Vector:Plasmid DNA preparation: 13th april

Preparation of Plasmid DNA

A plasmid preparation is a method used to extract and purify plasmid DNA. Many methods have been developed to purify plasmid DNA from bacteria. These methods basically involve three major steps

• Growth of the bacterial cell
• Harvesting and lysis of the bacteria
• Purification of plasmid DNA

Growth of the bacterial cell

Plasmids are almost always purified from liquid bacteria cultures, usually E-coli, which have been transformed and isolated. Virtually all plasmid vectors in common use encode one or more Antibiotic resistance genes as a selectable marker, which allows bacteria that, have been successfully transformed to multiply uninhibited.

Harvesting and lysis of the bacteria

When bacteria are lysed under alkaline conditions both DNA and proteins are precipitated. Some scientists reduce the concentration of NaOH used to 0.1M in order to reduce the occurrence of ssDNA. After the addition of acetate-containing neutralization buffer the large and less supercoiled chromosomal DNA and proteins precipitate, but the small bacterial DNA plasmids can renature and stay in solution.

Purification of plasmid DNA

Purification of plasmids from 2 cultures of bacteria involves the same procedure like preparation of total cell DNA but there is an important distinction between plasmid purification and preparation of total cell DNA. The plasmid preparation, we have the plasmid DNA from large amount of bacterial chromosomal DNA. Several methods are available for removal of the bacterial DNA during plasmid preparation

General method

During cell lysis, the shearing of chromosomal DNA is very less; hence the large size of the chromosomal DNA tends it to be removed along with the cell debris by centrifugation

There is another method on the basis of conformational difference between plasmids and bacterial DNA. Before going to this you must prepare a clear lysate (plasmid, DNA) by centrifugation in which plasmid and DNA are present. Most plasmid resides in supercoil state.

For the separation, two different methods are commonly used

1. alkaline Denaturation method
2. EtBr-CsCl (cesium chloride) density gradient centrifugation

Alkaline Denaturation method

The basic concept of this method is to maintain a very narrow pH range in which the non supercoiled DNA will be denatured but not the supercoiled plasmid.

Initially the pH of the solution containing both is adjusted at 12-12.5. This in turn denatures the open circular DNA or the non supercoiled DNA.

When a bit of acid (acidic potassium acetate) is added, it neutralizes the base.

At neutral pH, the genomic DNA renatures and is trapped in the SDS/lipid/protein precipitate and forms a lump.

The plasmid DNA renatures into double stranded molecules that remain in the solution.

Diagrammatic representation

Density gradient Method

A solution of cesium chloride when centrifuged at a very high speed a density gradient is produced. A high centrifugal force pulls the CsCl ions towards the bottom. This method is also called isopycnic centrifugation.

Why is CsCl used?

Caesium chloride is used because, at a concentration of 1.6-1.8 gm/ml, its density is almost similar to that of the DNA.

The CsCl and DNA mixture is kept in a centrifuge for several hours at a high speed to create a force of almost 10⁵ g. after sometime when a gradient of caesium ion is formed, it causes 2 forces to act simultaneously .i.e. the diffusion and the centrifugal force. The centrifugal force pushing the molecule outward where as diffusion pulls it towards the center. In turn, the denser molecules are pulled inside to the bottom of the tube. So, the density of the caesium ions moves in a decreasing order of density towards the surface. once the force has been created all the other ingredients of the misture are too stacked as per their decreaing densities.

In case of DNA, the GC bonding counts denser than the AT bonding. So, for 2 equal proportion of DNA run in a density gradient, the higher GC concentrated molecule will form the lower layer.

So if in case we have RNA, DNA, plasmid in a density gradient, then, the order of decreasing density would be

RNA > supercoiled plasmid DNA > DNA

Purification of total cell DNA: 7th april

Purification of DNA

DNA is the basic constituent of any living being, and some viruses too. The isolation and purification of DNA is a significant step in any molecular biological technique. We are to discuss about the mentioned three.

• Total cell DNA preparation
• Preparation of plasmid DNA
• Preparation of bacteriophage DNA

Preparation of Total Cell DNA

The procedure can be divided into the following 4 steps

Step 1: Bacterial culture growth and harvest

Step 2: Cell wall rupture and preparation of cell extract

Step 3: Purification of DNA from the cell extract

Step 4: Concentration of DNA solution

Bacterial culture and growth

Most of the bacteria are grown in or are preferred to grow in liquid media {broth culture}.

• The culture medium must provide 2 balanced mixtures of essential nutrients at concentrations that will allow the bacteria to grow and divide effectively
• Though the medium are of different types, we are here to discuss only about nutrient media.
• The media should consider the basic sources
• The most frequently used are
  • • Defined media

In this case we know the exact concentration of all the chemicals used in the media preparation. This media contains 2 mixtures of inorganic nutrients to provide essential elements like N, Mg, Ca as well as glucose. Additional growth factors like trace elements and vitamin may be added to provide additional nutrients to the bacteria depending upon the bacterial species.

