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Summer Research Fellowship Programme of India's Science Academies

Expression and optimization of Car9 tagged proteins

R. Durai Dhanya

University of Madras, Chennai 600085

DR. B. Anand

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

Abstract

Protein purification involves a series of processes to isolate one or more proteins from a complex mixture, usually cells, or whole organisms. It is a crucial step for studying the structure, activity, functions and interactions of the protein of interest. The process usually involves isolation of proteins by cell lysis and finally separating the protein of interest from other proteins, which is extremely laborious. The differences in size, binding affinity and biological activity of different proteins are exploited in the separation process. Affinity chromatography is a technique used in these cases where separation is based on the tagged polypeptide to the protein of interest and utilizes application specific resins that have ligands attached to them to which these tags show affinity to. It is an expensive procedure due to the consumables involved, such as the pre-packed ligand columns. Hence, the search for a viable alternative was initiated. Studies have found that Car9, a dodecapeptide that shows high affinity to silica, and the interaction can be disrupted with L-Lysine or L-Arginine. It has been observed that Car9 tagged proteins on either N or C terminals support affinity purification on silica beads column, which is a relatively cheaper alternative. Here, the objective of this project is to express Car9 tagged protein (T7 RNA polymerase) and eGFP (enhanced Green Fluorescent Protein) by introducing the Car9 cloning vector into competent cells of E. coli. The transformed cells will then be over-expressed in the presence of the inducer IPTG (Isopropyl β-D-1-thiogalactopyranoside). The protein obtained will be purified to homogeneity on a silica matrix, which is a highly economic alternative to recover proteins quickly and efficiently, compared to other affinity purification processes.

Keywords: purification, Car9, affinity chromatography, proteins, in vitro transcription

Abbreviations

Abbreviations
E. coli Escherichia coli
Min Minutes
TAE Tris Acetate EDTA
EDTA Ethylenediaminetetraacetic Acid
DNA Deoxyribo Nucleic Acid
IPTG Isopropyl β-D-1-Thiogalactopyranoside
SDS Sodium Dodecyl Sulphate
PAGE Poly Acrylamide Gel Electrophoresis
.ccDTT Dithiotheritol
APS Ammonium Per Sulphate
TEMED Tetramethylethylenediamine
PCR Polymerase Chain Reaction

INTRODUCTION

Affinity tags aid in converting a multistep protein purification into a single-step process which yields polypeptides with moderate purification. The primary function of a genetically or chemically affixed affinity tag is to temporarily and reversibly bind to a ligand with a stationary phase or to interact with the stationary phase itself. Some tags enhance solubility and help detect the protein to which they are fused to. Often, the tags are likely to abolish or compromise the native function of the protein due its interaction with the tag. Hence, they are excised by specific proteolysis or self-cleavage events.

One of the commonly used tags in laboratories is polyhistidine tag (His-tag) where histidine forms coordination bonds with immobilized metal ions (Ni2+ and Co2+) and this interaction can be competitively disturbed by imidazole, by which the proteins are recovered. His tag has a small size and it is likely to not interfere with the product. But studies have been found that His tagging may result in protein insolubility, alter the structure and function of the protein, and lead to product insolubility. The stripping of the product with imidazole may cause protein misfolding. His tagging is an expensive process at the laboratory level, due to the resin involved.

Silica (SiO2) is one of the most abundant elements on the earth and studies have shown that silica can act as a good stationary phase for preparative scale purification of proteins. Car9 has been reported to bind to the edges of carbonaceous materials and exhibit a high affinity to silica (Soto-Rodríguez, et.al., 2017). It has also been shown that Car9 can be competitively displaced from silica with high concentrations of Lysine and Arginine. In the following experiments, we attempt to optimize a simple, rapid and economically friendly protocol to purify Car9 tagged proteins to homogeneity on a disposable silica matrix at a small scale.

T7 RNA Polymerase

Bacteriophage T7 RNA polymerase is a DNA dependent RNA polymerase that is highly specific for T7 phage promoters. This enzyme catalyzes in vitro RNA synthesis from a cloned DNA of interest under T7 promoter. RNA produced in this method is useful for many applications in biotechnology. It is also used in preparation of radiolabeled RNA probe, expression control via anti-sense RNA, and RNA generation for studies of RNA structure, function and catalysis. (international.neb.com)

eGFP

Enhanced green florescent protein is a marker (tag) that fluoresces under UV light. It is commonly used as a reporter for gene expression. The protein absorbs UV light and emits a lower energy green light. GFP provides an opportunity to track activities in live cells at any given moment in time.

METHODOLOGY

Work plan

work plan.png
    Work plan followed

    Materials and Methods

    Isolation of plasmid for cloning

     The isolation of plasmid DNA from bacteria is a crucial technique in molecular biology and is an essential step in many procedures such as cloning, DNA sequencing, transfection, and gene therapy. These manipulations require the isolation of high purity plasmid DNA. The purified plasmid DNA can be used for immediate use in all molecular biology procedures such as digestion with restriction enzymes, cloning, PCR, transfection, in vitro translation, blotting and sequencing.

