What is selection and counter selection?

In contrast to selection markers, counter-selection markers serve to eliminate unwanted elements. These markers are often toxic or otherwise inhibitory to replication under certain conditions. Selective conditions often involve exposure to a specific substrates or shift in growth conditions.

How does sacB work?

sacB comes from the gram-positive bacteria Bacillus subtilis and encodes the enzyme levansucrase that converts sucrose into a toxic metabolite in gram-negative bacteria. Exchange of the sacB gene for the gene of interest allows the bacteria to grow on sucrose.

What is a counter selection?

Counterselection is a genetic engineering technique to eliminate specific genetic fragments containing selectable marker genes. Although the technique is widely used in bacterial genome engineering and plasmid curing experiments, the repertoire of the markers usable in Escherichia coli is limited.

What are antibiotic resistance markers?

Selectable markers are often antibiotic resistance genes (An antibiotic resistance marker is a gene that produces a protein that provides cells expressing this protein with resistance to an antibiotic.).

What is the difference between positive and negative selection?

Positive selection involves targeting the desired cell population with an antibody specific to a cell surface marker (CD4, CD8, etc.). Negative selection is when several cell types are removed, leaving the cell type of interest untouched.

Is the plasmid suitable for positive selection?

A number of selection systems have been developed for direct selection of recombinant plasmids in cloning experiments (positive selection). In this study, the commonly used LacZ-based α-complementation plasmid vectors have been used for designing a positive selection system for the selection of recombinants.

How do you do plasmid transformations?

DNA transformation protocol

1. Thaw all reagents completely on ice.
2. Add 1 µL of ligation reaction to thawed competent cells.
3. Gently mix by tapping tube of competent cells.
4. Incubate reaction on ice for 30 minutes.
5. Heat shock the competent cell mixture by incubation for 30 to 60 seconds in a 42°C water bath.

What is a Counterselection marker?

The most-used counterselectable markers are the genes that confer sucrose, streptomycin, or fusaric acid sensitivity. They have been used to construct mutants or vaccine strains in Mycobacterium tuberculosis, Helicobacter pylori, Bordetella pertussis, and many other bacteria.

What is counter selective marker?

Counter-selectable markers can be used in two-hybrid systems to search libraries for a protein or compound that interferes with a macromolecular interaction or to identify macromolecules from a population that cannot mediate a particular interaction.

What are examples of antibiotic resistance?

Examples of bacteria that are resistant to antibiotics include methicillin-resistant Staphylococcus aureus (MRSA), penicillin-resistant Enterococcus, and multidrug-resistant Mycobacterium tuberculosis (MDR-TB), which is resistant to two tuberculosis drugs, isoniazid and rifampicin.

What are the types of antibiotic resistance?

Bacteria resistant to antibiotics

• methicillin-resistant Staphylococcus aureus (MRSA)
• vancomycin-resistant Enterococcus (VRE)
• multi-drug-resistant Mycobacterium tuberculosis (MDR-TB)
• carbapenem-resistant Enterobacteriaceae (CRE) gut bacteria.

Is natural selection a negative process?

Natural selection can be of two types, based on its effect on the fate of genetic variations: purifying (negative) selection and positive (Darwinian) selection. The principal types of selection determining the distribution of traits across a population are directional, stabilizing, disruptive, and balancing selection.

Which is a counterselection tool used in cell culture?

Thymidine kinase (TK) is a popular counterselection tool used in mammalian cell culture. TK is involved in the salvaging of nucleotides for DNA synthesis through the phosphorylation of deoxythymidine to deoxythymidine 5′-phosphate (TMP).

How is URA3 used in selection and counterselection?

URA3 normally catalyzes the decarboxylation of orotidine 5-phosphate (OMP) to uridylic acid (UMP). The gene is especially useful as a marker in that it can be used for both selection and counterselection[5].

Why do you need a COunterselectable marker on a vector?

For this purpose, a counterselectable marker is included on the vector. Upon induction of the counterselectable condition, only those cells that have excised the vector sequence through a second recombination event will survive. Since this recombination event is usually rare the counterselection step is essential to find the desired mutants.