Example

Composition of M9 medium

Constituent	Amount	Source for?
NaH2PO4	6	Sodium source
KH2PO4	3	Potassium source
Nacl	0.5	Salt
NH4Cl	1.0
MgSO4	0.5
Glucose	2.0
CaCl2	0.015

Undefined Media

In this case the precised identity and the quantity of its components are not known. Ingredients like tryptone and yeast extract are used which are complicated mixture of unknown chemical compounds. It is known as tryptone contains small peptides and amino acids like whereas yeast extract supplement provides N₂ and glucose requirement to the bacteria. This complex media needs no further supplementation and support growth of wide range of bacterial species

Constituent	Amount req	Source
Tryptone	10
Yeast extract	5
Nacl	10

Preparation of cell extract

The bacterial cell is enclosed ni a cytoplasmic membrane and surrounded by 2 rigid cell wall. In some species like E.Coli, the cell wall itself may be engulfed by the second outer membrane. All these barriers have to be disrupted to release the cell components which can be done by 2 methods.

• Physical method (Use of beads and other physical methods to rupture the cell wall)
• Chemical method
  • Generally chemical method is used by making use of EDTA or lysozyme (secreted by saliva).
  • Lysozyme is present in secretion by saliva, tears egg-white which digests the polymeric compounds giving rigidity to the cell wall.
• EDTA (Ethylene Diamine Tetra-acetic Acid), it removes the Mg²⁺ ions that are essential for preserving the overall structure of the cell.

It also inhibits the cellular enzymes that could degrade the DNA.

• SDS (Sodium Dodecyl Sulphate).
  • In some cases it is also added, which helps in the process of lysis by removing lipid molecules. Generally mixture of EDTA and lysozyme is used.
  • This mixture is then centrifuged at a speed of 8000 rpm for 110 mins. It causes the precipitation of cell forming and the pellets in the test-tube.
  • Finally the cell extracts are extracted from cell debris, which are pellet down by centrifugation

Purification of DNA from cell extract

• The standard way to deproteinize a cell is to add phenol or a 1:1 mixture of phenol and chloroform.

• The organic solvents precipitate proteins but leave the nucleic acids (DNA and RNA) in an aqueous solution.
• The result is that is the cell extract is mixed gently with the solvent, and the layers then separated by centrifugation, precipitated protein molecules are left as a white coagulated mass at the interface between the aqueous and organic layers.
• The aqueous solution of nucleic acids can then be removed with a white pipette.
• Cell extract is treated with protease such as pronase or proteinase K before extraction.
• These enzymes will break polypeptides into smaller units thus making phenol easier to remove them.
• The only effective way to get rid of RNA is the use of Ribonuclease enzyme which will rapidly degrade thee molecules into ribonucleotide subunits.

Concentration of DNA samples

• The most frequently used method of concentration is ethanol precipitation.

• In the presence of salt and a temperature of -20^◦c or less absolute ethanol with efficiently precipitate polymeric nucleic acids.
• With 2 thick solution of DNA, the ethanol can be layered on the top of the sample.
• A spectacular trick is to push a glass rod through the ethanol into the DNA solution.
• When the rod is removed, DNA molecules will adhere and be pulled out of the solution in the form of long fiber.

for further reading click HERE

Vector: Bacteriophage lambda and M13: 7th april

Bacteriophage

Definition

They are one of the types of virus that attacks the bacteria and infects it.

It was first reported by Frederick Twort, a british biologist and later by Felix d’Herelle, a French microbiologist.

Diagram of phage

Characteristic features of a bacteriophage

1. They have a typical structure of outer protein capsid enclosing a genetic material
2. The genetic material can be DNA or RNA and might be ss or ds.
3. The size of the nucleotides can vary from 5000 to 500, 000 nucleotides
4. The genetic material can be circular or linear
5. They are much smaller than the bacteria typically of size 20nm to 200nm.
6. The prime source of these virions is the highly populated areas of bacteria, mostly the sea water where upto 9*10⁸ virion per ml is found.

The classification

They are classified by the International Committee on Taxonomy of Viruses (ICTV) according to the morphology and characteristics.

Replication

There are two stages of bacteriophage replication

1. lytic cycle
2. lysogenic cycle

Lytic cycle

In this case the phage particle infects the bacterial cell and inserts the viral genetic material into it. The viral genetic material if is RNA uses reverse transcriptase enzyme to convert it to DNA and thus translates other building materials for a potential virion. The number of this virions increase at an exponential rate to burst the bacterial cell and set free to attack other baterial cells in the vicinity. This is a pretty fast rate of replication.