    Alkaline lysis is a method used in molecular biology, to isolate plasmid DNA or other cell components such as proteins by breaking the cells open. Bacteria containing the plasmid of interest is first grown, and then allowed to lyse with an alkaline lysis buffer consisting of a detergent sodium dodecyl sulfate (SDS) and a strong base sodium hydroxide. The detergent cleaves the phospholipid bilayer of membrane and the alkali denatures the proteins which are involved in maintaining the structure of the cell membrane. Through a series of steps involving agitation, precipitation, centrifugation, and the removal of supernatant, cellular debris is removed and the plasmid is isolated and purified.

    Requirements

    Fresh overnight culture (15mL), resuspension buffer, lysis buffer, neutralization buffer, wash buffer, elution buffer, micro centrifuge tubes, glass milk (silica matrix), water bath, centrifuge, etc.

    1.1 Composition of buffers

    Resuspension Buffer

    50mM Tris-Cl, pH 8.0

    10mM EDTA

    100 µg/mL RNAase A

    Stored at 2-8 ºC

    Lysis Buffer

    200 mM NaOH

    1% SDS (w/v)

    Stored at 15-25 ºC

    Neutralization Buffer

    4.2M Gu-HCl

    0.9M Potassium Acetate

    pH 4.8

    Washing Buffer

    10mM Tris-HCl pH 7.5

    80% ethanol

    Elution buffer

    10mM Tris-Cl, pH 8.5

    Procedure

    Ø  15 mL of culture was pelleted in 3 micro centrifuge tubes.

    Ø  250 µL of resuspension buffer was added to each tube and gently mixed.

    Ø  250 µL of lysis buffer was added to each tube and the tubes were capped and inverted to mix.

    Ø  Similarly, 350 µL of neutralization buffer was added to each tube and gently mixed. The previous steps were done very quickly.

    Ø  The tubes were centrifuged for 10 minutes at 13,000 g.

    Ø  Meanwhile, a mix of silica beads, lysis buffer and neutralization buffer was made (equilibration of elution column). For every 5mL of culture volume pelleted, 20 µL of glass milk, 20 µL of lysis buffer and 30 µL of neutralization buffer is mixed together. Three such tubes were made, one for each pellet.

    Ø  The supernatant of the centrifuged cells was added to the silica beads and mixed for 5 minutes.

    Ø  This mixture was centrifuged for 5 minutes, and the supernatant was discarded.

    Ø  The cells were re-centrifuged for a dry spin.

    Ø  The beads were kept for drying at 31 ºC, until the solution droplets evaporated.

    Ø  30 µL of elution buffer was added to the beads and the beads were resuspended.

    Ø  The tubes were placed in a water bath of 50 ºC for 10-15 minutes.

    Ø  They were centrifuged for 1 minute at 13,000g to extract plasmid DNA.

    Ø  The clear supernatant was transferred to fresh tubes. Care was taken to avoid silica beads.

    Results: The plasmid was isolated and stored for further processing.

    vector 1.png
      Schema of plasmid vector

      Agarose gel electrophoresis

      Agarose Gel Electrophoresis is a technique used to separate nucleic acids (DNA, RNA) or proteins in a matrix under applied external electric field. The charged molecules move through the agarose matrix and separate based on their size. The separated bands can be viewed under UV, and DNA fragments can be extracted with relative ease. The gel can be prepared with various concentrations, according to the pore size required. The higher the concentration, the smaller the pore size is. This reduces migration speed and makes separating smaller fragments easier as smaller DNA fragments move faster than the larger ones.

      The DNA and RNA fragments are usually visualized by staining with ethidium bromide, which intercalates into the major grooves of DNA and fluoresces under UV light. The intercalation and complex-forming depends on the concentration of DNA present, hence a band of higher intensity indicates DNA of higher concentration.

      Requirements

      Agarose, TAE buffer (400mM Tris –pH 8.3, 200mM acetic acid, 10mM EDTA), Electrophoresis unit, micropipettes, EtBr, DNA ladder etc.

       Procedure

      Ø  1g agarose was weighed and mixed to 50 mL of distilled H2 O.

      Ø  1ml of 5X TAE buffer was added to the solution make 1X concentration.

      Ø  The solution was heated to boiling.

      Ø  2µL of EtBr is added to the solution once it has sufficiently cooled down.

      Ø  The gel is poured into the caster and we wait until it solidifies.

      Ø  The gel was then transferred to the electrophoresis tank with 1% TAE buffer.

      Ø  The comb was gently removed and the samples were loaded, along with DNA ladder.

      Ø  The electrodes were connected and the samples were run at 80 V, until they reached 60% of the length of the gel.

      Observations

      isolated plasmid_2.png
        Agarose gel electrophoresis of isolated plasmid

        Results: Plasmid was isolated and visualized using agarose gel. The expected size of the plasmid was approximately 4000 Bp.

        Preparation of competent cells and transformation of cells

        Competence is the property of a cell to take up plasmid DNA of interest. Competent cells are bacterial cells that can take up foreign DNA. To make bacteria permeable to DNA, usually chemical treatment is required. Cells can also be transformed by electroporation method, while some cells can be transformed by nature without any external treatment. In CaCl2 method, the competency is obtained by creating pores in bacterial cells by suspending them in a solution containing high concentration of calcium. DNA is then forced in to the host cell by heat shock treatment at 42 oC for the process of transformation. The treatment of the cells with divalent cations such as Mg2+ and Ca2+ results in the binding of the negatively charged plasmid DNA with the cations. The heat shock forms transient pores on the surface of the cell which allow the cells to take up the DNA. The pores close when the cells are put back in ice immediately after heat shock.

