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Insertional inactivation using pBR322

Insertional inactivation using pBR322


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I have questions regarding "selection using insertional inactivation".

In a typical DNA recombination experiment using plasmid pBR322 where a new gene in inserted in the place of tet gene, it is said that entities with the recombinant plasmid can be selected by growing all the bacteria in two different medium: one with tetracycline and one with ampicillin.

I know that pBR322 contains two genes that make it resistant to both tetracycline and ampicillin, and also that the newly created plasmid will only be resistant to ampicillin, since its tet gene is no longer functional.

So in the plate with tetracycline, only entities with the original plasmid will grow. In the plate with ampicillin, however, entities with the original plasmid and the modified plasmid will grow.

How can this be a selection when two different types of entities are growing in one plate?

Am I misunderstanding the meaning of "selection" in this case?

My explanation above seems very immature, so it would be great if someone can provide me with deeper understanding.


The ampicillin plate is selecting for bacteria transformed with the plasmid, either the original or recombinant, whereas the tetracycline plate is selecting only for bacteria transformed with the original plasmid. Since you are only interested in the recombinant plasmid, you need to do some form of replica plating and compare the two plates to infer which colonies contain the recombinant plasmid (those which grow on ampicillin but not on tetracycline).


  • Transposons contain signals to truncate expression of an interrupted gene, thus inactivating it.
  • Transposons are widely used tools in biology, frequently utilized for insertion mutagenesis, large-scale gene disruption studies, and gene tagging.
  • Transposon-mediated gene disruption experiments and promoter traps rely on promiscuous, undirected, and pseudo-random insertion of the transposon.
  • transposable: Able to be transposed (in any sense).
  • plasmid: A circle of double-stranded DNA that is separate from the chromosomes, which is found in bacteria and protozoa.

A transposable element (TE) is a DNA sequence that can change its relative position (self-transpose) within the genome of a single cell. The mechanism of transposition can be either &ldquocopy and paste&rdquo or &ldquocut and paste. &rdquo Transposition can create phenotypically significant mutations and alter the cell&rsquos genome size. Barbara McClintock&rsquos discovery of these jumping genes early in her career earned her a Nobel prize in 1983.

Transposons in bacteria usually carry an additional gene for function other than transposition&mdashoften for antibiotic resistance. In bacteria, transposons can jump from chromosomal DNA to plasmid DNA and back, allowing for the transfer and permanent addition of genes such as those encoding antibiotic resistance (multi-antibiotic resistant bacterial strains can be generated in this way). When the transposable elements lack additional genes, they are known as insertion sequences. Transposons are semi-parasitic DNA sequences that can replicate and spread through the host &lsquos genome. They can be harnessed as a genetic tool for analysis of gene and protein function. The use of transposons is well-developed in Drosophila (in which P elements are most commonly used) and in Thale cress (Arabidopsis thaliana) and bacteria such as Escherichia coli (E. coli ).

Synthetic DNA transposon system are constructed to introduce precisely defined DNA sequences into the chromosomes of vertebrate animals for the purposes of introducing new traits and to discover new genes and their functions (e.g. by establishing a loss-of-function phenotype or gene inactivation). Transposition is a precise process in which a defined DNA segment is excised from one DNA molecule and moved to another site in the same or different DNA molecule or genome.

Figure: Transposon System: The sleeping beauty transposon system applications.


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2nd PUC Biology Biotechnology: Principles and Processes One Mark Questions and Answers

Question 1.
Do eukaryotic cells have restriction endonucleases? Justify your answer.
Answer:
The eukaryotic cells do not have restriction endonucleases. The eukaryotic cells have some other means of defence i.e., immune system) against viral infection.

Question 2.
Which is the commonly used host cells in genetic engineering?
Answer:
E. coli.

Question 3.
Which are the common vectors used for cloning genes in plants?
Answer:
Tiplasmid present in Agrobacterium tumefaciens.

Question 4.
Who discovered the technique of PCR?
Answer:
Kary Mullis.

Question 5.
What is a bioreactor?
Answer:
It is an engineering setup designed to carry out biological reactions under aseptic conditions.

Question 6.
Name the technique by which DNA fragments can be separated?
Answer:
Gel Electrophoresis.

