Biotechnology: Gateway Cloning & Protein Localization

The process of determining where a protein resides within a cell or organism. This helps us understand its function.

A method where a fluorescent protein (GFP, YFP, etc.) is attached to another protein, allowing you to track its movement and location within a cell.

A transcription factor that belongs to the AP2/EREBP family, closely related to CBF1, CBF2, and CBF3. Its role in plants is not fully understood.

A system for recombining DNA fragments, used to insert genes into vectors. It utilizes specific enzymes for precise insertion.

A reaction that occurs in Gateway® cloning. It involves an 'entry clone' with the gene of interest and a donor vector. It creates an 'entry clone' for the next step.

A reaction that occurs in Gateway® cloning. It involves an 'entry clone' and an expression vector. It creates a 'destination clone' containing the gene of interest.

The specific location of a protein within a cell, such as the nucleus, cytoplasm, or organelles.

A type of protein fusion where a fluorescent protein is added to another protein during translation, creating a single fused protein.

Specific DNA sequences on the bacterial chromosome (attB) and the phage lambda genome (attP) that are involved in integration and excision of the phage DNA. They share a 15 base pair core sequence.

The process by which phage lambda DNA integrates into the bacterial host chromosome using the attB and attP sites. This is catalyzed by the Int protein.

The process by which the integrated phage lambda DNA is removed from the bacterial chromosome using the attL and attR sites. This is catalyzed by the Xis and Int proteins, and the IHF protein.

A DNA vector containing the gene of interest, generated in the BP reaction of Gateway cloning.

A molecular cloning technique that uses the BP Clonase enzyme to recombine a DNA fragment with an attB site (from a PCR product) into a pDONR vector containing an attP site. This creates an entry clone and a by-product.

A specific DNA sequence present in the PCR product that is recognized by the BP Clonase enzyme and essential for recombination.

A specific DNA sequence present in the pDONR vector that is recognized by the BP Clonase enzyme and essential for recombination.

A molecular cloning technique that uses LR Clonase enzyme to recombine a gene from an Entry Clone into a Destination Vector. This is the second step in the Gateway Cloning System.

The attB1 forward primer must begin with four guanines (GGGG), followed by the 25 bp attB1 site, and then 18-25 bp of gene-specific sequence.

To fuse a PCR product in-frame with an N-terminal tag, the forward primer needs two extra nucleotides to maintain the reading frame with the attB1 region. These nucleotides must not be AA, AG, or GA, as they create stop codons.

The attB2 reverse primer must begin with four guanines (GGGG), followed by the 25 bp attB2 site, and then 18-25 bp of gene-specific sequence.

For C-terminal fusions without a stop codon, the reverse primer needs one extra nucleotide to maintain the reading frame with the attB2 region.

If you don't want to fuse your PCR product with a C-terminal tag, your gene or the primer must contain a stop codon. This ensures the protein is properly terminated.

The gene-specific sequence in both forward and reverse primers ensures that the primer binds only to your desired target gene.

The reading frame refers to the way DNA is read in groups of three nucleotides (codons). Maintaining the correct reading frame is crucial for producing a functional protein.

Colony PCR is used to confirm successful recombination by amplifying the desired gene (CBF4) inserted into the pEarley101 vector using specific primers targeting the AttB1 and YFP regions.

The YFP-specific and AttB1-specific primers are designed to bind to specific regions flanking the insertion site in the pEarley101 vector. They won't bind to sequences within the ccdB gene, meaning they can't amplify it.

K-12 and B strains are common laboratory strains of E.coli with different traits. K-12 strains (like DH5alpha) are typically used for high-throughput cloning and general manipulations, while B strains (like BL21) are preferred for protein expression.

Mutations in E.coli strains can improve specific features for lab use, like faster growth, increased plasmid yield, and improved DNA quality. These mutations are designed to enhance a particular characteristic for specific applications.

Empty destination vectors (like pEarley) carry the ccdB gene, which is toxic to most E.coli strains. Using a CcdB-resistant strain, like DB3.1 with the gyrA462 mutation, allows the vector to be maintained without killing the bacteria.

The gyrA462 mutation confers CcdB resistance. Using a strain lacking this mutation, like DH5alpha, prevents empty destination vectors from growing after the LR reaction. Only cells containing the successfully recombined gene (like 35Sp:CBF4-YFP/pEarleyGate101) will be able to grow.

Common genetic changes include mutations that improve growth rate, plasmid yield, DNA quality, and other desirable traits for specific applications. These modifications provide desirable traits for different laboratory tasks.

Mutant alleles refer to altered versions of genes that confer specific phenotypes. Listing these alleles in the genotype highlights the specific genetic modifications that distinguish a particular strain from the wild type.

The ccdB gene encodes a toxic protein (CcdB) that acts as a DNA gyrase poison, leading to cell death.

CcdB binds to and inhibits DNA gyrase, a key enzyme involved in DNA replication. This blockage of DNA gyrase leads to the accumulation of broken DNA and eventually cell death.

ccdA encodes an antitoxin protein (CcdA) that protects the cell from the toxic effects of CcdB by neutralizing its activity.

The ccdB gene is often included in cloning vectors to select for desired clones. When a vector containing ccdB is introduced into a bacterial cell, it will only survive if the vector has incorporated the desired insert, usually disrupting the ccdB gene.

E.coli strains that contain the F plasmid (F' strains) are resistant to ccdB toxicity because they express ccdA, the antitoxin.

Most common cloning strains of E.coli are F- strains, meaning they lack the F plasmid and are not resistant to ccdB.

Gateway cloning offers several advantages: - It allows cloning of DNA fragments without the need for restriction enzyme digestion, purification, or ligation (fast, simple, and easy procedure). - It's versatile, allowing easy transfer of DNA fragments between different vectors (high-throughput cloning).

Researchers use Gateway cloning to clone and express genes in a variety of organisms, including plants, bacteria, vertebrates, and mammalian cell lines.