        Requirements

        Lb broth, test tubes, antibiotics (Kanamycin), micropipettes, micro centrifuge tubes, incubator, ice cold solution 1(0.08M MgCl2 + 0.02M CaCl2), ice cold solution 2 (0.1M CaCl2), antibiotic plates, glass beads.

        E. coli strain: BL21(DE3): The strain is deficient in Lon protease and OmpT protease, hence preferred for recombinant protein expression. DE3 designates the presence of λDE3 lysogen that carries the gene for T7 RNA Polymerase under control of LacUV5 promoter. IPTG is used to maximally induce the expression of T7 RNA polymerase and other recombinant genes located downstream of the T7 promoter.

        Procedure

        Competent cells

        Ø  150µL of BL21 (DE3) cells were inoculated in 15mL of LB broth.

        Ø  The tube was incubated at 37 oC until OD600 was 0.3.

        Ø  The media was transferred to a falcon tube taken in ice.

        Ø  The solution was centrifuged at 2710 g for 10 minutes, at 4 oC.

        Ø  The supernatant was discarded and 9mL of ice cold solution 1 was added.

        Ø  The pellet was gently swirled and resuspended, in ice.

        Ø  This solution was centrifuged at 2710 g for 10 minutes, at 4 oC.

        Ø  The supernatant was discarded, and 750µL of ice cold solution 2 was added and the pelleted cells were gently resuspended while in ice.

        Transformation

        Ø  250µL of competent cells were aliquoted into 2 micro centrifuge tubes of 1.5mL each. One was labelled + (positive) and the other – (negative).

        Ø  2µL of plasmid was added to the tube labelled +ve.

        Ø  The cells were incubated in ice for 30 minutes.

        Ø  After this, they were given heat shock treatment for 1 minute at 42 ºC.

        Ø  Immediately the cells were plunged in ice and left for 2 minutes.

        Ø  800µL of fresh LB broth media was added to both the tubes and the cells were incubated at 37 ºC for 45 minutes.

        Ø  Once the incubation period has finished, the cells were centrifuged at 13,000g for 1 minute. Most of the supernatant was discarded.

        Ø  The cells were resuspended in the few microliters of media and spread on to different LB agar antibiotic plates (kanamycin) using glass beads.

        Ø  The plates where incubated at 37 ºC for 12-16 hrs and observed for bacterial growth.

         Observations

        Transformed cells.jpg
          Cells transformed with T7 Car9 vector. Left: Positive plate Right: Negative plate

          Result: The cells were of good competency and showed good transformation efficiency.

          Expression check with IPTG

          IPTG (Isopropyl β-D-1-Thiogalactopyranoside) is a mimic of allolactose and is used to induce E. coli protein expression, when the gene of interest is under the control of lac operon. IPTG binds to lac repressor and releases a tetrametic repressor allosterically, allowing transcription of genes in the lac operon. But unlike allolactose, the Sulphur atom in IPTG creates a chemical bond that is not hydrolysable by the cell, thus preventing the cell from metabolizing the inducer. Thus, the concentration of IPTG remains constant over the duration of the induction. (D.C. Rio, 2011)

          Requirements: LB broth, 1M IPTG, antibiotics (kanamycin), micro-pipettes, micro-centrifuge tubes, lysis buffer (20mM Tris, 2mM EDTA, 7.5 pH), PMSF 100mM, β-mercaptoethanol, sonicator.

          Procedure

          Induction

          Ø  150µL of E. coli BL21 (DE3) cells with T7 Car9 plasmid were inoculated in 3 tubes of LB broth (with 0.5mM kanamycin) each and incubated at 37 ºC.

          Ø  Once the cells had reached OD600 of 0.6, the cells were induced with different concentrations of IPTG, namely 0.5M (7.5µL from stock solution) and 1M (15µL from stock solution). The third tube was labelled as uninduced.

          Ø  The tubes were placed back in 37 ºC for 4 hours.

          Ø  After the induction period, the cells were removed from the incubator and pelleted in micro centrifuge tubes separately at 13,000 g for one minute. The supernatant is completely decanted.

          Sonication

          Sonication is a method of cell lysis where ultrasound / high frequency energy is applied to the cells to agitate and disrupt the cell membranes, usually using an ultrasonic probe. This generates small bubbles and their violent implosion, known as cavitation, which causes cell rupture and ultimately cell lysis, which releases all the intracellular proteins.

          Ø  500µL of lysis buffer was added to each pellet and resuspended.

          Ø  To these cells, 5µL of PMSF and 0.21µL of β-mercaptoethanol was also added.

          Ø  The cells were sonicated for 45s each, where the pulse was on: 3s and off: 15s. The amplitude was 33

          Ø  Sonication was performed in ice as the process can generate large amounts of heat.

          Ø  Once the cells were lysed, they were centrifuged at 16,000 g, 4 ºC for 20-30 minutes.

           Result: The supernatant and pellet were separated and stored for further use.

           Checking protein expression by SDS PAGE

          When proteins are separated by electrophoresis in a gel matrix, smaller proteins migrate faster due to less resistance from the matrix. The rate of migration is also influenced by the structure and charge of the proteins.