Question 7.
Name the source of Taq polymerase?
Answer:
Thermus aquaticus.

Question 8.
What are recombinant proteins?
Answer:
Proteins produced from a recombinant DNA or transgenic organism are recombinant proteins.

Question 9.
Name the commonly used vector for transformation in plant cell?
Answer:
Agrobacterium tumefacien.

Question 10.
Who isolated Restriction enzyme for the first time?
Answer:
Warner Arber and Hamilton Smith.

Question 11.
Define a patent?
Answer:
Patent is the govemement protection to the inventor of biological material, securing to him for a specific time the exclusive right of manufacturing exploiting, using and selling an invention.

Question 12.
Define the term plasmid.
Answer:
Autonomously replicating circular extra chromosomal DNA (or) A circular double stranded, extra chromosomal DNA, present in the cytoplasm of bacteria.

Question 13.
Define Biotechnology.
Answer:
The integration of natural science and organisms, cells, parts thereof and molecular analogues for products and services is known as Biotechnology.

Question 14.
Name the compound used for visualizing DNA under UV radiation.
Answer:
Ethidium bromide.

2nd PUC Biology Biotechnology: Principles and Processes Two Mark Questions and Answers

Question 1.
Describe briefly the following:
(a) Origin of replication
(b) Bioreactors
(c) Downstream processing.
Answer:
(a) Origin of Replication: This is the sequence in DNA molecule, from where, replication starts and any piece of DNA when linked to this sequence can be made to replicate within the host cell.

(b) Bioreactors: These are culture vessels in which large volume (100-1000 liters) of culture can be processed to get large quantity of a product on a commercial scale.

(c) Downstream Processing: After the formation, a product, undergoes through some processes before a finished product is ready for marketing. These processes include separation and purification which are collectively called as downstream processing.

Question 2.
Explain briefly:
(a) PCR
(b) Restriction enzymes and DNA
(c) Chitinase
Answer:
(a) PCR: PCR stands for Polymerase Chain Reaction. In this reaction multiple copies of the gene (or DNA) of interest is synthesised vitro in, using sets of primers and the enzyme Taq DNA polymerase.

(b) Restriction Enzymes and DNA: These are the enzymes which restrict the growth of bacteriophages. These enzymes are present in many bacteria where they function as a part of their defence mechanism called Restriction Modification System. Restriction enzymes are of two types :
(a) Restriction endonuclease cuts DNA into pieces.
(b) Modification enzyme adds methyl group to DNA.

(c) Chitinase: It is an enzyme which breaks down the chitinous substance present in the wall of fungi.

Question 3.
Discuss with your teacher and find out how to distinguish between the following:
(a) Plasmid DNA and Chromosomal DNA
(b) RNA and DNA
(c) Exonuclease and Endonuclease.
Answer:
(a) Plasmid DNA and Chromosomal DNA: Plasmid DNA is a small double stranded circular DNA present in some bacteria. They carry genes for drug resistance, N2 fixation and fertility. The chromosomal DNA is much larger in size and carries genes for other traits of the cell.

(b) RNA and DNA: RNA is a single stranded polymer of ribonucleotides, which contain ribose sugar and adenine, guanine cytosine and uracil nitrogenous bases, whereas DNA is a double stranded polymer of deoxyribo-nucleotides containing deoxyribose sugar and adenine, guanine, cytosine and thymine nitrogenous bases.

(c) Exonuclease and Endonuclease: Exonucleases remove nucleotides from the ends of DNA, whereas endonucleases make cuts at specific positions within the DNA.

Question 4.
Mention the tools used in recombinant DNA technology.
Answer:
Desired gene, vector DNA, enzymes, host cell and Bioreactor.

Question 5.
What are palindromic sequences? Give an example.
Answer:
In a double stranded DNA molecule, if the two strands in a region are identical when read both in forward and backward direction, they are referred to as palindromic sequences.

Question 6.
How are restriction endonuclease named?
Answer:
The naming of restriction endonuclease is based on the following rules :

  • The first letter of the REN refers to the genus name of the bacteria from which it is derived and written in capitals.
  • The second and the third letters refer to the species name of the bacteria from which it is derived and is written as a small letter.
  • If a fourth letter is present it represents the name of the bacterial strain or subspecies.
  • The Roman number indicates the different REN’s derived from the same organism.
    Eg.: Hind III.

