Cloning
Working Principle:
- A desired sequence of DNA is inserted into a suitable vector, which results in a chimeric or recombinant DNA molecule. The vector transports the desired sequence of DNA into the host cell, usually a bacterium.
- Within the host cell, the vector along with the gene it carries, replicates. Then the host cells divide and further replications of the vector take place. After multiple divisions, identical host cells are produced, usually called “a colony” or “a clone”
- It consists of several steps, all of which are listed below along with their protocols.
Restriction digest
Working Principle:
- Restriction Digestion involves fragmenting DNA molecules into smaller pieces with special enzymes called Restriction Endonucleases commonly known as Restriction Enzymes (RE).
- These leave behind overhangs (sticky) or blunt ends. depending on the enzyme, in the DNA so that the vector and insert can ligate with corresponding ends to make a new plasmid.
- It consists of several steps, all of which are listed below along with their protocols.
Gel electrophoresis
Working Principle:
- Gel electrophoresis separates DNA fragments by size in a solid support medium (an agarose gel). DNA samples are pipetted into the sample wells. Application of an electric current at the top (anodal, negative) end causes the negatively-charged DNA to migrate (electrophorese) towards the bottom (cathodal, positive) end. The rate of migration is proportional to size: smaller fragments move more quickly, and wind up at the bottom of the gel.
- DNA is visualised by including in the gel an intercalating dye, Ethidium Bromide. DNA fragments take up the dye as they migrate through the gel. Illumination with ultraviolet light causes the intercalated dye to fluoresce with a pale pink colour.
- The digested pieces of DNA are run through the gel alongside a molecular weight ladder and we can easily visualise the parts we want in our new plasmid.
Gel extraction
Working Principle:
- DNA run on a gel needs to be extracted for further ligation steps.
- The cut gel slice is usually dissolved in buffers, and the DNA is precipitated by ethanol to elute digested DNA fragments.
Ligation
Working Principle:
- The cut vector DNA ends are ligated with the corresponding insert DNA ends with the help of ligases.
- After running the reaction, we transform the mixture into competent cells.
Transformation
Working Principle:
- Bacterial transformation is a process of horizontal gene transfer by which some bacteria take up foreign genetic material (naked DNA) from the environment.
- Not all bacteria are capable of taking up exogenous DNA from their environment. The practical approach to acquire competent cells is to make the bacterial cells artificially competent using chemicals or electrical pulses.
Confirmation
This is done using alkaline lysis. Alkaline Lysis to separate plasmid DNA from all other cell constituents including genomic DNA.
Working Principle:
- The cell pellets are resuspended in a buffer, usually EDTA or Tris. The cells are lysed using an alkali, like 10 N NaOH and a detergent like SDS that denatures most of the cell proteins.
- The cell debris is then precipitated using potassium acetate that helps renature the double stranded plasmid DNA. This is then dissolved and the dissolved DNA is eluted out after centrifuging.
- We now need to precipitate the DNA and clean it of the salts it was dissolved in. An ethanol/isopropanol extraction method precipitates the final plasmid DNA.
- A miniprep kit works on the same principle.
- Elute in low volumes, typically lower than the instruction manual gives, around 30uL in two rounds of 15uL each. Heat the elution buffer or NFW beforehand, and let it soak the membrane for 5-10 minutes before you spin it down.
Bacterial Expression
Chemical competency
Working Principle:
- The bacterial cells that can take up the foreign DNA from the surroundings by a process called transformation are known as competent cells.
- Competent cells have altered cell walls that allow the DNA to easily pass through it. Some cells need to be exposed to some chemical or electrical treatments to make them competent. Treatment with calcium ions is the standard method for the preparation of these cells. Bacterial cells can also take up DNA through a process called electroporation, which we didn’t use.
Streaking/Spreading
Working Principle:
- Bacteria from a culture or a plate need to be spread on an agar plate to form colonies.
- These colonies are then picked and grown in media with or without an antibiotic to make a primary culture.
Primary cultures
Working Principle:
- Bacterial colonies have to be grown in a media to form enough culture that can be further used to express our protein.
Secondary cultures
Working Principle:
- Secondary cultures are usually inoculated with primary cultures and further induced with a chemical like IPTG to enable the bacteria to synthesise our protein of interest.
- Secondary cultures are larger in size and can be scaled up according to the expression level of a certain protein.
Sonication
Working Principle:
- The sonication process uses ultrasonic sound waves. During the process, there is a production of thousands of microscopic vacuum bubbles in the solution due to applied pressure. The formed bubbles collapse into the solution during the process of cavitation.
- The collapsing of bubbles takes place in the cavitation field leading to the generation of enormous energy as there is a production of waves. This results in the disruption of the molecular interactions between the molecules of water. As there is a reduction in the molecular interactions, the particles start to separate and allow the mixing process to take place.
- There is a release of energy from the sound waves that result in friction in the solution. Ice cubes are used during and after the sonication process to prevent the sample from heating up.
- A buffer that can mimic the environment of the cell and the lysis method are important factors in the cell lysis protocol. Buffers commonly include salts, detergent and glycerol.
Protein Purification
Protein A chromatography
Working Principle:
- Protein A chromatography is the most frequently used affinity chromatography method in biomanufacturing. It is the standard technique for capturing recombinant monoclonal antibodies, which relies on the reversible and specific binding between the immobilised protein A ligand and antibodies.
