Recombinant selection with pBR322

Insertional inactivation of an antibiotic resistance gene

Insertional inactivation is a technique of recombinant DNA technology used to select bacteria that carry recombinant plasmids, a DNA fragment of interest is inserted into a restriction site within a gene conferring antibiotic resistance, causing that gene to become nonfunctional thus, inactivating the resistance gene. This technique is particularly valuable in selecting ampicillin (amp^R) and tetracycline (tet^S) transformed Escherichia coli (E. coli) carrying plasmid such pBR322; foreign DNA is cloned within the tet gene, disrupting the gene and rendering the cell tet^S (tetracycline sensitive) which enabled the selection when cells were plated onto amp and tet plates.

Insertional inactivation of an antibiotic resistance gene procedure.

• Antibiotic resistance can be used as an insertional inactivation system if DNA fragments are cloned into a restriction site within an antibiotic resistance gene.
• Foreign DNA is cloned within the tetracycline resistance gene of pBR322, disrupting the gene and making the cell sensitive to tetracycline but still resistant to ampicillin.
• Transformed cells are plated on an ampicillin-containing medium. Colonies that grow on ampicillin indicate successful transformation (amp^R).
• Transformed colonies are replica plated onto the tetracycline-containing medium. Some colonies will not grow on tetracycline, indicating disruption of the tetracycline resistance gene.
• Colonies that do not grow on tetracycline are recombinants (amp^R tet^S). These colonies have the tetracycline resistance gene disrupted by the inserted DNA. Once their positions are known, samples for further study can be recovered from the original ampicillin agar plate.
• The method involves a single step of plating on both ampicillin and tetracycline. Recombinants can be visually identified based on their inability to grow on tetracycline.
• Verify the presence of the inserted gene in the selected colonies using suitable molecular biology techniques such as PCR and DNA sequencing

Advantages and Limitations of Insertional Inactivation

Advantages

1. Insertional inactivation provides a selectable marker for identifying recombinant colonies. This allows for the specific identification of colonies where the tetracycline resistance gene has been inactivated by the insertion of foreign DNA.
2. The technique can be used in various applications of recombinant DNA technology, such as the production of vaccines, insulin, hormones, and genetically modified plant varieties.

Limitations

1. The size of the foreign DNA fragment that can be inserted without disrupting the plasmid’s essential functions is limited. Large insertions may interfere with the plasmid’s replication or stability.
2. The insertion of foreign DNA could lead to unintended effects on the plasmid, such as changes in its replication or compatibility with host cells.
3. In the case of pBR322, the use of tetracycline resistance as the sole marker for recombinants limits the ability to use tetracycline for other experimental purposes, as all non-recombinant colonies will also grow on tetracycline.

Insertional inactivation using LacZ.

Insertional inactivation, while effective for recombinant identification, poses challenges due to the requirement for two screenings. The first screening involves an antibiotic selecting for transformants, followed by a second screen, after replica-plating, with an antibiotic that distinguishes recombinants. To overcome this inconvenience, modern plasmid vectors adopt alternative systems. For instance, pUC8 incorporates a different approach to streamline the process.

pUC8 Modern Plasmid Vector

pUC8 carries both the ampicillin resistance gene and lacZ′, a gene encoding part of the enzyme β-galactosidase. Cloning with pUC8 involves insertional inactivation of the lacZ′ gene, and recombinants are identified by their inability to synthesize β-galactosidase.

The Role of β-galactosidase and LacZ′ Gene Modification

β-galactosidase is involved in lactose breakdown to glucose plus galactose. Some E. coli strains possess a modified lacZ gene lacking the lacZ′ segment, which codes for the α-peptide portion of β-galactosidase. These mutants can only synthesize the enzyme when harboring a plasmid like pUC8 that carries the missing lacZ′ segment.

pUC8 Screening and Identification

In a cloning experiment with pUC8, transformants are initially selected on ampicillin agar. Subsequently, screening for β-galactosidase activity is conducted to identify recombinants. Cells containing a normal pUC8 plasmid are ampicillin-resistant and synthesize β-galactosidase, while recombinants, although ampicillin-resistant, cannot produce β-galactosidase.

Lac Selection and Simplified Screening

The screening for β-galactosidase presence or absence is facilitated by using X-gal, a lactose analog. Non-recombinant colonies, synthesizing β-galactosidase, appear blue, whereas recombinants, with a disrupted lacZ′ gene, are white. This Lac selection system enables the testing of both ampicillin resistance and β-galactosidase production on a single agar plate. The streamlined process eliminates the need for a time-consuming replica-plating step associated with plasmids like pBR322.

FAQs

References

1. pBR322 Vectors Having Tetracycline-Dependent Replication – PMC (nih.gov)
2. https://collegedunia.com/exams/insertional-inactivation-biology-articleid-1544
3. https://www.sciencedirect.com/science/article/abs/pii/0378111983901907
4. Textbook of rDNA Technology, ISBN-13: 978-8127267452
5. Textbook: “The Basic Principles of Gene Cloning and DNA Analysis” by T. A. Brown