Question 7.
What are molecular scissors? Explain their role.
Answer:
The nuclear enzymes that break DNA at specific sites are called restriction endonucleases [REN]. The RENs are popularly known as Biomolecular scissors. REN may cut both the strands of a DNA at the same position or at different positions,
e.g.: Eco RI, Hind III, Sal I, Bam I.

Question 8.
Draw a neat labelled diagram of plasmid pBR322
Answer:

Question 9.
Write the applications of PCR technique.
Answer:

  • To amplify DNA and RNA strands.
  • To study the orientation and location of restriction fragments relative to one another.
  • Detection of mutations/presence of mutated genes in the chromosomes.
  • To identify DNA finger prints.

Question 10.
What are nucleases? Distinguish between exonucleases and endonucleases.
Answer:
Restriction enzymes are the nucleases. They are of two types namely exonucleases and
endonucleases.

Exonucleases remove nucleotides from the ends of the DNA and endonucleases can cut at specific points within the DNA itself

Question 11.
Draw a neat labeled diagram of stirred tank bio-reactor.
Answer:

Question 12.
What are ‘Selectable markers”? What is their use in genetic engineering?
Answer:
A selectable markers is a gene which helps in selecting those host cells which contain the vector and eliminating the non-transformants, e.g. gene encoding resistance to antibiotics are useful selectable markers as they allow selective growth of transformants only.

Question 13.
What is “Insertional inactivation”?
Answer:
If a recombinant DNA is inserted within the coding sequence of enzyme B – galactosidase, it results in the inactivation of the enzyme which is referred to as “Insertional inactivation”. The presence of chromogenic substrate gives blue coloured colonies if the plasmid in the bacteria does not have an insert presence of insert results in insertional inactivation and the colonies do not produce any colour.

Question 14.
Draw a neat labelled diagram of sparged stirred tank bioreactor.
Answer:

Question 15.
What are bioreactors? Name the most commonly used bioreactor in genetic engineering.
Answer:
It is an engineering setup designed to carry out biological reactions under aseptic conditions.

2nd PUC Biology Biotechnology: Principles and Processes Three Marks Questions and Answers

Question 1.
What are the two basic techniques involved in modern Biotechnology?
Answer:
The two basic techniques involved in modem Biotechnology are
(a) Genetic Engineering which is the technique of altering the nature of genetic material or introduction of it into another host organism to change its phenotype.

(b) Techniques to facilitate the growth and multiplication of only the desired microbes or cells in large number under sterile conditions for manufacture.

Question 2.
What are genetically modified organisms? Name two factors on which their behaviour depends?
Answer:
Those organisms whose genes have been altered by manipulation, are called genetically modified organisms or transgenic organisms. The two factors on which their behaviour depends are:

  • proper insertion of gene of interest.
  • proper harvesting of genetically modified organisms to produce the desired product.

Question 3.
What do you mean by “Biopiracy” Give an example?
Answer:
Biopiracy refers to the use of bio-resources by multinational companies and other oganizations without proper authorizations from the countries and people concerned eg. Basmati rice grown in India, is distinct for its unique flavour and aroma but an American company got patent rights on Basmati through US patent.

2nd PUC Biology Biotechnology: Principles and Processes Five Marks Questions and Answers

Question 1.
Explain the process of recombinant DNA technology.
Answer:
Process of genetic engineering :
(a) Isolation of genetic material (DNA): In recombinant DNA technology, it is essential to isolate DNA in pure form free from other macro molecules. Since DNA molecule is enclosed with the membrane in the cell, we have to break open the cell to release DNA along with other macromolecules like RNA, proteins, polysaccharides and lipids. This is carried out in bacterial cells, plant and animal cells with certain enzymes.

The other macro molecules can be removed by appropriate treatment with specific enzymes. Finally, the purified DNA molecules are precipitated out after the addition of chilled ethanol and this can be seen as collection of fine threads in the suspension.

