Plasmid DNA mediated Oligonucleotide Directed Mutagenesis in Protein Engineering

This post is about Oligonucleotide Directed Mutagenesis with plasmid DNA. Before going to discuss how Plasmid can be used to incorporate desired change in protein using oligonucleotide directed mutagenesis, in this post Plasmid will be discussed. Without knowing plasmid, it will be difficult to understand application of plasmid for incorporating desired change in protein. 

General Introduction

These are different procedures of Directed Mutagenesis. These are applied in protein engineering to achieve desired characteristics in protein of interest. In this post, we will study about “Oligonucleotide Directed Mutagenesis with plasmid DNA”. Follow our posts about updated knowledge with the theme of basic understanding of Protein Engineering to general audiences as well as to the specific reader of protein engineering.

Carefully studying this post, audience will be able to know about alternative approach to modify DNA using plasmid DNA as a template. These modifications are incorporated in DNA and consequently observe in protein of interest. Earlier we have posted some posts about how to use M13 ssDNA molecule to modify inserted gene using mismatch oligonucleotide primer against specific location in the sequence of gene of interest. So by studying this post, students or learner will be able to refresh some basic terminologies related to plasmid study and to know how we can modify gene of interest at protein level.

Introduction to Plasmid

The main question which triggers our mind at this stage is WHAT ARE PLASMIDS?Extrachromosomal Elements is the alternative name for plasmids. Think about this name This term predicts something that is resemble both in structure and function to chromosomes which are made of DNA and proteins, but they are not essential like chromosome for cell survival so we can call them extra chromosomal elements because prokaryotic cell can survive without extrachromosomal DNA.

Morphology of Plasmid

A small, circular, mostly supercoiled, extrachromosomal dsDNA molecules in cellular organisms (bacteria, yeast) which replicates independently of host chromosomal DNA is known as extrachromosoma elements or plasmid. In most cases they are smaller than chromosome. Discrete extrachromosomal genetic elements in bacteria

Discovery of Plasmid

In 1940s, plasmid was first observed during dysentery outbreak due to shigella spp. In Japan. That outbreak was not so scared like COVID-19 that have broken our daily routine, disturbed our regular classes and forced us to adapt distance learning. 

An American, Joshua Lederberg, expert in Molecular Biology, coined the term “Plasmid” in 1952 for the new observed DNA.

Component of Plasmid

Like DNA molecule, Plasmid also consists of some basic components such as

1. Replication site that helps in replication to amplify plasmid DNA before the reproductive cycle of bacteria.
2. Selectable marker are basically some extra or additional genes. The product of these genes provides some extra quality of life to the plasmid containing bacteria as compared to plasmid lacking bacteria of the same spp.
3. Like bacterial DNA, plasmid also have specific Restriction site, So, it can be easily converted into different size fragments using specific restriction enzyme.  

Plasmids Associated Genes

Plasmids consist of number of genes. they are involved to perform different types of useful functions for the host cells. Some of products of those genes are involved into transferring plasmid from one bacterium into another bacteria. Bacteria that have such type of plasmids are known as Donor Bacteria while bacteria which acquire plasmids during conjugation via mating through conjugative tube are known as Recipient Bacteria.

Classification of Plasmids

Based on transferring property, plasmids are divided into three types.

1. Conjugative plasmids possess tra genes and their product, Tra proteins are involved in transferring plasmid from donor bacteria into recipient bacteria. F, R and Col Plasmids are the examples of conjugative plasmids. F plasmid is the well-known example of conjugative plasmids. F stands for “fertility”. As you can see here in figure A, F+ve and F-ve bacteria come close to each other and initiate long-distance contact with each other by pilus formation. gradually, pilus depolyrizes near F+ve bacteria that result into closer contact between both bacteria. F pili is the main component of conjugating two bacteria. This F pili (singular Pilus) is produced by bacteria having F plasmid. Bacteria having F plasmid is commonly known as “F+” and bacteria lacking F plasmid is known as “F-” bacteria. Oncer F+ and F- bacteria come in contact due to F pili triggers rolling circle replication of Plasmid DNA. So one strand of plasmid starting transfer from F+ into F- bacteria. As the strand transferred, at this stage both bacteria having single strand of plasmid DNA. Simultaneously, both bacteria start synthesis of complementary strands. In this way both bacteria synthesized complementary strands. At the end of this process, F- bacteria become F+ bacteria. (Soon figure will be uploaded).
2. Non-conjugative plasmids lack tra genes therefore they are unable to transfer to recipient bacteria. E.g., R and Col Plasmids may be non-conjugative plasmids. When they haven’t tra genes. Keep in mind, nonconjugative plasmids can also be mobilizable to transfer into recipient bacteria. This process requires cointegration of nonconjugative plasmids into conjugative plasmids. For cointegration, both non conjugative and conjugative plasmids require copies of a transposable elements to perform homologous recombination. Cointegration is the combination of both non and conjugative plasmids. And this can be transferred into recipient bacteria by conjugation process. However non-conjugative can be released any time. the release of non-conjugative plasmid is shown here with up direction arrows (Note: figure will be uploaded soon)
3. There is an intermediate class of plasmids, known as mobilizable plasmids. They can be transferred into other bacteria in the presence of conjugative plasmids. Mobilizable plasmids parasitizes conjugative plasmids during their mobilizable phase.

Classification of Plasmids based on Functions

Another type of Plasmid classification is based on their specific and unique function. For example 

1. TOL Plasmid is degradative plasmids present in Pseudomonas putida involve in the digestion/degradation of unusual substance like toluene and/or salicylic acid.
2. Virulence Plasmids are those plasmids which possess vir gene. A host bacterium which acquires virulence plasmid can change their nonpathogenic status into pathogenic status. 
3. F Plasmid consists of tra genes. That are responsible for the transferring of F plasmid from donor bacteria into recipient bacteria via sex pilli in the process of conjugation.
4. Resistance Plasmids also known as R plasmids have different antibiotic genes for example amp, tetra, kana etc their products are responsible to protect host bacteria from the adverse effect of those antibiotic which are present in environment. For example, R plasmids, pRP4 plasmids.
5. Col Plasmids encode bacteriocins and are toxins to protect host bacteria from adverse effect of other bacteria and or animal.