Bacterial plasmid Flashcards
intro
Cloning a “gene of interest” into a bacterial expression plasmid is a fundamental technique in molecular biology used for the production of recombinant proteins. The process involves several key steps, including PCR amplification, primer design, ligation into a plasmid vector with a multiple cloning site (MCS), and verification of successful cloning. Each step ensures the accurate insertion of the gene and its subsequent expression in a bacterial host such as Escherichia coli.
First step - pcr amplification
The first step in the cloning process is the amplification of the gene of interest using Polymerase Chain Reaction (PCR). PCR is a method that allows for the specific amplification of a target DNA sequence, which is the gene of interest in this case. The primers used in PCR are short, single-stranded sequences of DNA that flank the region of interest and are designed to match the sequences at the 5’ and 3’ ends of the gene (Kumar et al., 2012). These primers are typically designed to include restriction enzyme recognition sites, which are essential for inserting the amplified gene into the plasmid vecto
Second step - primer design
Primer design is critical to ensuring the efficient and accurate amplification of the gene. Primers must be designed with care to avoid non-specific binding and primer-dimer formation. The forward primer typically matches the 5’ region of the gene, while the reverse primer complements the 3’ region. In addition to these, restriction enzyme sites are added to the primers. These sites enable the amplified gene to be cut with restriction enzymes, allowing for easy ligation into the plasmid vector’s multiple cloning site (MCS). The restriction enzyme sites chosen must be compatible with the MCS of the expression plasmid to ensure that the gene is inserted in the correct orientation for expression
Third step - ligation
After PCR amplification, the gene of interest is digested with restriction enzymes that cut the gene and the plasmid vector at the desired sites. The plasmid vector, typically a bacterial expression plasmid, is chosen based on the ability to promote high levels of protein expression. The plasmid contains an origin of replication, a selectable marker (such as an antibiotic resistance gene), and a multiple cloning site (MCS), which is a region with several restriction enzyme recognition sites that allows for the insertion of foreign DNA (
Once the gene is cut and the plasmid is prepared, the two are mixed and ligated using DNA ligase. This enzyme facilitates the formation of phosphodiester bonds between the plasmid and the gene, resulting in a recombinant plasmid that contains the gene of interest (Kumar et al., 2012).
4th step - transformation into ecoli
The recombinant plasmid is then introduced into E. coli cells through a process called transformation. In this process, chemically competent or electroporated E. coli cells are exposed to the recombinant plasmid, allowing them to take up the plasmid DNA. These cells are then plated onto agar plates containing an antibiotic to which the plasmid confers resistance. Only the E. coli cells that have successfully taken up the recombinant plasmid will grow on the antibiotic-containing medium, facilitating the identification of transformed cells (Kumar et al., 2012).V
verification methods
To confirm that the gene of interest has been successfully cloned into the plasmid, a blue/white screening method is often employed. This method relies on a colorimetric reaction that occurs when the plasmid’s MCS is inserted into a lacZ gene, which codes for beta-galactosidase. If the MCS is intact, the lacZ gene will produce a functional enzyme, causing colonies containing plasmids without inserts to turn blue on agar plates containing the substrate X-gal. However, when the gene of interest is inserted into the MCS, it disrupts the lacZ gene, preventing beta-galactosidase production, and the colonies remain white.
Alternatively, The most definitive way to confirm the correct insertion and sequence of the gene is through DNA sequencing. After amplifying plasmid DNA from individual colonies, the plasmid is sequenced to check for the correct gene sequence, ensuring no mutations have been introduced during the cloning process (Kumar et al., 2012). The sequence data can also confirm that the gene is in the correct orientation and frame for expression in E. coli.
conc
Cloning a gene of interest into a bacterial expression plasmid involves several precise steps, from PCR amplification and primer design to ligation into a plasmid, transformation into E. coli, and screening for successful insertion. Methods such as blue/white screening, restriction digest analysis, sequencing, and protein expression verification are crucial for confirming the correct cloning and successful expression of the gene. These steps are foundational in producing recombinant proteins for research, industrial, and pharmaceutical applications.