(b) Cutting of DNA at specific locations: The isolated purified DNA molecule is cut (cleaved) with the help of a suitable enzyme called restriction endonuclease, into segments with sticky ends.

(c) Gel electrophoresis: The cut DNA fragments are separated by gel electrophoresis using agarose gel. DNA is a negatively charged molecule, hence it moves towards the positive electrode (anode).

(d) Amplification of Gene of interest using PCR: Amplification of gene is ‘a process of making many copies of a gene’. It is achieved by using a technique called Polymerase Chain Reaction (PCR).

Procedure of PCR :
1. The DNA from the desired segment to be amplified, an excess of the two primer molecules, the four deoxyribose triphosphates and DNA polymerase are mixed together in a reaction mixture in a eppendorf tube with sufficient quantities of Mg ++ . The eppendorf tube is placed in the PCR unit and the following operations are performed sequentially.

2. Denaturation: The reaction mixture is first subjected to a temperature between 90 – 98°C (commonly 94°C). It results in the separation of two strands of DNA due to the breakage of hydrogen bonds. This is called denaturation. Each strand of DNA acts as a template strand for DNA synthesis. The duration of this step in the first cycle of PCR is usually 2 minutes at 94°C.

3. Annealing (anneal=join): The mixture is now cooled to a low temperature (40- 60°C). During this step, two oligonucleotide primers, complementary to a region of DNA, anneal (hybridize) one to each 3 end of DNA strand. The duration of annealing step is usually one minute during the first as well as the subsequent cycles of PCR.

4. Primer extension: During this step, the enzyme taq DNA polymerase extend the primers using nucleotides and DNA templates. The two primers extent towards each other in order to get two new strands of DNA (at 5) end. The duration of primer extension is usually 2 minutes at 72°C. The amplified fragment if required can now be used to ligate with Vector for further cloning. The taq DNA polymerase remains active, during the high temperature induced denaturation of double stranded DNA.

(e) Insertion of recombinant DNA into the host:
1. Electroporation: The bacterial cell is placed in a solution with cold CaCl2 solution followed by placing them at 42°C intermittently. This results in the development of pores in the cell membrane. Now the recombinant plasmid migrates into the host cell and the bacterial cell gets transformed.

2. Microinjection: It is direct injection of a desired gene into the nucleus of an animal cell by microsyringe.

3. Biolistics: Here a suitable plant-cell is bombarded with high velocity microparticles (2-2mm) of gold or tungsten coated with DNA in order to introduce the DNA into the cell.

(f) Obtaining the foreign gene product:

  • The transgene expresses itself in the form of protein (s) under appropriate conditions.
  • The product (s) can be extracted from the medium by employing a suitable procedure.
  • The transgenic cells may be cultured in the laboratory to obtain the transgene product on a small scale.
  • The transgenic cells can also be cultured/multiplied in a continuous culture system, in which the used medium is drained out from one side and fresh medium is added from the other side for the production of larger biomass and the desired product.
  • Bioreactors are used for processing large volumes of culture for obtaining the product of interest in sufficient quantities on a commercial scale.

(g) Downstream Processing: The products formed in a bioreactor have to be subjected ‘ through a series of processes before they are ready for marketing as finished products.
The various processes used for the recovery of useful products are collectively called downstream processing. The processes include separation and purification of the product, addition of suitable preservatives and a stringent quality control testing etc. Such formulation has to undergo strict clinical trials as in the case of drugs. These quality control testing and clinical trials vary from product to product.

Question 2.
With the help of diagram explain plasmid pBR322.
Answer:

pBR-322 is a natural plasmid and F + plasmid. It is about 4.3 kb in size. It is a plasmid with an ori site (origin of replication), two antibiotic resistance sites, selectable markers for Amphicillin resistance (Amp r ) and Tetracycline resistance (Tet r ). It has thirteen unique sites in different regions out of which seven are important. A unique site is a specific restriction enzyme (REN) recognition site called ECORI.

Vectors for cloning genes in plants and animals are Ti plasmid isolated from Agrobacterium tumefaciens in plants and retrovirus are now made non pathogenic and are used to deliver gene into animal cells.

Note: Insertional inactivation: When a gene or recombinant DNA is inserted within the coding sequence of a vector, the coding sequence responsible for an enzyme or a particular character becomes inactivated. This is known as Insertional inactivation.

