial and 2μl of plasmid A and plasmid B was added in respective vials.The third vial was used as a negative control(no plasmid was added).
          • The cells were incubated in ice for 30 mins.
          • Then heat shock was given to cells at 42ºC for 1 min.
          • Immediately the cells were cooled in ice for 2 min.
          • Then to each vial 800μl of fresh LB broth was added and incubated at 37ºC for 45 min.
          • After that, the cells were centrifuged at 13000g for 1 min in 25ºC.
          • The pellet was resuspended in the medium and were spread on the surface of LB agar antibiotic plates{Plasmid A to Ampicillin plate and Plasmid B to Kanamycin plate}.It was spread evenly using 3-4 silica beads.
          • The plates were then incubated at 37ºC for 10-2 hours.

          Observation

          ac_2.png
            Fig 5. Antibiotic plates with colonies of a) Plasmid A, b) Plasmid B. No colony in c) negative

            No colonies were observed in negative plates whereas numerous colonies were observed in the positive plates thereby consolidating the fact that transformation was efficient.

            Result

            The colonies in the positive plates would be further used for preparing primary and secondary culture to check expression.

            PREPARATION OF PRIMARY CULTURE

            Procedure

            • Two 5 ml LB broth for primary culture of cells containing plasmid A and plasmid B was prepared.
            • 5μl Ampicillin and 2.5μl Kanamycin was added to first and second test tubes espectively.
            • A single colony was picked from the agar plate using a pipette tip and transferred it to respective LB broth.
            • Then the cell cultures were incubated at 180 rpm in 37ºC till OD600 reaches 0.6.

            OVER-EXPRESSION OF PROTEINS AT DIFFERENT TEMPERATURES

            Materials Required

            LB broth, antibiotics (Ampicillin and Kanamycin), IPTG, incubator, centrifuge, test tubes, cotton balls, micro pipettes and tips.

            Procedure

            •  60 ml of LB media was prepared and 5 ml was aliquoted into 12 test tubes( 6 test tubes for each plasmid).
            • The test tubes were marked as induced and uninduced with different temperatures.
            • 5 μl of Ampicillin and 2.5 μl of Kanamycin was added with respect to the plasmid in the test tube.
            • Then, 50 μl of primary culture (i.e., Plasmid A and Plasmid B) was added to the respective test tubes.
            • The cell cultures were incubated at 180 rpm in 37ºC till OD600 reaches 0.6.
            • After OD reaches 0.6, 1μl of IPTG was added to all test tubes which is marked as ‘induced’.
            • Then both induced and uninduced cell cultures were incubated at 180 rpm in various temperatures (16ºC, 25ºC, 30ºC).
            • After incubation, the cell cultures were transferred to a 2 ml microcentrifuge tubes and centrifuge at 13000g for 2 min.
            • The above steps were repeated for other two temperatures.
            • Then the supernatant was discarded.
            • The pellets were resuspended in 1ml of pellet washing buffer
            • Cells were centrifuged at 13000g for 2 min in 4ºC.
            • Finally, supernatant was discarded and stored in -20ºC.

            SONICATION AND CENTRIFUGATION

            Principle

            Sonication is one of the non-mechanical method which is employed for cell lysis by applying high frequency ultrasonic waves (> 20 kHz) into the cell suspension. During sonication, the ultrasound waves passes through the fluid and creates alternative high pressure (compression) and low pressure (decompression) respectively. It creates bubbles and these bubbles gets collapsed into the solution by a process known as cavitation. The intracellular components are then collected by the process centrifugation. In order to prevent over-heating, the cell suspension is always kept inside ice box during and after sonication.

            Materials Required

            Sonicator, PMSF, centrifuge, ethanol, cell lysis buffer (50 mMTris-HCl pH (6.9), 500 mM NaCl, 5% glycerol).

            Procedure

            •  500 μl of cell lysis buffer was added to each cell pellet and resuspended.
            • Then, 5μl of PMSF was added to each vial.
            • The cells were lysed at 33% amplitude (3 seconds on and 15 seconds off) for 15 cycles.
            • After sonication, the lysate was centrifuged at 16000g for 45 min at 4ºC.
            • Then, the supernatant and pellet were collected separately and stored at 4ºC.

            PROTEIN EXPRESSION CHECK IN GRADIENT SDS PAGE (8%-18%)

            Principle

            PAGE is a technique used to separate the proteins according to their molecular masses. The polyacrylamide gel mainly consists of acrylamide and bisacrylamide that forms a molecular sieve by their copolymerization activity. Acrylamide forms a linear polymer, which is cross-linked by bis-acrylamide unit that forms a continuous network. The pore size of the gel depends on the ratio of acrylamide and bisacrylamide and the concentration of acrylamide taken.