          SDS PAGE is a denaturing PAGE which eliminates the influence of the structure and charge of the protein and they are separated based on the length of the polypeptide chain only. 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. Polymerized acrylamide (polyacrylamide) forms a mesh-like matrix suitable for the separation of proteins of typical size. Polyacrylamide gel electrophoresis of SDS-treated proteins allows separation of proteins based on their length in an easy, inexpensive, and relatively accurate manner.

          Stacking gel

          The stacking gel has a lower polyacrylamide gel concentration and is placed on a much concentrated resolving gel. It is used to improve the resolution of the electrophoresis by concentrating the proteins, by creating an ionic gradient that traps the molecules. Negatively charged chloride ions from Tris-HCl and positively charged glycine molecules create the gradient that keeps molecules moving together until they reach the separation gel in which pH changes that allows the molecules to then be separated based on size.

          Resolving gel

          The resolving gel or the separating gel has a higher pH and lower pore size compared to the stacking gel. The net charge density of glycine is also higher than that of protein in the resolving gel, thus Cl- ion moves fastest, followed by glycine. The proteins are not sandwiched between glycine and Cl- ions. The proteins all have equal charge densities due to treatment with SDS, the proteins are separated based on mass only.

          Loading buffer

          Before loading a protein sample in polyacrylamide gel it is boiled with loading buffer which contains sodium lauryl sulfate SDS (C12 H25 SO4Na), anionic surfactant and reducing agents like dithiothreitol (DTT) or β-mercaptoethanol. Most of the proteins binds SDS with constant-weight ratio (one SDS molecule per two amino acids) leading to identical charge densities. However, unless proteins are completely unfolded SDS cannot bind uniformly to protein amino acid residues as some amino acid residues may not be accessible due to folding of protein. Thus, reducing agent, which further denatures the proteins by reducing disulfide linkages and enables SDS to bind proteins uniformly. There are two more ingredients of loading buffer Bromophenol Blue and glycerol. Glycerol increases density of loading buffer so sample can settle in loading well while Bromophenol Blue is a colored dye and indicates progression of electrophoresis. Electrophoresis is performed at constant current till bromophenol blue band reaches bottom of the gel.

          Requirements

          29:1 acrylamide to bis-acrylamide solution, Tris-HCl buffer (pH 6.8 and pH 8.8), 10% SDS, APS, TEMED, dH2O, 1.5mm casting plates, 1.5 mm combs, casting frame, etc.

          Resolving gel: (for 40mL)

          Components of resolving gel
          30% Acrylamide 13.33mL
          Tris (pH 8.8) 10 mL
          10% SDS 0.4 mL
          Trichloroethanol 0.2 mL
          APS 0.4 mL
          TEMED 0.016 mL
          dH2O 15.654 mL

          Stacking gel: (for 20mL)

          Components of stacking gel
          30% Acrylamide 3.32 mL
          Tris (pH 6.8) 2.52 mL
          10% SDS 0.2 mL
          APS 0.2mL
          TEMED 0.02 mL
          dH2O 12.64 mL

          Procedure

          Ø  Casting plates were fixed and filled with water and allowed to stay for a while to check for leakage.

          Ø  Once the plates were determined to be leak-free, the water was drained and the resolving gel was poured into the plates until the level marked.

          Ø  A few drops of isopropanol was added to remove the bubbles that were formed.

          Ø  Once the resolving gel had solidified, the excess isopropanol was drained and the stacking gel was poured in. The combs were placed inside immediately and the gel was allowed to solidify.

          Ø  Once the gel had solidified the gel cassettes were removed from the casting tray for further use.

          Ø  The cassette was placed in electrophoresis tank and the tank was filled with Tris-Glycine fer.

          Ø  The comb was gently removed and the samples were mixed with the loading buffer and loaded into the wells, along with a protein ladder marker in one of the wells.

          Ø  The gel was run until the dye had run out and imaged.

          Observations

          Induction was observed in both 0.5mM and 1mM concentrations.

          Results

          The cells were further optimized using other variables for expression.

          Optimizing Car9 tagged T7 RNA Polymerase expression with different media

          Three different media were prepared to check expression of T7Car9:

          LB Media: Luria Bertani Broth – It is the most common media used for E. coli growth, and consists of Tryptone, yeast and NaCl.

          2XYT Media: It is a moderately rich medium for the growth of E. coli cells and contains tryptone and yeast in high amounts.

          Terrific Broth: It is a rich medium compared to LB and 2XYT. The medium is developed for higher density growth of E. coli cells and higher yield of plasmid DNA compared to LB and Tryptone broth.

          Requirements

          LB broth, 2XYT media, Terrific Broth, test tubes, cotton plugs, pipettes, antibiotic (kanamycin) distilled H2O, E. coli culture, IPTG, incubator.

          Procedure

          Ø  The media broths were prepared according to package instructions, 2 tubes of 5mL each for each media, in clean test tubes.

          Ø  The tubes were plugged and autoclaved.

          Ø  Once the media was ready, 2.5µL of Kanamycin was added to each tube. The two tubes of each media were labelled as “Induced” and “Uninduced” respectively.

          Ø  50µL of E. coli culture was inoculated in each tube and the tubes were plugged and placed in an incubator at 37 ºC, until OD600 reached 0.6.

          Ø  The tubes were then taken out and 1mM IPTG was added to all the tubes marked as “Induced”.