Question 3.
a) What is gel electrophoresis? Explain how DNA fragments are separated and detected using this technique.
b) What are plasmids? Mention any two features of an ideal plasmid.
Answer:
(a) The cutting of DNA by restriction endonucleases results in the fragments of DNA. These fragments can be separated by a technique known as gel electrophoresis. Since DNA fragments are negatively charged molecules they can be separated by forcing them to move towards the anode under an electric field through a medium/matrix.

Now a days the most commonly used matrix is agarose which is a natural polymer extracted from sea weeds. The DNA fragments separate (resolve) according to their size through sieving effect provided by the agarose gel. Hence, the smaller the fragment size, the farther it moves.

The separated DNA fragments can be visualised only after staining the DNA with a compound known as ethidium bromide followed by exposure to UV radiation (you cannot see pure DNA fragments in the visible light and without staining). You can see bright orange coloured bands of DNA in a ethidium bromide stained gel exposed to UV light.

The separated bands of DNA are cut out from the agarose gel and extracted from the gel piece. This step is known as elution. The DNA fragments purified in this way are used in constructing recombinant DNA by joining them with cloning vectors.

(b) The small circular and extra chromosomal double stranded DNA molecules present in the cytoplasm of bacteria are called plasmids. They show self replication. They contain 2 to. 8 thousands of nitrogen base pairs. They also contain some genes, like antibiotic resistant genes and genes for expression of some characters. They can be cut at specific sites using restriction enzymes and desired genes can be inserted into these plasmids.


Biotechnology : Principles and Processes | Study Notes | Class-12 Biology

Biotechnology – Field of biology that deals with the use of live organisms or biological systems to make products or processes that are useful to human beings.

✔ Biotechnology as per European Federation of Biotechnology (EFB) – The integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services.

Principles of Biotechnology

Genetic Engineering:

o Techniques to modify genetic material.

o It allows the introduction of these modified genetic material into the host organisms.

o Can change the phenotype of the organism.

Bioprocess Engineering:

o Maintenance of sterile condition to enable the growth of only desired microbes.

▪ Sterile: Free from microbial contamination.

o Prevents the growth of undesirable microbes.

Some Basic Concepts

o What happens when a DNA molecule is somehow introduced into a host cell?

▪ The DNA cannot replicate within the host cell.

▪ Most probably it will be degraded.

o How can you make the foreign DNA multiply or make copies of itself within the host cell?

▪ The foreign DNA must become part of the host organism’s genome by integrating with it.

● Genome: All the genetic material of an organism.

▪ The foreign DNA now gets multiplied along with the host chromosome as the host chromosome contains the origin of replication.

● The origin of replication is essential to initiate the process of DNA replication.

▪ This process is called the cloning or making multiple identical copies of any template DNA.

Construction and Cloning of the first artificial rDNA molecule:

o Scientists: Stanley Cohen and Herbert Boyer (1972)

o Native Plasmid of Salmonella typhimurium was linked to antibiotic resistant gene.

Plasmid : Double stranded, autonomously replicating, extra chromosomal circular DNA that provides additional features to the bacterial cell if present.

Antibiotic resistant gene : Gene that enables the bacteria to survive in a medium that contains antibiotics.

▪ Identification and cutting of a piece of DNA from a plasmid that provides resistance to antibiotics.

● The DNA was cut at a specific position.

● Enzyme involved: Restriction Enzymes – Molecular scissors.

▪ The cut piece of DNA was linked to the native plasmid of S. typhimurium.

● Enzyme involved: DNA Ligase

▪ This modified circular DNA is a recombinant DNA (rDNA) and acts as a vector.

● Vector – carrier of foreign DNA/gene of interest.

● rDNA – A DNA molecule that has been modified and may have a foreign DNA segment.

▪ Upon introduction of this rDNA into Escherichia coli bacteria, the rDNA was able to replicate within the new host (E. coli) and multiply in number using the new host’s DNA polymerase enzyme.

▪ As the original antibiotic resistant gene has been multiplied within the E. coli host, this process is referred to as the cloning of the antibiotic resistance gene in E. coli.