            A reducing agent DTT or β-ME breaks down the disulfide bridges that are responsible for protein folding and the detergent such as SDS imparts negative charge to the proteins thereby linearizing them into polypeptides. Tris-HCl maintains a constant pH. TEMED and APS induce the polymerization process by free radical mechanism. TEMED reacts with APS, splitting APS into sulfate free radicals which then initiates polymerization acrylamide and bis-acrylamide. The mobility of the charged proteins depends on the charge and mass.

            Stacking gel interactions

            Stacking which has low acrylamide concentration and low pH (6.8) helps in the migration of proteins regardless of its size. When voltage is applied, glycine molecules and cl- ions move through the gel towards the positive electrode. Glycine exist in the form of zwitterion and cl- ions move faster than glycine. Between the glycine and cl- ions exist all the proteins from the mixture.

            Resolving gel interactions

            On reaching the resolving gel, pH increases and pore size decreases. At pH 8.8 glycine becomes negatively charged and run ahead of proteins. So, both glycine and cl- ions run out quickly leaving the protein molecules all free for the separation on the basis of their mass to charge ratio.

            Gradient gel

            Gradient PAGE is composed of gradient resolving gel and stacking gel. The gradient of the resolving gel ranges from 8 to 18 % of acrylamide concentration from top to bottom. Polyacrylamide gradient gels sharpen the protein bands and allow complex mixtures of proteins to be separated on a single gel. It allows protein wih close molecular weight values to separate in a gradient gel than a linear gel.

            The apparatus consist of two reservoirs in which one contain 8% and the other 18% acrylamide solutions. Two reservoir are interconnected by a valve which opens to the gradient castor through a silicon tubing. Gradient is developed by mixing the two solutions at a constant speed such that gradient decreases from bottom to top. 1 μl of bromophenol blue can be added to 18% acrylamide solution to indicate gradient.

            Materials Required

            SDS loading dye, PCR tubes, micro-pipettes and tips, combs, glass plates (short plate and top plate), multicasting chamber, protein ladder, supernatant and pellets, acrylamide solutions, isopropanol, TG buffer, APS, TEMED, Tricholoroethanol, distilled water, Tris-HCl, 10% SDS.

             Resolving gel

            Table.2 Composition of Resolving gel
              Components   Volume  
            8% gel for 19 ml (ml) 18% gel for 18ml (ml)
            30% Acrylamide 5.06 (50%Acrylamide) 6.48
            10%SDS 4.75 4.5
            Tris – HCl 0.19 0.18
            5X TG buffer 3.8 3.6
            Trichloroethanol 0.095 0.09
            APS 0.19 0.18
            TEMED 0.01 0.01
            Water 4.905 2.96

            Stacking gel

            Table 3. Composition of stacking gel
              Components     Volume (20 ml)
            30% Acrylamide 3.32
            Tris-HCl (pH 6.8) 2.52
            10% SDS 0.2
            5X TG buffer 4
            APS 0.25
            TEMED 0.025
            Water 8.64

            Procedure

            Gel casting

            • Resolving gel was poured between two glass plates (one short plate and the other tall plate).
            • Bubbles are removed by adding isopropanol on the top of the gel and allowed to solidify.
            • After solidification, isopropanol was removed by tilting the plate.
            • Then, stacking was prepared and poured on the top of the glass plates.
            • Comb was placed above the stacking gel and allowed to solidify
            • Once it is solidified, comb was removed from the plate.

             Loading and running the gel

            • 5 μl of SDS loading dye was mixed with 20 μl of the sample.
            • Samples were heated at 95ºC for 15 min in PCR.
            • Then, 20 μl of each sample was loaded into the respective wells.
            • 8 μl of protein ladder was loaded into one well as a control.
            • Then the gel was run at 100 volts for 1:30 hrs

            Observation

            pv10-exp_3.png
              Fig 6.  Protein expression check of Plasmid A at diiferent temperatures vizualized in gradient SDS PAGE.
              pia-exp_4.png
                Fig 7. Protein expression check of Plasmid B at different temperatures vizualized in gradient SDS PAGE.

                Result

                Over – expression of E. coli RNAP was observed in gradient SDS PAGE setup. The bands were compared with the protein ladder and had a molecular weights of 150KDa, 15KDa, 36KDa, 10KDa. In the documented picture given below we see the bands of proteins which appeared only in induced samples at different temperatures i.e., 16ºC, 25ºC, 30ºC. No bands appeared in the uninduced samples. Hence, Plasmid A (RNAP core complex) was over- expressed at 16ºC and Plasmid B was over- expressed at 25ºC.