          Ø  The tubes were put back in the incubator at 37 ºC for 4 hours for induction.

          Ø  The cells were then pelleted separately. They were lysed by lysis buffer and subjected to SDS PAGE to analyze the expression of T7Car9 protein.

          Observations

          induction in different media.png
            SDS PAGE gel showing induction in supernatant and pellet of cells grown in different media

            Results: Induction was found to be optimal in LB broth. The expected size of the induced protein was around 95kDa.

            Small scale extraction using silica matrix

            Preparing silica matrix for elution using Car9

             Requirements

            Silica beads, distilled water, falcon tubes.

            Procedure

            Ø  5g of silica beads was taken in a falcon tube and 50 mL of distilled water was added to it.

            Ø  The solution was centrifuged at 3000g for 5 minutes and the supernatant was discarded.

            Ø  The above step was repeated twice.

            Small scale purification

            Requirements

            30mL LB broth, primary culture, antibiotic, IPTG, micropipette, sterile tips etc.

            Procedure

            Ø  Primary culture of 300 µL was inoculated in LB broth along with the required antibiotic.

            Ø  The cells were incubated until OD600 reached 0.6 and was induced with IPTG.

            Ø  Induction was done for 4 hours and the cells were pelleted and lysed using different buffers.

            Composition of lysis buffers and their respective elution buffers:

            Lysis buffer 1: 20mM Tris, 300mM NaCl, 6mM β-ME, pH 7.6

            Elution buffer 1: 1M Lysine, 20mM Tris, 300mM NaCl, 6mM β-ME, pH 7.6

            Lysis buffer 2: 20mM Tris, 300mM NaCl, 1mM DTT, pH 8.2

            Elution buffer 2: 1M lysine, 20mM Tris, 300mM NaCl, 1mM DTT, pH 8.2

            Elution of protein using silica beads:

            Requirements

            Eppendorf tubes, sonicated samples, lysis buffers, elution buffers, silica matrix, micro centrifuge.

            Procedure

            For sonication of samples with respective buffers,

            Ø  200µL of bead matrix was taken and centrifuged. The excess water was removed and the column was equilibriated with 200 µL of lysis buffer used.

            Ø  For every 500 µL of lysate, 200 µL of equilibriated column was used.

            Ø  The beads and lysate were mixed together for 30 minutes in 4ºC.

            Ø  The solution was then centrifuged at 13,000g for 2 minutes and the supernatant was stored as Flow Through.

            Ø  The beads were then resuspended in 500µL of lysis buffer and mixed for 30 minutes at 4 ºC.

            Ø  The solution was centrifuged and the supernatant was stored as wash. This step was repeated twice.

            Ø  200 µL of elution buffer was added and was mixed by pipetting 3-4 times. The sample was then centrifuged and the eluent separated.

            Ø  2 elution of gradually reducing volumes (150 µL, 100 µL) were done and stored as eluents.

            SDS PAGE was run to analyze the sample.

            Observations

            Buffer 1

            buffer 1_1.png
              SDS PAGE showing elution of T7Car9 protein with buffer 1

              Buffer 2

              Buffer 2.png
                SDS PAGE showing elution of T7Car9 protein with buffer 2

                Results: No binding was observed. The expected size of eluted protein was approximately 95 kDa.

                As T7 RNA polymerase was not showing the desired levels of expression and binding, the protein of interest that we are trying to express was changed to eGFP, as eGFP is considerably a smaller molecule as compared to T7 RNA polymerase, and is visible to the naked eye to a certain extent. The change in the intensity of the fluorescence can also be easily detected during elution.

                Plasmid and primer design

                Components of a Plasmid

                ORI (Origin of Replication): Has a DNA sequence that directs initiation of plasmid replication and expression, critical for amplification by bacteria.

                Antibiotic Resistance gene: Acts as a selection marker, enables selection of plasmid containing bacteria by providing survival advantage to host.

                MCS (Multiple Cloning Site): A multiple cloning site contains sequences for several restriction enzymes enabling easy insertion of DNA by digestion and ligation.

                Insert: The gene/ promoter/ DNA fragment being cloned into MCS.

                Promoter Region: The promoter region drives the transcription of interest, and is designed to recruit transcriptional machinery from a particular organism or a group of organisms. It can direct cell-specific expression by using a tissue specific promoter. The strength of the promoter is important for controlling level of insert expression.

                Selectable Marker: Which is different from antibiotic resistance gene which is to select cells that have taken up plasmid for replication.

                Primer Binding Site: It is a single stranded DNA sequence used as initiation point for PCR amplification or DNA sequencing. Primers are also used to verify sequence of inserts/ other regions of plasmid.

                Plasmid A, which exhibits resistance to the antibiotic Kanamycin was selected. Primers were designed according to the gene of interest, corresponding to the plasmid used. Two forward primers were designed to accommodate both eGFP and Car9. The reverse primer remained the same in both amplifications.

                Forward Primer

                Ø  The primer consisted of at least 20 bases.

                Ø  The percentage of G and C residues must be less than 50%.

                Ø  The vector overhang also consisted of at least 20 bases.

                Reverse Primer

                Ø  The reverse primer was selected from the 5’ end of the gene of interest and flipped to generate the anti-sense sequence.

                Ø  Other conditions are similar to that of the forward primer.

