Molecular Cloning and Biotech Basics

Naturally occurring, circular DNA molecules found in bacteria.

Enzymes that cut DNA at specific sequences.

The enzyme EcoRI is named after the bacteria it came from (E. coli), the strain (R) and the order discovered (I).

These are cuts in DNA where a restriction enzyme leaves unpaired bases at the ends.

The modification of DNA by adding a methyl group can prevent a restriction enzyme from cutting.

A DNA sequence that is recognized and cut by a specific restriction enzyme. It is often palindromic, meaning it reads the same forward and backward on opposite strands.

A type of restriction enzyme that cuts DNA at a specific sequence, producing sticky ends. Sticky ends are short, single-stranded overhangs that can base pair with complementary ends on other DNA fragments.

A type of restriction enzyme that cuts DNA at a specific sequence, producing blunt ends. Blunt ends have no overhangs and are less efficient for cloning than sticky ends.

A circular piece of DNA that can replicate independently of the host chromosome. They are often used as vectors in cloning experiments.

A region on a plasmid vector that contains multiple restriction enzyme recognition sites. This allows researchers to insert foreign DNA into the plasmid at a specific location.

A bacterial colony that lacks LacZ activity, indicating that a DNA fragment has been inserted into the lacZ gene within the plasmid.

A bacterial colony that expresses LacZ activity, indicating that the lacZ gene in the plasmid is intact and functional.

The addition of IPTG induces the expression of the lacZ gene, which is under the control of the lac promoter.

The use of ampicillin in the growth medium ensures that only bacteria containing the plasmid can grow.

This vector is designed to express a cloned gene in E. coli, with features like a strong promoter, a terminator, and a ribosome binding site.

A molecule that induces the expression of the lac operon in a bacterial cell. It mimics lactose but is not metabolized by the cell, ensuring continuous activation.

A method used in molecular cloning to identify bacteria that have been successfully transformed with a specific DNA insert. Colonies containing the insert appear white, while those without the insert appear blue.

A gene that encodes the enzyme β-galactosidase, which breaks down lactose into glucose and galactose. It is often used in molecular biology for gene expression studies and cloning.

A synthetic substrate used in blue-white screening that turns blue when cleaved by β-galactosidase. It is used to distinguish colonies with or without the lacZ gene.

A type of protein expression vector that uses the T7 RNA polymerase and a strong promoter to allow for high-level protein production in bacterial cells. It also contains a multiple cloning site for inserting genes of interest. This vector uses IPTG (Isopropyl β-D-1-thiogalactopyranoside) as an inducer for the production of T7 RNA polymerase, which then transcribes the cloned gene into mRNA for protein expression.

A method used to purify proteins from a bacterial cell lysate. It uses a Nickel resin, which binds to a specific amino acid tag (His-tag) attached to the target protein. All other proteins that do not contain the His-tag are washed away, leaving only the target protein bound to the resin.

A method for determining the exact sequence of nucleotides in a DNA molecule. It involves a special type of polymerase that can incorporate modified nucleotides that terminate DNA synthesis during sequencing reactions. These reactions generate DNA fragments of different lengths that are then separated by electrophoresis. By reading the sequence of fragments, the exact order of bases in the DNA can be determined.

A collection of DNA sequences from all the microorganisms found in a particular sample, such as a soil sample or a human gut. It allows us to study the diversity and function of these microorganisms without culturing them in the lab.

A system of bacterial defense against viral infection, involving short DNA sequences called spacers that are integrated into the bacterial genome and used to recognize and destroy invading viral DNA.

Short DNA sequences within the CRISPR locus that originate from previous viral infections. They serve as templates for recognizing and destroying future viral infections.

An enzyme that cuts DNA at specific sequences, guided by a CRISPR spacer. It plays a critical role in destroying viral DNA during CRISPR-Cas defense.

A molecular technique based on the CRISPR/Cas system that allows for targeted editing of DNA sequences. It uses a guide RNA to direct Cas9 to a specific location in the genome, where it can make precise changes to the DNA.

A short RNA molecule complementary to a specific DNA sequence that guides Cas9 to the target location in the genome.

A piece of DNA provided to the cell during CRISPR/Cas gene editing which acts as a template for repairing the DNA break made by Cas9. This template can be modified to introduce the desired changes.

A single-base change in the DNA sequence, causing a change in a protein's amino acid sequence.

The hemoglobin produced by the fetus that can compensate for mutated adult hemoglobin, potentially providing a therapeutic benefit for genetic diseases such as sickle cell anemia.