                PURIFICATION OF HISTIDINE TAGGED RNAP CORE COMPLEX BY Ni AFFINTIY CHROMATOGRAPHY

                Principle

                Immobilized metal affinity chromatography is a technique used to separate particles based on interactions between transition metal ions immobilized on a matrix and specific amino acid side chains. Iminodiacetic acid (IDA) is used as a matrix to chelate transition metals through three coordination sites. The most common ion for His-tag purification of recombinant protein is Ni2+. His-tag has high affinity for these metal ion and binds strongly to the IMAC column as the electron donor groups of histidine imidazole ring forms coordinate bonds with immobilized transition metal. Low concentration of imidazole is added to both binding and wash buffer to elute the wealy bounded proteins. Then high concentration of imidazole is added to the elution buffer to elute the His-tagged proteins Later the column is preserved by passing ethanol through it.

                Materials Required

                His-Trap IMAC 5ml column, LB broth, antibiotics (Ampicillin, Kanamycin), PMSF, incubator (16ºC, 25ºC), sonicator, centrifuge, 1.5 ml vials, inoculum, AKTA FPLC system.

                Reagents: 

                1.  Stripping buffer: 20mM Sodium phosphate, 0.5M NaCl, 50Mm EDTA, pH 7.4

                2.  Lysis buffer: 50mM Tris-HCl, pH 7, 500mM NaCl, 5% glycerol.

                3.  Binding buffer: 50mM Tris-HCl, pH 7, 500mM NaCl, 5% glycerol.

                4.  Elution buffer: 50mM Tris-HCl, pH 7, 500mM NaCl, 5%glycerol, 250mM Imidazole.

                Procedure

                Regeneration of His-Trap IMAC column

                • The column was washed with water at 2 ml/min for 15 mins (3-4 CV).
                • Then the column was washed with stripping buffer at 1 ml/min for 4CV.
                • 1M NaCl was passed for 5CV at 1 ml/min.
                • Then again the column was washed with water for 3-4CV at 2 ml/min.
                • Final, 20% ethanol was passed through the column for 4 CV at 1 ml/min and stored at 4ºC.

                 Charging of the column with Ni2+

                • The column was washed with water for 3-4 CV at 1 ml/min.
                • 0.1M Nickel sulphate was passed through the column at 1 ml/min for 3-4 CV.
                • Again washed with water for 3-4CV at 1 ml/min.
                • Then Ni binding buffer was passed through the column at 1ml/min for 4CV.

                 Binding of protein to the Ni-NTA column

                • The supernatant fractions(collected after sonication) was passed through the column for 3-4 hours at 1 ml/min in 4ºC.

                 Elution of protein by FPLC

                FPLC is a modern chromatographic technique used to purify proteins that helps to obtain different fractions of proteins by developing gradients of elution buffer passing through it. It consist of buffer mixer, sample inject valve, UV detector, restrictor, pH meter, fraction collector, etc.. The buffer mixer helps to mix the buffers to develop gradient during elution of proteins. The detectors measures the UV absorbance of eluate which helps us to collect the protein fractions. The restrictor maintains a constant column pressure. Finally through the outlet, relevant protein fractions can be collected. Here, we applied a gradient of 0 to 250mM imidazole elution buffer through the IMAC column for 60 minutes. Protein fractions are collected in 2ml vials by analyzing the UV absorbance at 280nm.

                Gradient SDS Page

                • After elution, the supernatant, flowthrough, pellet dissolved in lysis buffer and all eluted fractions (each 20 μl ) along with the protein ladder were loaded into each well.
                • Then the gel run at 100 V.

                Observation

                ninta-1 part_1.png
                  ninta2 part_1.png
                    Fig 8. Fractions of Ni- affinity chromatography after SDS PAGE.
                    ni nta_2.png
                       A chromatogram showing volume versus Absorbance at 280nm plot obtained after Ni- affinity chromatography.

                      Result

                      Ni-NTA column was used for purifying His-tagged RNAP. After running the fractions on a gradient SDS PAGE, 150KDa, 155 KDa, 36 KDa, 10 KDa from18th fraction to 25th fraction confirmed the presence of purified His-tagged RNAP core complex. This fractions were collected separately and used in further chromatographic techniques to obtain high purity.