                Amplification of gene of interest by polymerase chain reaction

                Polymerase Chain Reaction or PCR is a common technique used to make multiple copies of a specific region of DNA in vitro. It heavily relies on a thermostable DNA polymerase and primers that are specifically designed for the DNA region of interest. The reaction is cycled through a series of temperature changes, which allows production of multiple copies of the target region. PCR is routinely used for DNA cloning, medical diagnostics and forensic analysis of DNA.

                Pfu polymerase

                Pfu DNA polymerase is a thermostable enzyme isolated from Pyrococcus furiosus. In addition to 5’ to 3’ polymerase activity, it also contains 3’ to 5’ exonuclease (proofreading) activity. Pfu polymerase has a 6-fold accuracy than Taq DNA polymerase.

                PCR primers

                Primers are short pieces of single-stranded DNA about 20 nucleotides in length. Two primers are used in each reaction, so that they flank the target region. The primers bind to the template (the region to be amplified) by complementary base pairing, from which the polymerase will recruit dNTPs and extend the primers to complete the chain.

                Basic steps involved in PCR

                Denaturation (96ºC): Heat the reaction strongly to separate, or denature, the DNA strands. This provides single-stranded template for the next step.

                Annealing (55-60ºC): Cool the reaction so the primers can bind to their complementary sequences on the single-stranded template DNA.

                Extension (72ºC): Raise the reaction temperatures so that a thermostable polymerase extends the primers, synthesizing new strands of DNA.

                Procedure

                Ø  Two forward primers and one reverse primer were used for extension as the length of the gene was long.

                Ø  First amplification was done with FP2, after which the amplified product was used as template for amplification of gene of interest with FP1. The RP was the same in both reactions.

                Ø  The following reaction mix was put up in 8 PCR tubes-with a gradient annealing temperature to determine the optimal annealing temperature.

                Reaction mix (per 10µL)

                Components of a PCR reaction mixture
                FP 1µL
                RP 1µL
                Template 0.2 µL
                dNTP 1 µL
                5X buffer 1 µL
                Pfu pol 0.2 µL
                Water 5.6 µL

                PCR protocol

                The following were the steps to run the PCR

                Steps involved in a PCR reaction
                  Temperature Time
                Step 1 95 ºC 5 min
                Step 2 95 ºC 30 sec
                Step 3 Gradient 48 – 60 ºC 45 sec
                Step 4 72 ºC 2 min
                  Repeat steps 2-4 X 35
                Step 5 72 ºC 8 min

                 Gradient temp.values:

                1. 48 ºC

                2. 49.1 ºC

                3. 50.7 ºC

                4. 52.7 ºC

                5. 55.5 ºC

                6. 57.7 ºC

                7. 59.1 ºC

                8. 60 ºC

                The samples were run on a 1% agarose gel and visualized.

                Observations

                pcr amplification.png
                  PCR amplification of eGFP tagged with Car9

                  Results: All temperatures showed amplification. The expected size of amplified product was approximately 290 Bp.

                  Restriction digestion of plasmid A

                  Restriction digestion is the process of cutting DNA into smaller fragments using special enzymes, known as restriction endonucleases. They recognize specifics sequences in the DNA and nick the DNA strand there. It also linearizes the plasmid which can be used for cloning.

                  Requirements: Restriction Enzymes, Plasmid A, Cut Smart Buffer, Micropipettes, Tips etc.

                  Restriction Enzymes used: NcoI, SspI

                  Reaction volume: 50 µL

                  Plasmid : 30 µL

                  Elution Buffer : 20 µL

                  Enzyme 1 (NcoI): 3 µL

                  Enzyme 2 (SspI): 1.5 µL

                  CutSmart Buffer: 6.05 µL

                  The reaction mixture was mixed in a PCR tube and incubated in 37ºC for 4 hours.

                  The restriction digestion was checked by subjecting the samples to Agarose Gel Electrophoresis.

                  Observations

                  restriction digestion.png
                    Agarose gel showing restriction digestion of plasmid

                    Results: The vector was linearized by restriction digestion. The size of the linearized vector was approximately 3190 Bp.

                    Gel extraction of linearized plasmid and gene of interest

                    Gel extraction is a technique used to isolate a desired fragment of DNA from agarose gel after agarose gel electrophoresis. After the samples are run on agarose gel, the fragments are physically extracted after visualizing with UV light using a razor blade. Further this fragment is chemically treated and DNA is eluted.

                    Requirements

                    Qiagen extraction column, Agarose gel electrophoresis apparatus, DNA samples, Eppendorf tubes, isopropanol, elution buffer, wash buffer, micro centrifuge, UV transilluminator.

                    Procedure

                    Ø  The DNA sample was run on agarose gel and placed on UV transilluminator.

                    Ø  The UV light was switched on to determine the location of the DNA to be extracted. Care was taken to not over expose the sample to UV light and damage it.

                    Ø  DNA was cut along with the gel using a lancet and dropped in a centrifuge tube.

                    Ø  The weight of the gel piece was determined and 3X volume of QG buffer was added.

                    Ø  The vial was kept in a tumbler until the gel melted and the sample had completely dissolved.

                    Ø  To this solution, 1X (the weight of the gel) volume of isopropanol was added.

                    Ø  The sample was thoroughly mixed and the solution was transferred to a Qiagen spin column.

                    Ø  The column was centrifuged at 13,000 g for 1 minute.