Three basic steps in genetically modifying an organism

I. Identification of DNA with desirable genes

II. Introduction of the identified DNA into the host

III. Maintenance of introduced DNA in the host and transfer of the DNA to its progeny.

TOOLS OF RECOMBINANT DNA TECHNOLOGY

Restriction Enzymes

o 1963 : Two enzymes were discovered that could restrict the growth of bacteriophage in E. coli.

▪ One enzyme added methyl group to DNA.

▪ The other enzyme could cut the DNA- restriction endonuclease.

o The first identified restriction endonuclease is Hind II.

o Restriction enzymes comes under Nucleases (larger class of enzyme).

o Removes nucleotides from the periphery/ends of the DNA.

o It cuts the DNA at specific positions away from the periphery within the DNA.

o The restriction endonuclease cuts the DNA at a specific position by recognizing a specific sequence of DNA referred to as recognition sequence.

▪ Each restriction endonuclease recognizes a specific palindromic nucleotide sequences in the DNA.

Palindrome in DNA is a sequence of base pairs that reads same on the two strands when orientation of reading is kept the same.

● For example, the following sequences reads the same on the two strands in 5' → 3' direction. This is also true if read in the 3' → 5' direction.

o Upon identification of the recognition sequence the RE binds with the DNA and cuts each of the two strands of the DNA double helix at specific points in their sugar-phosphate backbones.

Blunt end and Sticky end

o Many RE make a simple double-stranded cut in the middle of the recognition sequence resulting in a blunt end.

▪ Examples of RE producing blunt end: PvuII and AluI.

o In other RE the two DNA strands are not cut at exactly the same position. Instead the cut is staggered, usually by two or four nucleotides, so that the resulting DNA fragments have short single-stranded overhangs at each end.

▪ These are called sticky ends, as base pairing between them can stick the DNA molecule back together again.

▪ This stickiness of the ends facilitates the action of the enzyme DNA ligase.




Nomenclature of Restriction Enzymes

o Rules of nomenclature of RE :

▪ The first letter of the name comes from the genus name.

▪ The second two letters come from the species of the prokaryotic cell from which they were isolated

▪ Following the three letters, the strain is sometimes represented.

▪ Following the strain, the Roman numbers following the names indicate the order in which the enzymes were isolated from that strain of bacteria.

o Examples: (need to remember the EcoRI nomenclature only)

Genus name of Source organism

Species name of Source organism

Order of isolation from the bacteria strain

Steps in formation of recombinant DNA by action of restriction endonuclease enzyme – EcoRI

Recombinant DNA (rDNA)

o RE are used in genetic engineering to form ‘recombinant’ molecules of DNA, which are composed of DNA from different sources/genomes.

o When cut by the same restriction enzyme, the resultant DNA fragments have the same kind of ‘sticky-ends’ and, these can be joined together (end-to-end) using DNA ligases.

o Unless one cuts the vector and the source DNA with the same restriction enzyme, the recombinant vector molecule cannot be created.

Agarose Gel Electrophoresis

o Used for the separation and isolation of DNA fragments that is formed after a restriction digestion.

o Gel electrophoresis separates DNA molecules according to their size.

o DNA molecules are negatively charged.

▪ due to the presence of phosphate moiety .

o DNA molecules can be separated by forcing them to move towards anode under the influence of an electric field through a medium/matrix.

o Matrix used: Agarose

▪ Agarose is extracted from sea weeds.

o The DNA fragments separate (resolve) according to their size through sieving effect provided by the agarose gel.

▪ Smaller DNA fragments: Moves the farthest.

▪ Larger DNA fragments: Moves the least and typically will be nearer to the loading wells of the agarose gel.

Visualization of separated DNA fragments

o DNA is not visible under normal light without staining with a compound that makes the DNA visible.

o Ethidium bromide (EtBr) is used for staining the DNA in the agarose gel.

o The different DNA bands in the gel is clearly visible under ultraviolet light after staining with EtBr.