                      BUFFER EXCHANGE

                       Principle

                      Buffer exchange refers to the replacement of one set of buffer salts with another set. It is performed by first equilibriating the column resin with the buffer the sample should end up in. The buffer constituents carrying the sample into the column will be replaced by the solution with which the colmn is pre-equilibriated. Here, we equilibriated our desalting column with 75mM NaCl buffer. From the above obtained results, we collected 18 – 25 th fractions (around 15 ml ) and stored in a centrifuge tube. To that 6.3 μl of β-ME was added and passed into the desalting column during the process.

                      Materials Required 

                      Desalting column, protein sample (collected after Ni- Affinity chromatography), centrifuge tube, water, ethanol, syringe, AKTA FPLC system.

                       Buffer A: 50mM Tris-HCl (pH 6.9), 0.5mM EDTA, 5% glycerol, 6mM β-ME.

                       Buffer B: 50mM Tris-HCl (pH 6.9), 1.5M NaCl, 0.5mM EDTA, 6mMβ-ME, 5%glycerol.

                      Procedure 

                      • First the system pumps (pump A and pump B) was washed with water at a flow rate of 5 ml/min.
                      • Then water was passed through the column at a flow rate of 5 ml/min and system alarm was set as 0.15 MPa.
                      • After that the system pumps was washed with buffer A and buffer B by passing it through tube A and tube B respectively to pass 2 CV of buffer containing 75mM NaCl.
                      • 7 ml of sample was injected into the column using a syringe at a flow rate of 5 ml/min.
                      • Then buffer exchanged fractions was collected by observing a steep increase in absorbance at 280nm. 

                      HEPARIN AFFINITY CHROMATOGRAPHY

                      Principle

                      The heparin affinity chromatography is a very effective and simple method to purify a wide range of proteins. It has a high purification potential and its columns are compatible with oxidizing agents, reducing agents and chelators. The heparin affinity chromatography is not dependent on an affinity-tag in contrast to other affinity chromatography such as IMAC. Heparin is a negatively charged polydispersed linear polysaccharide which have the ability to bind with wide range of biomolecules such as enzymes, serine protease inhibitors, growth factors, DNA modifying enzymes and extracellular matrix proteins. (Xiong et.al., 2009).

                       Here, Our RNAP is nucleic acid binding protein that can reversibly absorb over heparin which helps in further purification. It can be eluted by passing buffer having very high salt concentration.

                      Materials Required 

                      HiTrapTM Heparin HP(5 ml) column, 2 ml eppondrof tubes, peristaltic pump, water, AKTA FPLC system.

                       Buffer A: 50mM Tris-HCl (pH 6.9), 0.5mM EDTA, 6mM β-ME, 5% glycerol, 75mM NaCl

                       Buffer B: 50mM Tris-HCl (pH 6.9), 0.5mM EDTA, 6mM β-ME, 1.5M NaCl, 5% glycerol

                      Procedure

                       Regenerating HiTrap Heparin HP column

                      • The column was washed with water for 3-4 CV at 2 ml/min for 15 min.
                      • Then buffer B was passed through the column at 1 ml/min for 20 min (4 CV).
                      • Again water was passed at 2 ml/min for 15 min (3-4CV).
                      • Then buffer A was passed through the column at 1 ml/min for 30 mins (4 CV).

                      Binding of RNAP core complex

                      • The RNAP core complex solution (obtained after buffer exchange) was passed through the column at a flow rate of 1 ml/min for 4 hours.
                      • Then buffer A was passed at 1 ml/min for 30 min.

                       Elution of RNAP core complex by FPLC

                      • First, the system pumps were washed with buffer A and buffer B at 5 ml/min.
                      • Then the heparin column was connected to the system.
                      • A gradient of 75mM NaCl to 1.5M NaCl was applied to it for 200 min at a flow rate of 1 ml/min.
                      • Finally, protein fractions are collected by analyzing the UV absorbance at 280nm.

                       Gradient SDS gel

                      • After elution, all the eluted fractions, protein sample collected after buffer exchange, flowthrough and protein ladder were loaded in the respective wells.
                      • The gel was run at 100 V.

                      Observation

                      hep_1.png
                        heparin affinty chtomatogrphy.png
                          Fig 9. Fractions of Heparin- affinity chromatography after SDS PAGE.
                          hep-1_1.png
                            A chromatogram showing volume versus Absorbance at 280nm,after HAC

                            Result

                            The RNAP core complex were observed from 21st to 25th fractions. The bands were compared with the protein ladder and found around 155KDa, 150KDa, 36KDa. This purified fractions was further used for buffer exchange and other purification techniques.