- Each protein A molecule has five immunoglobulin-binding domains, and each domain can bind proteins from many mammalian species, most notably Immunoglobulin G (IgG). The primary binding site is the heavy chain within the Fc region of most IgG. Through such a binding between protein A and the Fc region of IgG, antibodies are retained on the column, which later can be eluted in a purified and concentrated form.
- The binding strength of protein A to IgG depends upon the source species of the immunoglobulin. The dynamic binding capacity depends upon the binding strength and factors such as flow rate during sample application.
Ni-NTA chromatography
Working Principle
- The Ni-NTA Purification System is designed for purification of 6xHis-tagged recombinant proteins expressed in bacteria, insect, and mammalian cells.
- The system is designed around the high affinity and selectivity of Ni-NTA Agarose for recombinant fusion proteins that are tagged with six tandem histidine residues and exhibits high affinity and selectivity for 6xHis-tagged recombinant fusion proteins.
- Proteins can be purified under native, denaturing, or hybrid conditions using the Ni-NTA Agarose. Proteins bound to the resin are eluted with a low pH buffer or by competition with imidazole or histidine. The resulting proteins are ready for use in target applications.
- Ni-NTA Agarose uses nitrilotriacetic acid (NTA), a tetradentate chelating ligand, in a highly cross-linked 6% agarose matrix. NTA binds Ni2+ ions by four coordination sites.
SDS-PAGE
Working Principle:
- SDS PAGE or Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis is a technique used for the separation of proteins based on their molecular weight. A charged molecule migrates to the electrode with the opposite sign when placed in an electric field. The separation of the charged molecules depends upon the relative mobility of charged species. The smaller molecules migrate faster due to less resistance during electrophoresis. The structure and the charge of the proteins also influence the rate of migration.
- SDS is a detergent present in the SDS-PAGE sample buffer. SDS along with some reducing agents function to break the disulphide bonds of proteins disrupting the tertiary structure of proteins. It makes the amount of negatively charged protein far exceed its original charge, masking the natural charge difference between various protein molecules. Therefore, the mobility of various protein-SDS complexes during electrophoresis is no longer affected by the original charge and molecular shape, but only depends on the relative molecular mass.
- Polyacrylamide gel is composed of acrylamide and cross-linking agent N, N'-methylenebisacrylamide under the action of catalysts ammonium persulfate (APS) and N, N, N', N'-Tetramethylethylenediamine (TEMED).
- The polyacrylamide gel is usually composed of a stacking gel in the upper layer and a separating gel in the lower layer. The difference between the upper and lower gels is the concentration of acrylamide and the pH of Tris-HCl.
- During electrophoresis, an electric field is applied to the gel, and negatively charged proteins migrate across the gel from the negative electrode to the positive electrode. The most common electrophoresis buffer consists of Tris and glycine. The pH in the stacking gel is 6.8, and only a few glycine molecules dissociate. Therefore, the SDS-treated protein molecules move between the upper glycine molecule and the lower Cl- ion. This process compresses the protein sample in the gel into bands that are much thinner than the volume initially loaded.
- As the electrophoresis progresses, the protein moves to the separating gel (pH 8.8), where the glycine molecules dissociate. The speed of the movement increases and exceeds the protein. In the separating gel, the speed of movement of each protein depends on its molecular weight. Proteins with small molecular weights can pass through the pores in the gel easily, while those with large molecular weights have more difficulty passing through. After a period of time, proteins reach different distances according to the sizes, achieving the purpose of protein separation.
Bradford Assay
Working Principle:
- The Bradford protein assay is used to measure the concentration of total protein in a sample.
- The assay is based on the observation that the absorbance maximum for an acidic solution of Coomassie Brilliant Blue G-250 shifts from 465 nm to 595 nm when binding to protein occurs. Both hydrophobic and ionic interactions stabilise the anionic form of the dye, causing a visible colour change.
Western Blot
Working Principle:
- Western Blot refers to the electrophoretic transfer of proteins from sodium dodecyl sulphate polyacrylamide gels to sheets of PVDF or nitrocellulose membrane, followed by immunodetection of proteins using antibodies with fluorescent or chemiluminescent detection.
- Bradford Assay can be used to determine protein concentration and thus determine equal volumes of protein to load for binding in a Western Blot.
Protein Concentration
Working Principle:
- Proteins are concentrated using a centricon which has a 10kDa membrane, allowing salts to pass through but not proteins.
- Proteins are eluted in an Imidazole buffer which will give readings in a Nanodrop. So, proteins must be dialyzed before taking nanodrop readings. Dialysis buffer is the elution buffer without Imidazole.
- After successive washes with the dialysis buffer, most salts pass through the membrane and we get about 200uL of concentrated protein from a 2mL sample.
Thermal Shift Assay
Working Principle:
- SYPRO Orange dye binds non-specifically to exposed hydrophobic residues in a protein. If the protein is unstable in a given buffer, SYPRO will bind to its hydrophobic regions and give out a fluorescence signal at lower temperatures. Getting a signal at a higher temperature means that the protein is stable in the given buffer.
- Protein samples are gradually heated to find their melting point.
VLP Fusion Assay
Working Principle:
- It involves vesicles concentrated with dye to the extent that the dye is quenched. Dengue virus-like particles are added to these vesicles - and the pH is lowered to make the particles fuse with the vesicles.
- As the vesicles grow upon fusion - the dye becomes less concentrated and visible, allowing for a simple colorimetric readout for the extent of fusion.
Miscellaneous Protocols/Buffer Recepies
There were some common lab protocols that we followed and are illustrated below. The buffers used in the above protocols are also mentioned.