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A DNA fragment (called an open reading frame, or ORF) must be inserted into an expression vector, typically a plasmid vector, and then this vector must be transformed into bacterial cells for recombinant protein to be expressed in bacteria. The target protein is subsequently forced into expression by cultivating the cells. The samples are prepared, the cells are extracted by centrifugation, and the proteins are identified by polyacrylamide gel electrophoresis. The gel is then stained with Coomassie Brilliant Blue, silver stain, or by immunoblotting (1).
The procedure of gene cloning
Following mRNA extraction from the tissue that has the highest concentration of the desired protein, reverse transcription is utilised to create cDNA from the various mRNAs. Open reading frame, or ORF, of the cDNA encoding the target protein is amplified by PCR using gene-specific primers, and at the same time, extra sequences are introduced that contain restriction sites for cloning the DNA into a vector. Utilising restriction enzymes, the vector and the PCR fragment are both digested before being combined in a ligation step (2). After the amplification of the DNA template, the DNA was separated through agarose gel electrophoresis. The separated DNA sample was subjected to cloning inside the bacterial plasmid.
Consider the lac promoter, which only starts transcription when lactose or the IPTG (isopropyl-D-1-thiogalactopyranoside), a non-hydrolysable lactose counterpart, is given to the culture. The gene governed by the lac promoter is programmed into mRNA after IPTG induction and is subsequently transformed into protein. The late promoter of the bacteriophage T7 is a highly well-liked promoter. Due to the involvement of two levels of amplification, the T7 promoter is part of an expression system that is more intricate.
First, bacterial strains that have undergone genetic modification and have the lac promoter-controlled gene expressing T7 RNA polymerase from the bacteriophage T7 in their genomes are employed (3). The expression plasmid can be multiplied in appropriate E. coli strains before plasmid purification and before transfecting the plasmid onto the expression host cells to get these quantities. The expression of the target genes (genes related to the protein of interest) is induced by supplying a source of T7 RNA polymerase in the host (4).
In order to create several copies of a recombinant DNA molecule, bacterial transformation is an important step in the molecular cloning process. Traditional cloning fundamentals address the first stages for making recombinant plasmids, which entail inserting an interesting DNA sequence into a vector backbone. A competent variety of bacteria is injected with the DNA (often in the shape of a plasmid) during transformation, enabling the bacteria to replicate the desired sequence in quantities appropriate for additional research and/or manipulation (5).
Process of transformation
LB agar plates was prepared and settled at room temperature. A suitable antibiotic is added to the LB agar depending on the antibiotic marker found in the plasmid DNA. After addition of antibiotic, the LB agar plates for kept overnight in the shaking incubator. For blue/white screening for recombinants, 500 g/mL ferric ammonium citrate, 300 g/mL S-Gal, or 40 g/mL X-Gal are mixed into the LB agar along with 1 mM IPTG.
In a microcentrifuge or falcon tube, 1 to 5 microlitre of DNA (10 pg to 100 ng) was mixed with 20 to 50 microlitre of competent cells.
The most frequent bacterial variety employed in a cloning workflow's transformation step is E. coli. The cells must be rendered competent for conversion by heat shock or by the electroporation because E. coli's native competency is extremely low or even non-existent.
Depending on whether transformation will occur through heat shock or electroporation, different methods must be followed to create competent cells. In both situations, a single, brand-new colonies of the appropriate strain are removed off an agar plate and injected into a liquid medium to create a starter culture. By continuously measuring optical density at 600 nm using spectrophotometer, this starter culture and the ensuing bigger culture are closely watched for active growth.
Prior to chemical transformation, purification is typically not needed when a ligation mixture is employed as the transforming DNA (commonly 1–5 microlitre is adequate). It is crucial to keep in mind that ligation combinations may produce transformation efficiencies that are as low as 1–10% when compared to transformation using a supercoiled undamaged plasmid DNA (6).
According on the DNA type and bacterial strain utilised, heat shock is carried out at 37–42 °C for 25–45 seconds. The heat-shock interval, which is determined by the surface-to-volume ratio of the cell suspension, ought to be shortened when cells are contained in lower volumes or smaller tubes. Then, for around two minutes before the following step, heat-shocked cells are placed back on ice (7).
The transformed cells containing gene of interest was lysed using sonicator. The lysed cell was centrifuged using centrifuge or ultra centrifuge at 4֯C for 10-30mins 4000xg (low temperature- protects the protein) for the collection of the expressed protein.
- Langlais C, Korn B. Recombinant protein expression in bacteria. Encyclopedic Reference of Genomics and Proteomics in Molecular Medicine, Springer, Berlin Heidelberg. 2006:1609-16.
- Konczal J, Gray CH. Streamlining workflow and automation to accelerate laboratory scale protein production. Protein Expression and Purification. 2017 May 1;133:160-9.
- Hausjell J, Kutscha R, Gesson JD, Reinisch D, Spadiut O. The effects of lactose induction on a plasmid-free E. coli T7 expression system. Bioengineering. 2020 Jan 6;7(1):8.
- Greenwich JL, Alakavuklar MA, Fuqua C. An Inducible T7 Polymerase System for High-Level Protein Expression in Diverse Gram-Negative Bacteria. Microbiology Resource Announcements. 2023 Jan 16:e01119-22.
- Arai T, Aikawa S, Sudesh K, Kondo T, Kosugi A. Electrotransformation of thermophilic bacterium Caldimonas manganoxidans. Journal of Microbiological Methods. 2022 Jan 1;192:106375.
- Green MR, Sambrook J. Cloning and transformation with plasmid vectors. Cold Spring Harbor Protocols. 2021 Nov 1;2021(11):pdb-top101170.
- Green MR, Sambrook J. The Inoue method for preparation and transformation of competent Escherichia coli:“Ultracompetent” cells. Cold Spring Harbor Protocols. 2020 Jun 1;2020(6):pdb-rot101196.