                            BUFFER EXCHANGE

                            • From the above results, 21-25 fractions were collected separately in a centrifuge tube and stored at 4ºC. The above mentioned procedure was carried out again with buffer A and buffer B respectively.
                            • The fraction was then collected by analyzing the UV absorbance at 280nm.  

                            ION- EXCHANGE CHROMATOGRAPHY

                            Principle 

                            Ion exchange chromatography separates molecules based on their respective charged groups. There are two types of ion exchanger, namely cation and anion exchangers. Cation exchangers possess negatively charged groups and these will attract positively charged groups and will attract positively charged cations. Anion exchangers have positively charged groups that will attract negatively charged anions.In a buffer with a pH greater than the pI of the protein of interest, the protein will carry a net negative charge. Here, our RNAP is a negatively charged protein so we used an anion exchanger to separate this protein. Anion exchange resins are regenerated by treatment with NaOH, then washing with water. The charged salt ions compete with bound proteins for the charged resin functional groups. Proteins with few charged groups will elute at low concentrations, whereas proteins with more charged groups will have greater retention time and elute at high salt concentrations.

                            Materials Required 

                            HiTrapTM Q HP column, protein fractions (collected after buffer exchange), water, AKTA FPLC system, buffer A (50mM Tris- HCl (pH 6.9), 75mM NaCl, 0.5mM EDTA, 5% glycerol, 6mM β-ME), buffer B (50mM Tris-HCl, 1.5M NaCl, 0.5mM EDTA, 5%glycerol, 6mM β-ME), microcentrifuge tubes.

                            Procedure 

                            Regenerating HiTrapTM Q HP column

                            • First water was passed through the Q sepharose column at 2 ml/min for 15 min.
                            • Then buffer B was passed through the column at 1ml/min for 20 min.
                            • After that, buffer A was passed to equilibrate the column at a flow rate of 1 ml/min for 30 min.

                            Binding of protein to the Q sepharose column

                            • The RNAP core complex solution (obtained after buffer exchange) was passed through the column at a flow rate of 1 ml/min for 4 hours.
                            • Then the column was washed with buffer A for 30 min at 1ml/min.

                            Elution of RNAP core complex

                            • First, the system pumps were washed with buffer A and buffer B at 5 ml/min .
                            • Then the Q sepharose column was fixed with the system.
                            • A gradient of 75mM NaCl to 1.5mM NaCl was applied to it for 200 min at a flow rate of 1 ml/min.
                            • Finally, protein fractions are collected by analyzing the UV absorbance at 280nm.

                             Gradient SDS gel

                            • After elution, all the eluted fractions, protein sample collected after buffer exchange, flowthrough and protein ladder were loaded in the respective wells.
                            • The gel was run at 100 V.

                             Staining SDS gel

                            • The gel was incubated with water for 5min.
                            • Then water was released and Coomassie Brilliant Blue G250 dye solution was added to it.
                            • Then the gel was heated for 10 min and then the dye was removed.
                            • The gel was then transferred to water and heated several times till the bands become visible.
                            • Finally, the gel was viewed in BIO-RAD chemi-doc.

                            Observation

                            iec_1.png
                              Fig 10. Fractions of IEC after SDS PAGE.
                              iec after staimimg_1.png
                                Fig 11. Fractions of IEC visualized after staining with coomassie Brilliant Blue dye.

                                iex-i_1.png
                                   A chromatogram showing volume versus Absorbance at 280nm,after IEC.

                                  Result

                                  Purified RNAP core complex were observed from 7th fraction to 10th fraction. Then these fractions were further used to obtain highly purified protein via gel filtration chromatography.

                                  GEL FILTRATION CHROMATOGRAPHY

                                  Principle

                                  Gel filtration or size exclusion chromatography separates molecules based on their size by filtration through a gel. The gel consist of spherical beads containing pores of a specific size distribution. Molecules that are too large to enter pores stay in the mobile phase and move through the column with the flow of the buffer. Smaller molecules that are able to move into pores enter the stationary phase and move through the column by a longer path through the pores of the beads. Here, we have used superdexTM 200pg column as our size of protein is very large (~440KDa). Superdex is a composite of cross-linked agarose and dextran. It gives high resolutions with short run times and good recovery.

                                  Materials Required

                                  SuperdexTM 200pg column, buffer A (50mM Tris-HCl (pH 6.9), 75mMNaCl, 0.5mM EDTA, 5% glycerol, 6mM β-ME), water, AKTA FPLC system, protein sample (collected after buffer exchange), microcentrifuge tubes.