Lysogenic cycle

In this case the viral genetic material is incorporated into the bacteria’s own chromosome and thus is transferred to its progeny. When integrated with the host DNA the formation is called a prophage. And the phages able to cause lysogeny are called temperate phages. The virus remains dormant until the host conition deteriorates and once the stage reaches the viral DNA becomes active and thus causes lysis of the cell.

The prophages sometimes add more functions to the host cell. This is called lysogenic conversion. One famous example is the conversion of harmless viobrio cholerae by a phage into a highly virulent one. This is one reason why temperate phages are not used for phage therapy.

Diagram explaining the lytic and lysogenic cycle of a bacteriophage

More information at

click HERE

One more classification of bacteriophage is a m13 bacteriophage

M13 Bacteriophage

1. It is a filamentous bacteriophage composed of circular single stranded DNA
2. The length is 6407 nucleotides
3. It is encapsulated in 2700 copies of the major coat protein P8, capped with 5 copies of two different minor coat proteins (P9, P6, and P3).
4. The minor P3 attaches to the receptor at the tip of the host E.Coli.
5. It is not lethal but causes plaques in the bacterial cell
6. It is a non-lytic virus
7. The M13 phage is used for many recombinant DNA processes due to its extreme size and the virus has also been studied for its uses in nanostructures and nanotechnology

Diagram

The M13 infection cycle, the replication process

1. In this cycle the DNA is put into the bacteria through the F-pilus.
2. Once inside the cell the single stranded molecule acts as the template for the synthesis of a complementary strand, resulting in normal double stranded DNA.
3. This molecule is not inserted in to bacterial genome but instead replicates until over 1000 copies are present in the cell.
4. When the bacterium divides, each daughter receives copies of the phage genome. This continues to replicate thereby maintaining its overall numbers per cell.
5. The new phage particles are continuously assembled and released.

Diagrammatic representation

For further information about M13 bacteriophage click HERE

Plasmids: lecture notes on 17th march

Plasmids

It is an extra chromosomal DNA molecule separate from the chromosomal DNA which is able to replicate independently of the chromosomal DNA.

• They vary from 1kb to 1000kb
• The term plasmid was first introduced by the American molecular biologist Joshua Lederberg in 1952

• The plasmid doesnot contribute to the genome of the bacterial cell it is present in but often translates proteins of significance. For example
  • It contains the antibiotic resistance gene that helps the bacteria survive from that specific antibiotic.
  • It can provide the bacteria with an ability to fix elemental nitrogen or to degrade calcitrant organic compounds which provide an advantage under conditions of nutrient deprivation.

  A small diagram representing the function of the plasmid in gene cloning

http://bioweb.wku.edu/courses/biol350/CloningVectEuk9/Review.html

For a better understanding of plasmids, integrative replication and about Ag Tumefaciens.

Replication of plasmid

Non Integrative Replication

In this type of replication the plasmid DNA once introduced into the cell grows as per the copy number and the multiplication of the cells

Integrative Plasmid

Episome

Under certain conditions some plasmids may integrate into the bacterial chromosome. They are called episome or integrative plasmids. At this stage they replicate along with the bacterial chromosome.

The plasmids in this way are classified into 2 types

Relaxed plasmids

They are the ones which are normally maintained at multiple copies per cell.

Stringent plasmids

They are the ones which have a limited number of copies per cell.

In this case of plasmid replication, the plasmid DNA is integrated in the bacterial chromosome and grows along with the cell. It uses the bacterial machinery for division A good example of this type of replication is Ti Plasmid, often used in agricultural genetic engineering experiments. It completely uses the cell genetic mechanism to grow.

In this case integration and excision of a plasmid is given. Please refer the animation below for better understanding.

This animation link given below will help to gain a better understanding of the integration and excision of the vector gene from the chromosomal DNA

http://www.sutree.com/LoadVideo.aspx?s=5073&url=http%3a%2f%2fhighered.mcgraw-hill.com

Types of plasmid

The plasmids are classified as per the conjugation properties as in it was done warlier, but plasmids are now classified using functions

1. Fertility plasmid:

That contains the tra genes required for conjugation else known as F plasmids. For example F plasmids of E coli

1. Resistance Plasmid or R plasmids

Carry geens of resistance to one or more antibacterial agents such as ampicilin, mercury etc

1. Pseudomonas Plasmid:

Such types of plasmids that help in treatment of bacterial infection

1. Col plasmid:

It contains genes for production of bacteriocins, proteins that kill other bacteria example col E1

1. Degradative plasmid:

It produces proteins for degradation of unusual substance like toluene and salicylic acid. Example TOI of pseudomonas