                    Ø  The flow through was discarded.

                    Ø  Dry spin of the column was done.

                    Ø  The top of the column (with beads) was removed and placed in a 1.5mL vial for elution.

                    Ø  Pre-heated elution buffer (80ºC) of 20 µL volume was added to the column.

                    Ø  After 2 minutes, the vial, along with the column was centrifuged at 13,000g for 2 minutes and the eluent was collected in the vial and stored. This contained the DNA sample.

                    Ø  Extraction was performed separately for linearized vector and gene of interest.

                    Results: The DNA samples were extracted and stored for further processing.

                    Gibson cloning

                    Gibson cloning assembly is a molecular cloning method that allows the joining of multiple DNA fragments in a single isothermal reaction. It is preferred as it involves few components with minor manipulations. It can combine up to 15 DNA fragments in a single reaction. It requires that the fragments have at least 20 overlapping base pairs, with the adjacent DNA fragments.

                    The fragments are added to a reaction mix of 3 enzymes and other buffering agents. The enzymes used are:

                    Exonuclease: The exonuclease chews back DNA from the 5’ end, thus not inhibiting polymerase activity and allowing the reaction to occur in one single process. The resulting single-stranded regions on adjacent DNA fragments can anneal.

                    DNA polymerase: The DNA polymerase incorporates nucleotides to fill in any gaps.

                    DNA ligase: The DNA ligase covalently joins the DNA of adjacent segments, thereby removing any nicks in the DNA.

                    The product is multiple DNA fragments joined into one. The molecule assembly can be linear or closed.

                    Requirements

                    Gibson mix, gene of interest, digested vector, micro pipette, tips, PCR tubes, thermocycler etc.

                    Composition of Gibson Mix

                    Components of Gibson mix
                    5X Iso Buffer 32 µL
                    T5 Exonuclease (10X dilution) 0.64 µL
                    Taq Polymerase 1.5 µL
                    Taq DNA Ligase (10X dilution) 0.16 µL
                    ddH2O 85.7µL

                    Procedure

                    Ø  PCR tubes were taken and kept in ice.

                    Ø  15µL of Gibson mix and 5µL of nucleic acid mix were mixed together.

                    Ø  The nucleic acid mix consists of 3.5µL of the gene of interest and 1.5 µL of digested vector. The gene was taken in higher concentration to ensure higher chances of insertion into the vector.

                    Ø  The reaction mix was mixed with a pipette and incubated in a thermocycler at 50 ºC for 1 hour.

                    Results: Gibson clones were prepared and stored for further processing.

                    Transformation of Gibson Clone in E. coli TOP 10 cells

                    Strain: E. coli Top 10: A strain of E. coli used for cloning and plasmid preparation. The cells have a high transformation efficiency and the genes recA and endA (a recombinase and DNAse) are knocked out, which lower plasmid yield. Thus the strains are optimized for cloning. 

                    Procedure

                    Ø  Competent Top 10 Cells are mixed with 20 µL of the Gibson reaction and transformed.

                    Ø  The cells are plated and incubated for 12-16 hours or until growth of colonies is observed.

                    Observations and results: The cells were transformed and growth of colonies was observed.

                    Colony streaking

                    This method is a qualitative isolation method. It involves isolation of discrete colonies thereby reducing the population size. It is essentially a diluting technique that ensures that individual cells are sufficiently far apart for picking.

                    Requirements

                    Antibiotic agar plate (kanamycin), inoculation loop, transformed culture, sterile conditions etc.

                    Procedure

                    Ø  From the transformed cells, 10 colonies were picked randomly.

                    Ø  A fresh antibiotic plate was divided into 10 segments with a marker, to identify the spots for the 10 different colonies.

                    Ø  Inoculation loops were heat sterilized and allowed to cool down.

                    Ø  The selected colonies were picked and streaked onto the fresh plate in the respective boxes.

                    Ø  The plate was then incubated at 37 ºC for 12-16 hours for colony growth.

                    Observations and results: Colony growth was observed and the plates were stored for further processing.

                    Colony PCR

                    Colony PCR is a convenient high-throughput method for determining the presence or absence of insert DNA in plasmid constructs. Individual transformants can either be lysed in water with a short heating step or added directly to the PCR reaction and lysed during the initial heating step. This initial heating step causes the release of the plasmid DNA from the cell, so it can serve as template for the amplification reaction. Primers designed to specifically target the insert DNA can be used to determine if the construct contains the DNA fragment of interest. Alternatively, primers targeting vector DNA flanking the insert can be used to determine whether or not the insert is the correct molecular size. Insert specific primers can provide information on both the specificity and size of the insert DNA while the use of vector specific primers allows screening of multiple constructs simultaneously. Colony PCR can also be used to determine insert orientation. PCR amplification of the plasmid using an insert specific primer paired with a vector specific primer can be designed to produce an amplicon of a specific size only if the insert is in the correct orientation. In all experimental designs, presence or absence of a PCR amplicon and size of the product are determined by electrophoresis alongside a DNA size marker on an agarose gel. 

                    Requirements

                    T7 forward primer, T7 reverse primer, dNTPs, Templates (from streaked colonies), 10X buffer, PFU polymerase, ddH2O, PCR tubes, micropipettes, sterile tips etc.

