Short Notes On Gateway Cloning Technology (Life Science) - Download PDF!

By Renuka Miglani|Updated : July 4th, 2022

Are you an Aspirant of CSIR-NET and looking for some short and reliable notes for Life Sciences to strong your base for preparations? We have got you covered!

Candidates preparing for their CSIR NET exam can really make their preparation journey easier with the help of some reliable study notes that covers the topics in the most simple way. We at BYJU'S Exam Prep have come up with the idea of providing short notes on Gateway Cloning Technology, which comes under the Methods in Biology of the Life Science syllabus. 

The short note on Gateway Cloning Technology is developed by our experienced subject-matter experts to provide you with the most standard and authentic set of study materials to be focused upon. The students need the best resources for their preparation to clear the CSIR NET examination, Here are the most reliable study Notes to make the topics easier for you and also help you to save your time for the preparations for the upcoming CSIR-NET 2022 exam.

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Study Notes On Gateway Cloning Technology

Gateway cloning technique is a cloning technique that removes many steps of cloning that can limit the success of cloning which normally occurs in traditional cloning method like restriction enzyme this enhance the workflow of cloning. some examples are like many restriction enzymes are not usable because they might cut within the gene of interest and disrupt the insert which makes the gene used less for downstream expression. So for low-efficiency recovery of recombinants from cloning large fragments is experience additional clean-up steps are also needed, time is wasted to screen colonies to find the clone of interest. All of the abovesaid steps take considerable time and effort, and no success is guaranteed.  While In Gateway recombination cloning technology circumvents these cloning limitations which enable to access virtually any expression system.

The Gateway cloning method is an in vitro version of the integration and excision recombination reactions and this reaction takes place when lambda phage infects bacteria. In vivo, condition facilitation of these recombination reactions occurs by the recombination of attachment sites from the phage (attP) and the bacteria (attB). As a result of recombination reactions between the attP and attB sites, the phage integrates into the bacterial genome flanked by two new recombination sites. In specific certain conditions, the attL and attR sites can recombine which leads to the excision of the phage from the bacterial chromosome and the regeneration of attP and attB sites.

The Gateway cloning system is a recombinational cloning system that was developed for cloning multiple DNA fragments in parallel in a standardized manner and by using the same enzyme. This technique is purely based on the highly specific integration and excision reactions of bacteriophage λ into and out of the Escherichia coli genome. Because of the sites of much longer recombination sites (“att” sites) than restriction sites, they are extremely unlikely to occur by chance in DNA fragments. so, the same recombination enzyme can be used to robustly clone many different fragments of variable size in parallel reactions.

Basic Protocol of Gateway Cloning Technique:

1. Entry Clone Determination:

At the first step of cloning, it is determined how and where the experiment starts and entry of clone is done, as it contains the gene of interest or DNA fragment flanked by attL sequences, these sequences are then recombined with attR sequences for the creation of the desired expression in the clone. So at first  Choose appropriate cloning vectors to create an Entry clone, or purchase a premade clone from our validated Ultimate ORF Clone Collection.

Generation of Entry Clone in Gateway Cloning

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2. Reaction Mediation with Clonase Enzymes:

When the Entry clone is ready, the gene of interest is lucidly shuttled to a secondary plasmid,  which is known as the Destination vector. This reaction is mediated by a particular enzyme which is known as the Clonase enzyme these enzyme contains the protein machinery which is necessary to excision the gene of interest from the Entry clone and integrate it into the Destination vector, then it becomes an expression clone.

Reaction for Preparation of Vectors

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3. Destination Vector Selection:

When a cloned gene of interest or DNA fragment reaches into a Gateway vector, the process can shuttle it to as many Destination vectors as you require.  their as many Destination vectors for application for example From proteins expression in E. coli, yeast, insect, or mammalian cells to RNAi studies, from crystallography to protein-protein interaction functional studies, And also for those applications which require a specialized or customized vector, the Gateway Vector Conversion System can convert any vector into a Gateway cloning–compatible vector.

Reaction of Gateway Cloning

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Gateway Vector Technology Overview

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Advantages and Disadvantages of Different Recombination-Based Cloning Methods

Cloning system(s)

Gateway

Univector Creator

In-Fusion, MAGIC, SEFC

Method Involve

In this technique λ-based BP and LR recombinases that use att sites, it is a Completely in vitro technique.

P1-based Cre recombinase that uses loxP sites, it is

Completely in vitro technique.

The homologous recombination method is used

 

Advantages

In this technique, no gain/loss of nucleotides occurs during the transfer

No digestion required

Recombination-reversible

Uses Entry clones

Dozens of Destination vectors are available

Multisite transfers possible

Wide variety of recombination sites.

No gain/loss of nucleotides during transfer

No digestion required

Uses Entry clones

No recombination sites

No enzyme purchase necessary, Multisite transfers possible

Any vector can be used without conversion

Disadvantages

There is a Need to purchase enzymes

Recombination sites may interfere with the experiment

Need to make the desired vector compatible.

Need to purchase enzymes

Recombination sites may interfere with the experiment

Recombination irreversible

Few Destination vectors are available

Multisite transfers not possible

Limited variety of recombination sites

Need to make the desired vector compatible

Occasional gain/loss of nucleotides during transfer

Need to generate insert by either PCR or digest before recombination

Do not use Entry clones.

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