1. Virulent plasmids:

Which in turn convert bacteria into a pathogen. For example, the Ti plasmid of agrobacterium tumefaciens induces crown gal disease on dicot of the palnts

1. Yeast plasmids:

They are normally 2 micrometer circular plasmids present in yeast cells and other eukaryotic cells

Incase you need a list of plasmids and their sources click       HERE

For more information on plasmid please click                               HERE

Size of some plasmids as vectors

Plasmid	Size	Mol. Wt in kb	Marker	Organism
puc 8	2.1	2.7	Apr	Escherichia coli K12 JM83
col E1	6.4	6.6	E1 imm	Escherichia coli JC411
rP4	54.0	56.4	Apr, Kmr, Tcr	Escherichia coli K12 J53
ToL	117.0	78.0		pseudomonas
pTiach 5	213.0	142.0		Ag. Tumefaciens

Vector part I: 17th march

Vector

In molecular biology, a vector is a DNA molecule used as a vehicle to transfer foreign genetic material into another cell. The four major types of vectors are plasmids, bacteriophages and other viruses, cosmids, and artificial chromosomes. Common to all engineered vectors are an origin of replication, a multicloning site, and a selectable marker.

Features of an Ideal Vector

Size

It must be relatively smaller size(10 kbp) because larger molecules tend to break down and very small ones may not allow the insertion of the gene

Copy number

It is the no. of molecules of plasmid  present in a cell. The copy number should be higher.

Independent existence

The only molecule should be having independent existence within the host cell. (e.g plasmid within bacteria)

Use of host mechanism

It should be able to use the host cell enzymer machinery for process of replication

Selectability/presence of antibiotic resistance gene

Antibiotic resistance genes hould be present because these are helpful in selecting the host cell containing recombinant DNA

Origin of replication

There should be a presence of origin of replication

Presence of restriction sites

Please check in for futher notes
click here

Basics of gene cloning: 17th march

The basic steps in Gene Cloning

1. Preparation of Pure sample of DNA from the organism of desire
2. Cutting of the DNA molecule using Restriction enzymes to form fragments
3. Analysis of DNA fragments using electrophoresis
4. Identification of suitable vectors for insertion of the gene (DNA molecule).
5. Joining of the two DNA molecules, I.e the fragmented DNA here and the plasmid.
6. Introduction of the DNA molecule, i.e the recombinant vector or the chimera, into the host cell.
7. Identification of the cell containing the recombinant DNA, through screening techniques

An animation of Gene cloning…

For further notes,

Genetic engineering introduction

What is Genetic Engineering?

Genetic engineering, recombinant DNA technology, genetic modification or manipulation (GM) and gene splicing are terms that apply to the direct manipulation of an organism’s genes.

The term “genetic engineering” was coined in Jack Williamson’s science fiction novel Dragon’s Island, published in 1951, two years before James Watson and Francis Crick showed that DNA could be the medium of transmission of genetic information.

Later on Arber in 1950 discovered enzymes that degrade bacterial viruses and Smith in (1970) purified the enzymes and characterized them

The founding fathers of genetic engineering were Stanley Cohen and Herbert Boyer who in 1973 in vented the technique of DNA cloning

STEPS INVOLVED IN SEQUENCING

Separation of DNA

In this step, preparation of genomic DNA from the cell line of the desired organism is done.

Fragmentation

Fragments of DNA are obtained using various types of restriction enzymes. This is due to the presence of various restriction sites in the sequence.

Polymerization

The fragments to be sequenced are cloned in a vector plasmid or cosmid (to get sufficient quantity of fragments to be sequenced). Polymerization of the DNA sequence can be done by using a PCR naturally or artificially.

Labeling

The fragments are then labeled according to the requirement. It might be radioactive labeling or fluorescent labeling. The labeling is done basically for identification purposes.

Sequencing reactions

Many of the methodologies of sequencing have been used. For e.g Maxam Gilbert sequencing, Sanger’s chain termination methods etc . The later method  is widely used and is the base of modern sequencers.

Gel electrophoresis

The colonies of DNA fragments are run in the gel to identify and infer the results. Four different lanes for different nucleotides are made. Analysis in modern way are done in capillary electrophoresis unit.

Visualization

The fragments are now visualized using staining method, and fluorescent readings.

Computer analysis

The overlapping regions of the fragments are determined using computer analysis. The overlapping sequences are assumed into continuous sequence. This is a base method in modern sequencers which are completely computerized.

Annotation

Conquering the pieces of DNA with known sequences and database which helps in annotation and identification of unknown gene.

Further reading:

DNA sequencing
**

How do we sequence DNA?

An animation of sanger’s method for DNA sequencing

How do we obtain the graph?

For my notes and detailed procedure of DNA click    HERE