Flowering on Demand: Integrating the FT Gene into the pCAMBIA1391Z Vector using Gateway Cloning
By Izabela Ninu
Table of Contents
Introduction
Background
Objective
Part 1: Overview and used technique
Part 2: integrating the FT gene into the pCAMBIA1391Z vector
Introduction:
In this article, we study the integration of the Flowering locus T (FT) gene into the pCAMBIA1391Z vector via Gateway cloning and floral dipping methods. The FT gene, a regulator of flowering in plants, is incorporated into the plant genome to manipulate flowering time, a crucial factor for plant breeding and crop production.
Background:
The FT gene, also known as AT1G65480 in Arabidopsis thaliana, plays a vital role in controlling the flowering process. It encodes the FT protein, a key component of florigen, a mobile signal that triggers flowering by traveling from leaves to the shoot apical meristem. This gene’s conservation across plant species underscores its significance in regulating flowering time, essential for environmental adaptation and successful reproduction. (See the Tair website where the gene was taken from)
Objective:
The primary aim of integrating the FT gene into the pCAMBIA1391Z vector is to study and modulate flowering time in plants. By altering flowering time, we aim to enhance agricultural productivity and develop crops better suited to varying climates and environmental conditions.
Part 1: Overview and used technique
A. Gateway Cloning Method
An overview in nature:
Gateway cloning is a genetic engineering technique characterized by site-specific recombination. It involves two sequential reactions: the BP and LR reactions. Initially observed in a phage infecting E. coli, this method offers advantages over traditional cloning techniques, primarily due to its efficiency and directional nature.
In the Gateway cloning method, the integration of phage DNA into the E. coli genome is facilitated by site-specific recombination. The phage DNA contains attachment sites known as attP, while E. coli has corresponding attB sites. These sites are capable of recombining, allowing for the incorporation of phage DNA into the bacterial genome.
This method is preferred over traditional plasmid-based cloning, which often relies on restriction enzymes and ligase for gene integration — a process prone to failure. The Gateway cloning technique offers a more reliable alternative.
The process begins with the BP clonase reaction, where the gene of interest is flanked by attB sites and combined with BP clonase enzyme and a donor vector. This mixture results in the integration of the gene into the vector through recombination.
Subsequently, the LR clonase reaction takes place. This involves mixing LR clonase with the destination vector and the donor vector containing the gene of interest. During this reaction, the gene is transferred from the donor to the destination vector. The final product is a vector with attL sites, while the destination vector acquires attR sites, enabling further site-specific recombination.
B. How Gateway Cloning Works:
The att sequences
The att sequences are unique regions within the DNA that facilitate the site-specific recombination process. Unlike the sites recognized by restriction enzymes, att sequences are non-palindromic and impart directionality to the recombination event. This is crucial for ensuring the correct orientation of the inserted DNA.
The attB and attP sequences specifically pair with each other due to their shared nucleotide regions. The ‘L’ and ‘R’ designations refer to the left and right arms of the attP sequence, which are necessary for the recombination process.
The BP Clonase enzyme complex, which includes Integrase (Int) and the Integration Host Factor (IHF), catalyzes the forward reaction. This reaction combines attB and attP into attL and attR, facilitating the integration of DNA into the genome.
Conversely, the LR Clonase enzyme complex, composed of Int, IHF, and Xis, drives the reverse reaction. This reaction is used to excise viral DNA from the host genome.
The recombination reaction is enabled by a protein-DNA complex formation. Each enzyme monomer attaches to one side of a recognition site, creating a dimer at each att site. The 3D structures of these dimers differ between the two sites, allowing for correct pairing.
Recombination method
- Formation of DNA-Protein Complex: The cycle begins with the formation of a complex between the DNA and the recombination proteins.
- Cleavage and Covalent Attachment: A serine residue within the active site of the protein monomers cleaves the DNA’s phosphate backbone, creating sticky ends and forming a temporary covalent bond with the DNA.
(More details: During the cleavage phase, a serine residue located in the active site of the recombinase enzyme acts as a nucleophile — a molecule or substance that has a tendency to donate electrons or react at electron-poor sites such as protons. This serine attacks the phosphodiester bond in the DNA’s backbone, specifically targeting the phosphate group.
This attack results in two significant events:
- Creation of Sticky Ends: The cleavage of the DNA backbone generates two single-stranded overhangs, commonly referred to as “sticky ends.” These ends are complementary to each other, which allows them to anneal or “stick” together, facilitating the recombination process.
- Formation of a Covalent Protein-DNA Intermediate: Concurrently with the creation of sticky ends, the serine residue forms a transient covalent bond with the phosphate group of the DNA. This bond is through a phosphoserine linkage, which is essential for the next steps in the recombination process.) The covalent bond is a temporary and holds the enzyme and DNA close. It keeps the energy from the cut DNA bond, which is important for the next part of the process. This stored energy means the reaction can happen without needing extra energy like ATP. Next, the end of the DNA strand with a ‘OH’ group (5’ hydroxyl group) comes in and attacks the covalent bond. This breaks the temporary glue, and at the same time, it ties the DNA strands back together. - DNA Backbone Re-ligation: The final step resembles the action of a ligase enzyme, where the DNA backbones are rejoined. The end of the DNA strand with a ‘OH’ group (5’ hydroxyl group) comes in and breaks the covalent bond. This breaks the temporary glue, and at the same time, it ties the DNA strands back together. This concludes the recombination, releasing the enzyme from its covalent attachment to the DNA so it can perform other recombination at other sites.
