CELL TRANSFORMATION
Cell transformation is a process by which cells take up exogenous genetic material directly into them and hence transforming the characteristics of the cell. In synthetic biology, transformation is used to is used to create genetic circuits which can be manipulated to get the desired product.
Living cells can be manipulated by synthetically made DNA constructs to obtain new phenotypes of interest. Some methods by which cells can be artificially transformed are:
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(i) Physical Methods:
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(a) Electroporation:
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This method involves the application of a series of electric pulses (~200 V/cm for tens of milliseconds) in order to induce a transient increase in membrane permeability. The electric pulses induce the formation of hydrophilic pores in the cell membrane and the subsequent passive passage of DNA through these pores. Major problems of electroporation include induction of tissue damage due to the electric pulses and transient expression of the internalized plasmid DNA.
The efficiency of gene transfer can be increased by the administration of hyaluronidase that degrades hyaluronic acid in the ECM and thus increases gene transfer efficiency by favoring diffusion of the nucleic acids.
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(b) Gene Gun:
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This method involves delivering DNA into the cells by bombarding them with micron-sized beads carrying plasmid DNA adsorbed onto their surface. These particles can easily cross the cell and nuclear membranes and release the DNA adsorbed on their surface into the nucleus. This method is widely used for gene transfer into plants, which has a rigid cell wall .
It now finds application for gene therapy of accessible tissues, such as skin, where once delivered, the microparticles are taken up by APCs, which can thus process the encoded antigens and present them to T lymphocytes for immune stimulation.
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(ii) Chemical Methods:
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(a) Liposomes and Cationic Lipids (Lipofection):
Liposomes are closed vesicles formed by one or more lipid bilayers surrounding a core aqueous compartment. Liposomes were originally developed in the 1960s and are now extensively used to carry different types of molecules. When liposomes form in a solution containing a drug or a nucleic acid, the aqueous core of the liposome traps the cargo, which can eventually get transported into the cell.
Once in contact with a cell, liposomes can directly fuse with the plasma membrane, thus liberating their cargo into the cytosol, or be actively endocytosed. Liposomes are broadly classified based on the polar head groups into anionic, cationic, zwitterionic, and non-ionic liposomes.
(b) Divalent Cations:
The use of divalent cations has been the most effective chemical treatment to bring about transformation. Among various cations, divalent calcium cation (Ca2+) has proven to be the most effective one both alone and in various combinations.
A combination of divalent and monovalent ions, such as calcium and magnesium, calcium and manganese , calcium, rubidium, and dimethyl sulfoxide and other alkali metals with a prolonged incubation at 0°C has also been reported to be effective.
(iii) Viral Methods:
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A viral genome that is modified to accommodate an exogenous sequence of interest is called a vector . The generation of different viral vectors from their parental genomes consist of the following steps:
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(a) Eemoval, from the viral genome, of most genes coding for viral proteins and, in particular, of those that are potentially pathogenic.
(b) Maintenance of the cis-acting sequences of the viral genomes required for viral replication; in particular, those determining inclusion of the genomes within the viral particles (packaging signal, ψ).
(c) Expression of the viral genes required for viral replication within the virus-producing cells (called packaging cells) encoded by transiently transfected plasmids, or expressed in the context of a helper virus simultaneously infecting the packaging cells, or directly contained inside the packaging cell genome which was previously engineered.
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Proteins are some of the most important biological macromolecules in living systems with multifaceted structural and functional roles. One of the biggest features of proteins is to work in in-vitro conditions which makes them an important and powerful tool in synthetic biology. As per the 2014 research report released by Markets & Markets, the protein engineering market was $610 million and is expected to grow to $1,460 million by 2020.
With the fast evolving arena of protein engineering, we are able to produce proteins of our one choice which have immense therapeutic and industrial implications. Properties of proteins are mostly determined by their primary amino acid sequence in them. Protein Engineering aims to alter these sequences and generate synthetic proteins with viable functions. Some goals of protein engineering are the production of proteins with:
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(i) Better Kinetic Properties
(ii) Thermo stability
(iii) Stability and activity of the enzyme
(iv) Substrate and Reaction specificity
(v) Cofactor Requirement
(vi) Optimum pH
There are many ways by which a protein can be engineered. The most classical approach is “site-directed mutagenesis”. This can be done in two ways:
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(i) Overlap Extension:
This method uses 4 primers and a series of two PCRs. Two of the 4 primers contain the mutagenic code. After denaturation and annealing, two hetroduplexes are produced with each one containing the desired mutagenic code.
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(ii) Whole Plasmid single round PCR:
In this method, two sets of primers complimentary to a plasmid DNA with the mutagenic code is used, which then undergoes PCR. Both strands are replicated without displacing the primer and mutated plasmid is obtained. The gene of interest is then nicked out using restriction enzymes followed by transformation into cells.
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However, then disadvantage with these methods are that the structure and function of the proteins should be well known. However that is not the case most of the times. For this purpose, we use “random mutagenesis”.
The only important factor here is deciding a suitable selection scheme for the desired property. It can be done in two ways:
(i) Saturation Mutagenesis:
It involves the replacement of a single amino acid within a protein with each of the natural amino acids and provides all possible variations at that site.
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(ii) Localized or region specific Mutagenesis:
It is a combination of rational and random approaches of protein engineering. It includes the simultaneous replacement of a few amino acid residues in a specific region, to obtain proteins with new specificities.
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Along with these methods, Homology modelling of protein structures, NMR of large proteins, molecular dynamics simulations of protein structures, and simulation of electrostatic effects (such as pH-dependent effects) are important to determine various additional information about the synthesised protein.
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Peptidomimetics is the mimicking or blocking the activity of enzymes or natural peptides upon design and synthesis of peptide analogs that are metabolically stable. Peptidomimetics is an important approach for bioorganic and medical chemistry. It also holds importance in protein engineering.