                                  Procedure

                                  • The column was equilibrated with buffer A at a flow rate of 1.5 ml/min for 2 hours in manual load.
                                  • After that the column washed for 15 min in inject mode also.
                                  • Then the protein sample (fractions collected after Ion exchange chromatography) was slowly injected into the loop in inject mode.
                                  • Following this, buffer A was passed through the column in manual load mode.
                                  • Finally, the protein samples were collected by analyzing the UV absorbance at 280nm.

                                  Observation 

                                  gfc-puri 1_1.png
                                    obs-gfc-2_1.png
                                      Fig 12. Fractions of Gel filtration chromatography after SDS PAGE.
                                      gfc-1_2.png
                                         A chromatogram showing volume versus Absorbance at 280nm ,after gel filtration chromatography.

                                        Result

                                        The desired protein was observed in protein fractions from 12-19.Later, the fractions were pooled up and concentration was measured using Bradford assay {described below}. Since the concentration of protein was less, we further concentrated protein using Vivaspin 20(5K MVCO). The concentrated protein was stored in buffer containing 100mM NaCl, 0.1mM EDTA, 20mM Tris-HCl (pH 7.5), 1mM DTT, 50% glycerol at -20ºC.

                                        BRADFORD ASSAY:ESTIMATION OF CONCENTRATION OF PROTEIN

                                        Principle

                                        The Bradford assay is used to measure total concentration of protein in a solution. The method is based on the proportional binding of the Coomassie blue dye to proteins. Under acidic conditions, Coomassie G-250 is cationic and is red, whereas in neutral conditions the dye is green and in anionic form it is blue. The Bradford reagent is an acidified solution of Coomassie G-250. The change in the color of Coomassie G-250 from red to blue upon binding protein is measured spectroscopically. In the absence of protein, when the dye is red, Bradford reagent has an absorbance maximum of 470nm. In the presence of protein, the change to the anionic blue form of the dye shifts the absorbance to 595 nm. Most commonly used standards in Bradford assay is BSA.

                                        Materials Required 

                                        Spectrophotometer (595nm), 96 well plates, buffer C (100mM NaCl, 0.1mM EDTA, 1 mM DTT, 20mMTris-HCl (pH 7.5), 50% glycerol), protein samples (RNAP), micropipettes, Comassie Brilliant Blue dye, protein standard (BSA) solution, PCR tubes.

                                        Procedure 

                                        • 11 PCR tubes were taken and marked from 0 – 100.
                                        • A series of protein standards was diluted with buffer to a final concentration of 0 - 1000 μg/ml in PCR tubes.
                                        • Then 80 μl of Coomassie Brilliant Blue-G 250 dye was added into each well in a 96 well plate.
                                        • A standard graph of known protein concentration versus OD595 was plotted as shown below
                                        • The RNAP was diluted 100 and 50 times and its concentration was measured by comparing OD595 with standard curve. 
                                        BSA ( μg/ml) (20μl volume) Secondary stock Buffer (μl)
                                        0 0 20
                                        10 2 18
                                        20 4 16
                                        30 6 14
                                        40 8 12
                                        50 10 10
                                        60 12 8
                                        70 14 6
                                        80 16 4
                                        90 18 2
                                        100 20 0

                                        Observation

                                        bsa_1.png
                                           Graph of OD versus Concentration of protein standard BSA. 
                                          Table.4  Bradford assay data for the determination of unknown concentration of protein sample based on line of best fit of the above standard curve.
                                          Sample Absorbance (595nm) Concentration (μg/ml) Dilution Final concentration(μg/ml) Average (μg/ml)
                                          1. 0.0205 4.500001 100 450.0001431 220.0002
                                          2. 0.0067 -0.1 100 -9.999696414  
                                          3. 0.0251 6.033341 50 301.6670426 275.8336
                                          4. 0.022 5.000005 50 250.0002464  

                                          Result 

                                          The unknown concentration of protein sample (RNAP) was determined to be 5.5 mg/ml using Bradford assay.

                                          RNA SYNTHESIS BY IN VITRO TRANSCRIPTION

                                          Principle

                                          In vitro transcription is a simple procedure that allows for template-directed synthesis of RNA molecules of any sequence from short oligonucleotides to those of several kilobases in μg to mg quantities (Bertrand Beckert et.al). It requires a purified linear DNA template containing a promoter, ribonucleotide triphosphates, a buffer system that includes DTT and magnesium ions and phage RNA polymerase. But here we have used E. coli RNAP as an enzyme.

                                          Materials Required

                                          PCR vials, template, enzyme (RNAP), NTP’s (ATP, UTP, GTP, CTP), RNase inhibitor, RNase free water, incubator, water bath, RNaseA, DNase 1.