                    Procedure

                    Ø  PCR tubes labelled 1-10 were taken and to each tube, 20µL of ddH2O was added.

                    Ø  The respective streaked colonies were picked and dissolved in 20µL of ddH2O.

                    Ø  The tubes were heated at 95 ºC for 15 min to lyse the cells and release DNA and plasmid.

                    Ø  Another set of PCR tubes marked from 1-16 along with + and – tubes. The 1-10 tubes contain template from the lysed cells, whereas the + and - were taken as controls with empty vector.

                    The following reaction mixture was prepared for each tube:

                    Components of one reaction mixture
                    T7 forward primer 1µL
                    T7 reverse primer 1µL
                    dNTPs 3µL
                    Template (from dissolved colonies) 0.2µL
                    10X Buffer 1µL
                    PFU pol 0.2µL
                    ddH2O 5.6µL

                     Ø  PCR was run once the reactions were mixed. The conditions were as follows: 

                    Colony PCR check conditions
                      Temperature Time
                    Step 1 95ºC 5 min
                    Step 2 95ºC 0:30 sec
                    Step 3 57 ºC 0:45 sec
                    Step 4 72 ºC 6 min
                    Step 5 Repeat steps 2-4 30 times
                    Step 6 72 ºC 12    min

                    Ø  When colony PCR had finished, 0.8% agarose gel was prepared and the samples were run and visualized.

                    Observations

                    Colony PCR.png
                      Agarose gel showing colony PCR clone check

                      Results: All the colonies except colony 8 were found to contain clones. The approximate size of amplified product was 900 Bp.

                      Transformation of clones in E. coli BL21(DE3) and expression check

                      Requirements

                      Plasmid with eGFP Car9, E. coli BL21(DE3) competent cells, antibiotic plates, IPTG, micropipettes, tips.

                      Procedure

                      Ø  Plasmid with eGFP Car9 was isolated (refer to section 9).

                      Ø  Competent BL21(DE3) cells were transformed with recombinant plasmid.

                      Ø  The cells were plated on antibiotic plates with 0.5mM IPTG to check expression as eGFP exhibits fluorescence under UV.

                      Ø  The plates were incubated at 37 ºC overnight for colony growth.

                      Observations

                      The transformed colonies were observed under UV transilluminator.

                      gfp car9.jpg
                        IPTG and antibiotic plates with E. coli cells expressing eGFP and Car9

                         Results: eGFP Car9 was expressed in E. coli BL21(DE3) cells.

                        Small scale purification of Car9 tagged eGFP

                         Requirements

                        Induced culture of E. coli BL21(DE3) cells with recombinant vector, Lysis buffer 2 (pH 8.2), Elution buffer 2, sonicator, SDS PAGE apparatus, micro pipettes, tips etc.

                         Procedure

                        Ø  Cells induced with IPTG were pelleted and sonicated with lysis buffer 2.

                        Ø  The samples were subjected to an SDS PAGE apparatus.

                        Observations

                        gfp elution.png
                          SDS PAGE of eGFP Car9 elutions

                          Results: Eluted fractions show presence of eGFP+Car9 tag. The approximate size of eluted product was 110 kDa.

                          CONCLUSION AND DISCUSSION

                          Here, we have used the Car9 tag on two different proteins. T7 RNA Polymerase is promoter specific and transcribes RNA downstream of the T7 promoter and is extensively used in in vitro transcription. eGFP is a fluoresent tag that is used as a visual marker for gene expressions.

                          In these experiments we show that Car9 is an efficient tag that can be used for protein purification and is quick and cost-efficient.

                          Further optimization with regards to elution can be done to avoid contaminants in the eluted fractions.

                          REFERENCES

                          1.      Soto-Rodríguez, Coyle BL, Samuelson A, Aravagiri K, Baneyx F (2017) Affinity purification of Car9-tagged proteins on silica matrices: Optimization of a rapid and inexpensive protein purification technology Protein Expr Purif. 2017 Jul;135:70-77.

                          2.      Rio DC (2013) Expression and purification of active recombinant T7 RNA polymerase from E. coli. Cold Spring Harb Protoc. 2013 Nov 1.

                          3.      Coyle BL & Baneyx F (2014) A cleavable silica-binding affinity tag for rapid and inexpensive protein purification Biotechnol Bioeng. 2014 Oct.

                          4.      www.openwetware.org

                          5.      www.khanacademy.org

                          6.      www.fpbase.org

                          7.      www.neb.com

                          ACKNOWLEDGEMENTS

                          I thank the Indian Academy of Sciences, Bangalore, for giving me this wonderful opportunity to work as a summer research fellow.

                          My sincere gratitude to Dr. B. Anand, Associate Professor, Dept. of Biosciences and Bioengineering, Indian Institute of Technology, Guwahati, for allowing me to work on this project and providing a wholesome environment.

                          I also thank the lab members Sunanda Chhetry, Yoganand K.N.R, Manasasri Muralidharan, Himanshu Sharma, Siddharth Nimkar, Rohan Pal, Perwez Bhakt and Pratyusha Chakraborty for their constant support, guidance, insights and encouragement.

                          Thanks to my parents and the faculty and staff of CAS in Botany, University of Madras, for giving me a chance to pursue my dreams and explore new opportunities. Special thanks to Dr. N. Radhakrishnan, Assistant Professor, CAS in Botany, for guiding me on the right path.

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