▪ This procedure is very hazardous because ethidium bromide is a powerful mutagen.

o The DNA bands appear as bright orange colored bands.




o The process of cutting out the required separated bands of DNA from the agarose gel and extraction of the DNA from the gel piece after the gel electrophoresis is called elution.

o This purified DNA fragments then is used in constructing recombinant DNA by joining them with cloning vectors.

o Plasmids and bacteriophages have the ability to replicate within bacterial cells independent of the control of chromosomal DNA.

Copy Number in Vector

o The copy number refers to the number of molecules of an individual plasmid/phage that are normally found in a single bacterial cell.

o Some plasmids may have only one or two copies per cell whereas others may have 15-100 copies per cell.

o Bacteriophages because of their high number per cell, have very high copy numbers of their genome within the bacterial cells.

o If the foreign DNA (alien DNA) can be linked to this plasmid DNA or bacteriophage, the number of foreign DNA becomes equal to the copy number of the plasmid or bacteriophage.

o In other words higher the copy number, more will be the gene expression, and hence more will be product obtained.

Features of a Cloning Vector

o Certain features are essential for the plasmid for the cloning to take place. These are as follows:

1. Origin of Replication (ori)

o Sequence where replication starts.

o If a DNA sequence is linked with the ‘ori’, it gets replicated.

o ‘Ori’ also regulates copy number of this linked DNA.

2. Selectable Marker

o It helps in identifying the transformants from the non-transformants that can be eliminated.

o It helps in selectively growing the transformants.

▪ Transformation – The process through which a foreign DNA (plasmid/vector/rDNA) is introduced into a host bacterial cell.

▪ Transformants – Bacterial cells that have successfully undergone the process of transformation and contains the foreign DNA.










▪ Antibiotic resistant gene

● Ampicillin resistant gene (amp R)

● Tetracycline resistant gene (tet R)

● Chloramphenicol resistant gene

Use of selectable Marker (Just for your Understanding!!)

o It helps to distinguish a cell that has taken up a plasmid (transformant) from the many thousands that have not taken up the plasmid (non-transformants).

o E. coli cells are normally sensitive to the antibiotics ampicillin and tetracycline.

o However, cells that contain the plasmid pBR322 (one of the first cloning vectors to be developed) are resistant to these antibiotics.

o This is because pBR322 carries genes that make the host cell (E. coli) resistant to ampicillin and tetracycline when expressed.

o After transformation with pBR322, only those E. coli cells that have taken up a plasmid are amp R tet R and able to form colonies on an agar medium that contains ampicillin or tetracycline.

o Non-transformants, which does not contain the pBR322, cannot express the antibiotic resistant genes, hence do not produce colonies on the agar medium that contains ampicillin or tetracycline.

o Transformants and non-transformants are therefore easily distinguished.

3. Cloning Sites




o It refers to the segment of DNA in the plasmid where the alien (foreign) DNA can be inserted.

o The vector/plasmid should ideally have one or very few recognition sites for the commonly used RE.




o For the process of cloning, an RE is chosen that is generally part of the selection marker.

▪ For example a foreign DNA can be ligated at the BamH I site of tetracycline resistance gene in the vector pBR322.

Insertional Inactivation

o When we grow bacterial cells on a selective medium ( agar medium that contains ampicillin or tetracycline), we can differentiate between the transformants and non-transformants.

o But we still have no idea if the transformants contains the recombinant plasmid DNA or the original plasmid DNA.

o This technique is used to identify the recombinants from the non-recombinants.

▪ Recombinants – Plasmid DNA with the inserted foreign/alien/target DNA.

o The insertion of a foreign DNA fragment into the plasmid destroys the integrity of one of the genes (selectable marker gene) present on the molecule.




o Recombinants can therefore be identified because the characteristic coded by the inactivated gene is no longer displayed by the host cells.

Insertional inactivation of an antibiotic resistance gene

o When a foreign DNA at the BamH I site of tetracycline resistance gene in the vector pBR322 is ligated, the recombinant plasmids will lose tetracycline resistance due to insertion of foreign DNA.

o It can still be selected out from non-recombinant ones by plating the transformants on tetracycline containing medium.

o The transformants growing on ampicillin containing medium are then transferred on a medium containing tetracycline.

o The recombinants will grow in ampicillin containing medium but not on that containing tetracycline.

o But non- recombinants will grow on the medium containing both the antibiotics.




