Biotechnology: Gateway Cloning & Protein Localization

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Questions and Answers

What is the role of the int, IHF, and Xis proteins during the excision process?

They facilitate the integration of phage DNA.

They prevent the recombination of attP and attB.

They catalyze the excision process. (correct)

They stabilize the non-homologous sequences.

Which sites are reformed when excision occurs between attL and attR?

attE and attF

attC and attD

attL and attR

attP and attB (correct)

What are the two main reactions involved in the Gateway cloning process?

BP reaction and LR reaction (correct)

Entry clone formation and expression clone formation

Transformation reaction and transfection reaction

Integration reaction and excision reaction

What is the ultimate goal of the Gateway reactions?

To make an expression clone for later analysis Signup and view all the answers

What is the first step in the Gateway cloning process?

Cloning the gene of interest into a donor vector Signup and view all the answers

What type of reaction is described for creating an entry clone from an attB-PCR product?

BP reaction Signup and view all the answers

Which sequence is invariant during the recombination process in the BP reaction?

GTACAAA Signup and view all the answers

What is the outcome of the BP recombination reaction?

An entry clone and a by-product Signup and view all the answers

What defines the composition of the attL sites in the recombination region?

Sequences from the attB and attP sites, plus an invariant region Signup and view all the answers

What is the next step after creating the entry clone in the BP reaction?

Subcloning into a destination vector via the LR reaction Signup and view all the answers

Which E.coli strain should be used to propagate vectors with the lethal ccdB gene?

DB3.1 Signup and view all the answers

What is the specific mutation carried by the DB3.1 strain that provides resistance to ccdB?

gyrA462 Signup and view all the answers

What will happen to E.coli cells transformed with empty pEarley vectors lacking the gyrA462 mutation?

They will be unable to grow. Signup and view all the answers

Which characteristic is common among most commercial E.coli strains used in molecular biology?

Fast growth rates Signup and view all the answers

Which of these strains is directly descended from the K-12 strain?

DH5alpha Signup and view all the answers

What is the primary purpose of the common commercial E.coli strains in molecular biology?

To enhance plasmid yield and DNA quality Signup and view all the answers

What kind of amplifications does the colony PCR using AttB1-specific and YFP-specific primers target?

Amplification of CBF4 gene Signup and view all the answers

Which of the following is NOT a main characteristic of commercial E.coli strains?

Resistance to all toxins Signup and view all the answers

What does the ccdB gene code for?

A toxic protein associated with cell death Signup and view all the answers

Which strains of E.coli can be used to propagate ccdB-containing vectors?

F’ strains such as JM109 and XL1 Blue Signup and view all the answers

What is a disadvantage mentioned regarding the Gateway cloning system?

There are no disadvantages mentioned Signup and view all the answers

What happens to cells that lack ccdA?

They succumb to the toxicity of CcdB Signup and view all the answers

What is the primary role of the ccdA gene in the ccd operon?

To produce a protein that neutralizes CcdB toxicity Signup and view all the answers

In the context of cloning, what is a key feature of the Gateway system?

It permits the cloning of fragments without purification Signup and view all the answers

What type of protein is CcdB?

A toxin that interferes with cellular processes Signup and view all the answers

Why are common cloning strains of E.coli considered F-?

They lack the F plasmid required for ccdA Signup and view all the answers

What is the primary purpose of tagging proteins with fluorescent proteins in gene studies?

To enable tracking of subcellular localization Signup and view all the answers

Which technique can be used alongside fluorescent protein tagging to study proteins?

Immunolocalization Signup and view all the answers

What is the significance of CBF4 belonging to the AP2/EREBP transcription factor family?

It shows its involvement in cold stress response Signup and view all the answers

Which component is NOT involved in the Gateway® recombination cloning technology?

Restriction enzyme digestion Signup and view all the answers

In the context of protein-reporter fusions, which protein is commonly used as a fluorescent marker?

GFP (Green Fluorescent Protein) Signup and view all the answers

What is one limitation of using fluorescent protein fusions for tracking protein localization?

They can alter the protein's native function Signup and view all the answers

What is the result of generating a CBF4-YFP fusion protein?

Localization tracking of CBF4 in cells Signup and view all the answers

What common feature exists between CBF1, CBF2, and CBF3?

They are all known cold stress response regulators Signup and view all the answers

What is the required sequence at the 5' end of the forward PCR primer for Gateway® cloning?

Four guanine residues (GGGG) Signup and view all the answers

What must follow the four guanine residues in the forward PCR primer?

25 bp attB1 site Signup and view all the answers

What is the minimum length of template-specific sequences required in the forward PCR primer?

18-25 bp Signup and view all the answers

Which nucleotides are NOT allowed as the two additional nucleotides in the forward primer when fusing with an N-terminal tag?

AA, AG, GA Signup and view all the answers

What is the structure of a reverse PCR primer necessary for Gateway® cloning?

Four guanine residues followed by an attB2 site Signup and view all the answers

If a C-terminal tag is included in the reverse PCR primer, what must be incorporated?

One additional nucleotide Signup and view all the answers

What should be ensured if a stop codon is present in the reverse PCR primer?

It should be in-frame with the gene of interest or the primer Signup and view all the answers

What should be removed if there are stop codons between the attB2 site and the gene of interest for C-terminal fusions?

Any in-frame stop codons Signup and view all the answers

Study Notes

Gateway® Recombinational Cloning & Fluorescent Protein Fusion Constructs

The Gateway® system is based on site-specific recombination by the lambda phage.
The lambda phage integrates its DNA into the E. coli chromosome using recombination.
Both the phage and the bacterium have specific recombination sites, attP in the phage and attB in the bacteria.
The integration process, lysogeny, is catalyzed by the two enzymes, Int (Integrase), and IHF (Integration Host Factor).