                                          E. coli RNAP reaction buffer: 40mMTris- HCl (pH 7.5), 150mM KCl, 1mM DTT, 10mM MgCl2, 0.01% TritonX-100.

                                          Procedure 

                                          • Three PCR vials was taken and marked as set 1, set 2,set 3.
                                          • 60 μl reaction was prepared in each set and consist of the following the components:
                                            COMPONENTS   SET 1 (μl)   SET 2 (μl)   SET 3 (μl)
                                          Template - 6 6
                                          Buffer 12 12 12
                                          NTP’s 9.6 9.6 9.6
                                          RNAP 0.48 - 0.48
                                          RNase free Water 37.67 32.15 31.67
                                          RNase inhibitor 0.25 0.25 0.25
                                          • All three sets were incubated at 37ºC for 1 hour.
                                          • Then, all three sets are heated at 95ºC for 20 min.
                                          • After that, each set is divided into three subsets (20μl each) i.e., 1a, 1b, 1c, 2a, 2b, 2c, 3a, 3b and 3c.
                                          • 1 μl of RNase A was added to 1b, 2b, 3b vials and incubated at 37ºC for 2 hours.
                                          • Similarly, 2μl of DNase buffer and 1 μl of DNase was added to 1c, 2c, 3c vials.
                                          • Then all the above three vials were incubated at 37ºC for 2 hours.
                                          • After incubation, 20 μl of RNA loading dye was added to all the tubes and heated at 95ºC for 5 min and samples were visualizied using 12% Urea PAGE.

                                          Urea PAGE 

                                          Materials required

                                          Small and large glass plates, combs, micropipettes, magnetic stirrer, water, electrophoresis chamber. 

                                          Components Volume (for 20 ml ) ml
                                          12% Acrylamide 8
                                          10X TBE buffer 2
                                          Urea ( 8M) 9.60
                                          APS 0.14
                                          TEMED 0.01

                                          Procedure

                                          • Before loading the samples, the gel was pre-run at 28mA for 40 min.
                                          • After heating, samples are loaded in separate wells and at last well RNA loading dye was loaded.
                                          • Then the gel was run at 12mA for 45 min.
                                          • After gel run, the gel was stained with EtBr and viewed in BIO-RAD chemi-Doc.

                                          Observation

                                          rna.png
                                            Fig 13. RNAP activity check visualized after Urea PAGE.

                                            Result

                                            We could observe RNA in the reaction containing RNAP and DNA template(2a). Unlike DNase,we could not observe any bands when the reaction was treated with RNaseA which suggest the sample contained RNA. However we could not observe a single RNA band probably due to non-homogeneity of RNA synthesis. Further optimization may be required to get a prominent RNA band.

                                            DISSCUSION

                                            Many researchers have started utilizing T7 RNAP for structural and functional studies. A shortcoming in using T7 RNAP is because its improper RNA folding during in vitro transcription. This challenge can be answered by using E. coli RNAP which fits the above necessities. This is achieved by optimizing the protocol of expression and purification of E. coli RNAP. The plasmid were first isolated and transformed into BL21 (DE3) cells. Then, the protein was over-expressed and the expression was confirmed. It was found that the genes expressed better at 16ºC and 25ºC when induced with 0.2mM IPTG. So we scaled up the protein expression upto 1 L in shaking flasks. The cells were lysed by sonication and the supernatant was collected by centrifugation. We confirmed the presence of RNAP subunits after ever step by running gradient SDS PAGE. We purified the enzyme by performing a series of chromatographic techniques that includes Ni-Affinity chromatography, Heparin-affinity chromatography, Ion-exchange chromatography followed by Gel-filtration chromatography. Later, the concentration of protein sample was determined using Bradford assay method. Then it was used to synthesize RNA and check its activity by in vitro transcription. But we couldn’t observe a single RNA band and hence further optimization is required to get a single RNA band. Thus, the purified E. coli RNAP enzyme will be useful for mechanistic analysis of transcriptional machinery and may lead to development of antibiotics to treat infections. Alongside doing my project work, I got familiar with lab techniques like cloning, PCR, Streak plate method.

                                            ACKNOWLEDGEMENTS

                                            I would take this moment to express my sincere gratitude to Dr. B. Anand, Associate professor, Department of Bioscience and Bioengineering, Indian Institute of Technology, Guwahati for allowing me to work in his lab and for providing me an excellent platform to enrich my scientific temper.

