Biomolecular Engineering Solutions for Renewable Specialty Chemicals. Группа авторов
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called transfection.
1.2.3.2.2 Electroporation
An electric current is used to create transient microscopic pores in the recipient host cell membrane allowing rDNA to enter.
1.2.3.2.3 Microinjection
Exogenous DNA can also be brought immediately into animal and plant cells without using eukaryotic vectors in the system of microinjection, foreign DNA is directly injected into recipient cells the use of a quality micro syringe under a section contrast microscope to useful resource imaginative and prescient.
1.2.3.2.4 Biolistics
An excellent technique that has been developed to introduce foreign DNA into in particular plant cells is by means of using a gene or particle gun. Microscopic particles of gold or tungsten are covered with the DNA of interest and bombarded onto cells with a device just like a particle gun. Subsequently, the time period biolistic is used.
1.2.4 Selection of Transformants
The artwork of cloning is to determine the precise transformed cell that consists of the cloning vector with the gene of interest (referred to as a recombinant cell). It is also very important to find the recombinant cells from the culturing transformed petri plate where nonrecombinant (containing the vector without insert DNA) transformed cells are also present. If the final goal is achieved, that means our desired gene of interest is inserted into the vector then it will be called as clone. Direct selection and selection from gene library are the two basic concepts for selection procedures. Direct selection involves designing of experiment in such a way that transformation takes place only for the clones containing specific gene of interest. Selection happens on the plating‐out stage of the transformation where the colonies are grown on agar plates. It is the preferred technique because of the ease and unambiguous in nature. Selection from gene library involves initial “shotgun” cloning test, to produce a clone library representing all or most of the genes present inside the cell, observed through evaluation of each individual clones to identify the correct one.
1.2.4.1 Direct Selection
Majority of cloning vectors are designed in such a way that inserting gene of interest in them inactivates the gene already present in the vector. This leads to insertional inactivation of gene. Two of the common examples of insertional inactivation, i.e., direct antibiotic resistance screening and blue white screening are discussed below.
1.2.4.1.1 Direct Antibiotic Resistance Screening
Cloning vectors are planned in such a way that insertion sites are present in between the genes in the vectors, often antibiotic resistance gene. Vector carrying ampicillin resistance gene confers resistance to ampicillin to the cells in which it will be transformed to. Transformed cells when grown in agar plates containing ampicillin shows resistant to the antibiotic and are capable to grow. But this does not guarantee the presence of gene of interest in the vector or the vector is recombinant. Ampicillin resistance gene become disrupted in vector having gene of interest. Thus, the transformed cells having self‐ligated vector grows on ampicillin agar plate, whereas the cells having recombinant vector does not show growth. The recombinant cells can be selected from the non‐recombinant one through replica plating method.
1.2.4.1.2 Blue–White Color Screening
Blue‐white screening is also based on insertional inactivation of a gene giving blue color compound as a visual representation of the result. The gene that plays a key role in this screening is lacZ gene which codes for the enzyme β‐galactosidase is under the control of the inducible lac promoter. lacZ gene is repressed by lac repressor and is induced by IPTG (isopropyl‐β‐D‐thiogalactopyranoside). The enzyme formed can breakdown X‐gal (five‐bromo‐4‐chlore‐3‐indolyl‐β‐D‐galactopyranoside) to give a blue color compound.
Insertional inactivation of lacZ gene due to insertion of desired gene prevents the formation of the enzyme, breakdown of X‐gal, and hence no blue color is obtained. The cells are plated on agar plates having IPTG, X‐gal, and an antibiotic giving combination of blue and white colonies. The transformed cells with religated vector having functional lacZ gene shows blue color colonies. Whereas the cells with gene of interest and disrupted lacZ gene forms colorless colonies. Further the colorless colonies can be picked and plated again.
1.2.4.2 Identification of the Clone from a Gene Library
Screening a positive clone from gene library involves typical techniques such as nucleic acid hybridization, functional screening, and chromosome walking. Nucleic acid hybridization needs preliminary information of both the gene of interest and area of gene to be cloned. Contrary, functional screening entails the information about the vector.
1.2.4.2.1 Nucleic Acid Hybridization
Nucleic acid hybridization utilises short synthetic radiolabeled oligonucleotides called as probe. These probes are used for locating complementary sequences in individual cells or phages containing an insert. The success of the hybridization experiment relies upon the probe. Therefore, probe should be designed with care and should have some part complementary to the sequence of the cloned gene. If there is no information about the gene, then the sequences from the related protein can be derived and degenerate probes can be synthesized to be used. So, in this technique first the colonies are transferred to a nylon membrane and then lysis of cells will be done. The released DNA will be denatured through an alkali treatment. The denatured single‐stranded DNA will be heat treated so that they could bind to the membrane. After that the membrane will be submerged into solution containing DNA probes and incubated for a certain time to get hybridized by complementary base pairing. Finally, the hybridized radiolabeled probes will be identified by autoradiography.
1.2.4.2.2 Functional Screening
If the gene encodes for a product that has a specific role, then the expression library is by means of cloning DNA (cDNA in case of eukaryotes). These libraries are prepared using unique cloning vectors for expression of cloned genes. The prepared library can be used to detect the product or the clones generated by using antibodies or other ligands. These antibodies or ligands binds with the encoded product and clones.
1.2.4.2.3 Chromosome Walking
Usually a genomic clone may not be includ all of the sequences for a specific gene so it is required to isolate overlapping clones that protect the genomic segment of interest. This process is known as chromosome walking.
1.3 Beneficial Biocommodities Produced Through Engineered Microbial Factories
Earlier biocommodities (up to some extent now also) were extracted from plants and animals leading to their overexploitation (Brower, 2008). Plants are the great source of secondary metabolites that have complex structures and used by humans, e.g. medicines, terpenes, flavoring and coloring agents, etc. (Facchini et al., 2012). Their yields are greatly affected by the time, climate, and other factors, which affect circadian rhythm of the plants (Li and Vederas, 2009). Moreover, the yields cannot satiate the current demand of the population. Being structurally complex in nature relying on chemical synthesis of the secondary metabolites is not feasible. Chemical synthesis also leads to toxicity of the end products and environmental pollution (Du et al., 2011). To overcome these challenges, the principles of genetic engineering explained in the earlier section are used to engineer microorganisms to become microbial cell factories to produce essential biocommodities such as bioplastic,