Protein Localization with Fluorescent Proteins

Protein localization studies are often part of gene function and knockout studies.
Fluorescent proteins (e.g., GFP, YFP) are often used to tag proteins.
These techniques can help researchers track the subcellular and spatiotemporal localization of a protein in living cells.
The coding region of the gene is fused to the DNA marker gene like GFP to generate protein-reporter fusions.

Subcellular Localization of CBF4

CBF4 is related to CBF1, CBF2, and CBF3 and belongs to the AP2/EREBP family of transcription factors.
CBF1/2/3 bind to the promoter region of genes that are regulated by cold.
Recombinational cloning (Gateway® Technology, Invitrogen) will be used to generate a CBF4-YFP fusion protein.
The CBF4 gene will be cloned into a plant expression vector carrying a strong 35S promoter and YFP to generate 35Sp:CBF4-YFP.
This construct will be introduced into plants to determine the subcellular localization of the CBF4 protein.

Experiment Outline

Clone CBF4 into a plant expression vector containing YFP via Gateway recombination cloning (LR reaction).
Transform the LR reaction into E. coli, followed by colony PCR.
Isolate and sequence the plasmid.
Transform the plasmid into Agrobacterium.
Infect plant leaves (N. benthamiana) for transient expression.
Localize protein using confocal microscopy.

Invitrogen Gateway® Recombinational Cloning Technology

The Gateway® cloning is a two-step process (BP reaction and LR reaction).
The BP reaction produces the entry clone.
The LR reaction creates the expression clone.
The reactions are in vitro versions of the integration and excision reactions used in lambda phage integration.

Types of Clonase Enzymes

Gateway BP Clonase Enzyme Mix contains Int (Integrase) and IHF (Integration Host Factor).
Gateway LR Clonase Enzyme Mix contains Int (Integrase), IHF (Integration Host Factor), and Xis (Excisionase).

Donor Vector and the ccdB Gene

The donor vector (pDONR207) carries the aacC1 gene (Gent') which provides gentamycin resistance.
pDONR207 also has the ccdB gene, which is toxic to E. coli. This aids in selecting for the correct plasmids, making cloning easier.
The ccdB gene targets the E. coli gyrase, inhibiting the cell's growth.
Using E. coli strains resistant to ccdB, ensures that only transformed bacteria survive and allows high-efficiency recovery of the desired clones.

Recovery of Entry Clone Following BP Reaction

After the BP reaction, the ccdB gene is released and replaced by the inserted gene (CBF4).
Laboratory strains of E. coli sensitive to ccdB (e.g., DH5α) are then used, facilitating recovery of desired clones.

Generating the CBF4 Expression Clone using Gateway Cloning

The entry clone (carrying the CBF4 gene) and the destination vector are mixed with LR clonase.
This results in two constructs: the CBF4 expression clone and a byproduct that contains the CmR and ccdB genes.
The LR reaction is then transformed into E. coli—specifically those lacking the gyrase mutation (i.e., DH5α).
Only cells containing the correct recombined construct will survive and grow.

pEarleyGate Destination Vector

The pEarleyGate101 vector contains a promoter to express the gene in the intended host.
Other useful genes include a selectable marker, polyadenylation and termination sequences.

pEarleyGate: Selectable Markers

The pEarleyGate 101 vector has selectable markers including nptll (KanR or Km) for kanamycin or neomycin resistance.
Also, cmla (CmR) gene for chloramphenicol resistance, and BAR for resistance to the herbicide Basta.

Colony PCR Primer Design

Primers containing four guanine residues at the 5' end are often used for colony PCR.
Additional gene-specific sequences are necessary to generate the desired product.

Confirming Cloned DNA Fragment Sequences

Cloning sequences are confirmed using sequencing to ensure correct orientation, reading frame, etc.
Plasmids can be purified for sequencing.
Sequencing results can be analyzed using chromatograms to confirm the correct sequence.

Gateway Cloning: Advantages and Disadvantages

Advantages: fast, simple cloning procedure, versatility for various uses, facilitates cloning multiple genes into the same vector (for high throughput).
Disadvantages: recombination products can contain "scar" regions, cost of Clonase enzymes is relatively higher compared to restriction/ligation.

Common E.coli Strains Used in the Lab

Many laboratory strains are descended from the K-12 and B strains.
Some strains have mutations increasing plasmid yield.
Strains have been developed for specific purposes.

The CcdA-CcdB Toxin-Antitoxin System

ccdB aids in identifying clones with the correct insert by eliminating those that lack the insert.
The ccd operon is a toxin-antitoxin system important in plasmid maintenance during cell division.
ccdB encodes a toxic protein (CcdB) which inactivates DNA gyrase.
ccdA encodes an antitoxin that protects from the toxic effects of ccdB.

CcdB Resistant E. coli Strains

DB3.1 strain is frequently used for cloning experiments in the presence of ccdB.
Other strains (e.g., JM109, XL1 Blue, XL10 Gold) might also be used depending on the protocol, but should not be used as the primary strain for ccdB selection.

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Description

This quiz covers key concepts related to the Gateway® recombinational cloning system and the use of fluorescent proteins in protein localization studies. Topics include the mechanisms of DNA integration by lambda phage and the application of GFP and YFP in tracking protein localization in living cells. Test your knowledge on these fundamental biotechnological techniques.