DNA Recognition, Strand Selectivity, and Cleavage Mode during Integrase Family Site-specific Recombination — PMC (nih.gov) — take a look for more info
Review of the method:
Advantages of Gateway Cloning:
- Directionality: The system ensures the correct orientation of the inserted gene.
- Speed: The entire process can be completed in approximately one hour.
- High Efficiency: The method boasts a success rate of 99%, making it highly reliable for cloning applications.
Part 2: integrating the FT gene into the pCAMBIA1391Z vector
My approach:
- PCR Amplification: We amplify the FT gene using primers (designed automatically by Snap Gene)
- BP Reaction: The PCR product undergoes a BP clonase reaction, integrating it into a donor vector (chosen from the proposed vectors in Snap Gene)
- LR Reaction: The entry clone containing the FT gene is mixed with the destination vector, pCAMBIA1391Z, in an LR clonase reaction.
- Transformation and Selection: The resulting expression clone is transformed into Agrobacterium tumefaciens, which subsequently transforms plant cells by floral dipping. Transformed cells are selected based on markers present in the pCAMBIA1391Z vector.
Floral dipping method
The floral dipping method is a technique used for the genetic transformation of Arabidopsis thaliana, a model organism in plant biology. This method is favored for its simplicity and efficiency, as it eliminates the need for tissue culture. During the process, the flowering parts of the plant are submerged in a solution containing Agrobacterium tumefaciens, which is genetically engineered to carry the desired DNA. The pCAMBIA1391Z vector is often used in this context.
The pCAMBIA1391Z vector is an Agrobacterium vector designed for plant transformation. It contains multiple features that facilitate genetic manipulation:
- Hygromycin and Kanamycin Resistance: These markers allow for the selection of successfully transformed plants by providing resistance to these antibiotics.
- pUC9 Multiple Cloning Site (MCS) that provide a versatile region where various DNA sequences can be inserted. The MCS is flanked by numerous restriction sites, making it compatible with many cloning techniques.
The floral dipping solution also contains sucrose, which serves as an energy source for the bacteria, and a surfactant to reduce the surface tension, allowing better contact between the Agrobacterium and plant tissues. This method is highly efficient, with transformation rates close to 100%, and can be completed within a few hours.
Snap Gene protocol
When working with Snap Gene to amplify a DNA fragment such as the FT fragment, the software can be utilized to design PCR primers that are specific to the sequence of interest. By inputting the att sequence as the starting point, Snap Gene generates primers that flank the target region. This ensures that the primers will bind efficiently during the PCR process, allowing for the successful amplification of the FT fragment.
The primer design process in Snap Gene considers several factors to optimize the primers for PCR, such as the annealing temperature (calculates the melting temperatures to ensure that both primers anneal to the template at a similar temperature for uniform amplification). The primer length is also important (suggests primers of an optimal length, usually between 18 to 25 nucleotides, to balance specificity and binding efficiency), as well as the GC Content (checks the GC content to ensure it’s within an ideal range, which promotes strong binding without forming secondary structures).
Once the primers are designed, they can be synthesized and used in a PCR reaction to amplify the FT fragment. The resulting product is then used for further cloning.
After successfully amplifying the PCR fragment, the next step is to proceed with the BP cloning reaction. This reaction is part of the Gateway cloning system, which allows for the efficient transfer of DNA fragments into plasmid vectors.
BP Cloning Reaction (in the lab — theoretically)
- Preparation: The amplified PCR product, which contains the FT gene flanked by attB sites, is mixed with a donor vector that has attP sites (as you can see in the screenshot of Snap Gene below)
- Enzyme Addition: BP Clonase II enzyme mix is added to the mixture. This enzyme facilitates the recombination between the attB sites on the PCR product and the attP sites on the donor vector.
- Incubation: The reaction is incubated at 25°C for about one hour, allowing the recombination to occur.
- Termination: Proteinase K is added to terminate the reaction, followed by a brief incubation at 37°C.
Once the BP cloning reaction is complete, the product is an entry clone containing the FT gene flanked by attL sites (as you can observe below in the Entry Clone FT). This entry clone is then ready for the LR recombination step, where it will be combined with a destination vector containing attR sites. The LR reaction will transfer the FT gene from the entry clone into the destination vector, creating an expression clone ready for further FT protein production.
LR Cloning Reaction
Then, we will perform the LR reaction. However, in order to do that, we need to chose a destination vector: and that, in my case, is the pCAMBIA1391Z vector. (below the map of this chosen vector, imported from the Snap Gene plasmid database.)
The selection of the pCAMBIA1391Z vector as the destination vector is a strategic choice due to its high cloning efficiency and the presence of a reporter gene, which simplifies the screening of successful LR recombination events. Once the LR reaction is initiated, the FT gene will integrate into the pCAMBIA1391Z vector through site-specific recombination facilitated by the LR Clonase enzyme.
Post-LR reaction, the expression clones (see below the end result, I colored the FT gene in yellow) can be introduced into Agrobacterium tumefaciens via transformation. The transformed Agrobacterium can then be used to deliver the FT gene into plant cells through a process of floral dipping, a widely used method for plant transformation. The successful integration of the FT gene into the plant genome will be confirmed through PCR and sequencing, followed by the analysis of FT protein expression levels in the transgenic plants.
The ultimate goal of this cloning strategy is to produce plants with enhanced traits conferred by the FT gene, such as improved flowering time or stress resistance.
Additionally, you can take a look at the videos below for an overview and of the Snap Gene technique.