                                            I am indebted to Indian Academy of Science for giving me the opportunity to explore the fields of molecular biology and microbiology. The internship has allowed me to learn new techniques and hone my skills in a laboratory setting.

                                            I would also like to sincerely thank Dr. S. Sivaramakrishnan, Head of the Department, Department of Biotechnology and Genetic Engineering, Bharathidasan University for granting me the opportunity to apply for this internship which will help to shape my career in the field of science.

                                            I express my heartfelt thanks to Dr. P. Sobana Piriya, Guest Lecturer and Dr. S. Sridharan, Visiting Professor, Department of Biotechnology and Genetic Engineering, Bharathidasan University, for their timely help and encouraged me to apply for this prestigious internship. In addition, I am thankful to all the faculties of my department for their valuable advice and co-operation.

                                            I am extremely grateful to my mentor Rohan Pal for his guidance and support. In spite of his busy schedule he took time out to help me in my research pursuit.

                                            I am also thankful to all my seniors in Mechanistic Approaches to Biology lab, Yoganand K N R, SunandaChhetry, Siddharth Nimkar, Manasasri M, HimanshuSharma, Rohan Pal, PerwezBakht and Pratyusha Chakraborty, for their valuable suggestions and help during the whole span of my internship. It has been my privilege to work with these extremely talented and great minds. Their valuable advice and suggestions enhanced my performance in lab and allowed me to pursue my research objectives with a lot of clarity. I would also like to thank my fellow trainees Dhanya Ramadurai and SaumyaRanjanSatrusal for being helpful and supportive throughout the tenure of my project.

                                            Last but not least, I would like to thank the almighty and my parents for their moral support and good wishes.

                                            REFERENCES

                                            • Murakami, K.S.(2013).X-ray crystal structure of Escherichiacoli RNA polymerase σ70 holoenzyme.Journal of bacteriology, 200(12), e00159-18.
                                            • JESS A. VASINA and FRANCOIS BANEYX,1996.Recombinant Protein Expression at Low Temperatures under the Transcriptional control of the major Escherichia coli Cold Shock Promoter cspA.APPLIED AND ENVIRONMENTAL MICROBIOLOGY,p. 1444-1447.
                                            • Svetlov, V and Artsimovitch, I,2015.Purification of bacterial RNA polymerase : Tools and protocols.In Bacterial Transcriptional control (pp. 13-29).Human Press, New York, NY.
                                            • Darst, S.A.Kubalek, E.W., and Kornberg, R.D, 1989.Three-dimensional structure of Escherichia coli RNA polymerase holoenzyme determined by electron crystallography. Nature, 340(6236), 730.
                                            • Zuo, y., Wang, Y., Steitz, T.A.(2013).The mechanism of RNA polymerase regulation by ppGpp is suggested by the structure of their complex.Mol.Cell 50: 430-436.
                                            • Vrentas CE, Gaal T, Ross W (2005).Response of RNA polymerase to ppGpp:requirement for the ω subunit and relief of this requirement by DksA.Genes Dev.19(21):2644.
                                            • Dombroski AJ, Walter WA, Gross CA.(1993).Amino-terminal amino acids modulate sigma-factor DNA-binding activity.PubMed.gov, 7(12):2446-55.
                                            • Anna Maciag, Clelia Peano,[…], and Paolo Landini, 2011.In vitro transcription profiling of the σs subunit of bacterial RNA polymerase-definition of the σs regulon and identification of σs- specific promoter sequence elements.NucleicAcids Research;39(13):5338-5355.Oxford University press.
                                            • Maxim V.Sukhodolets and Susan Garges,2003.Interaction of Escherichia coli RNA polymerase with the Ribosomal Protein S1 and the Sm-like ATPase Hfq.American chemical society (ACS), 42, 46, 8022-8034.
                                            • Ju-Sim Kim, Lin Liu […], and Andres Vazquez- Torres(2018).DksA-DnaJ redox interactions provide a signal for the activation of bacterial RNA polymerase.PNAS.org.115(50):E11780-E11789.
                                            • Milligan, J.F., Uhlenbeck, O .C.(1989).Synthesis of small RNAs using T7 RNA polymerase. Methods Enzymol 180,51-62.
                                            • Bertrand Beckert and Benoit Masquida(2011). Synthesis of RNA by in vitro transcription. Methods in MolBiol:703:29-41.
                                            • Artsimovitch I, Svetlov V, Murakami KS, LandrickR(2003). Co-overexpresion of Escherichia coli RNA polymerase subunits allows isolation and analysisof mutant enzymes lacking lineage-specific sequence insertions.Journal of Biochemisty.278:12344-12355.
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