Insertional inactivation without antibiotic resistance gene (β-galactosidase gene)

o Here the recombinants and the non-recombinants are differentiated based on the basis of their ability to produce color in the presence of a chromogenic substrate.

o In this method the foreign/target DNA is inserted within the coding sequence of an enzyme, β-galactosidase.

o This inactivated the β-galactosidase gene expression.




o When chromogenic substrate (X-gal) is added, bacterial colonies with functional β-galactosidase gives blue color, while bacterial colonies without the functional β-galactosidase gives no color.










Nomenclature of pBR322 (Extra information)

o “p” indicates that this is indeed a plasmid.

o “BR” identifies the laboratory in which the vector was originally constructed (BR stands for Bolivar and Rodriguez, the two researchers who developed pBR322).

o �” distinguishes this plasmid from others developed in the same laboratory (there are also plasmids called pBR325, pBR327, pBR328, etc.).

Vector for Cloning genes in Plants and Animals

Agrobacterium tumifaciens : It delivers ‘T – DNA’ in the several dicot plants and transforms the normal cells into tumor and direct these tumor cells to produce the chemicals required by the pathogen.

▪ The ‘Ti’ Plasmid of A tumifaciens has been modified into a cloning vector.

● It is no more pathogenic to plants.

● It can deliver the foreign gene into a large number of plants.

Retroviruses : It can transform the normal animal cells into cancerous cells.

▪ It now has been modified as follows:

● It is no more pathogenic to animal cells.

● It can deliver the foreign gene into animal cells.

Introduction of Alien DNA into Host Cell

Bacterial Transformation

o Process of uptake of DNA by bacteria.

Competent Cells

o As DNA is hydrophilic in nature, it cannot pass through the cell membranes.

o All bacterial cells can not take up the desired DNA.

o Only competent bacterial cells can take up the DNA.

▪ These cells are prepared by treating them with a specific concentration of a divalent cation, such as calcium.[ 50 mM calcium chloride (CaCl2)]

▪ It increases the efficiency with which DNA enters the bacterium through pores in its cell wall.

Process of Transformation (Heat Shock Treatment)

o Competent cells are incubated along with the rDNA (foreign/target DNA) in an ice-cold condition.

o A heat shock is given to the cells by briefly placing them at 42 0 C.

o The cells are then again placed back on the ice.

o This allows the competent cells to take up the foreign DNA.

Microinjection

o Here the rDNA is directly injected into the nucleus of the host cell.

o It makes use of a very fine pipette to inject DNA molecules.

o This method is generally used for the animal cells.

Biolistic / Gene gun

o The host cells are bombarded with high velocity microprojectiles, usually particles of gold or tungsten coated with DNA.

o This method is more suitable for the plant cells.




PROCESSES OF RECOMBINANT DNA TECHNOLOGY

o Recombinant DNA technology involves several steps in specific sequence such as isolation of DNA, fragmentation of DNA by restriction endonucleases, isolation of a desired DNA fragment, ligation of the DNA fragment into a vector, transferring the recombinant DNA into the host, culturing the host cells in a medium at large scale and extraction of the desired product.

Isolation of DNA

o DNA is isolated by first degrading all the membranes enclosing it with specific enzymes

▪ For bacterial cell – Lysozyme

▪ For plant cells – Cellulase

▪ For fungal cells – Chitinase

o RNA is removed by treatment with RNA digesting enzymes – Ribonuclease.

o Proteins are removed by treatment with protein digesting enzymes – Protease.

o Other biomolecules are removed by appropriate treatments.

o Finally, the DNA is precipitated out by the addition of chilled ethanol.

▪ This is seen as collection of fine threads in the suspension.

▪ This DNA can be removed by spooling.

Cutting of DNA at Specific Locations

o DNA can be cut at specific location by digesting the purified DNA with specific Restriction enzyme.

o Agarose gel electrophoresis can be used to check the progress of restriction digestion.

o After the source DNA and the vector DNA have been cut with a specific RE, the gene of interest is cut out and is ligated with the cut vector DNA (plasmid).

▪ The ligation of the gene of interest and the vector DNA is mediated by an enzyme named – DNA ligase.

Amplification of Gene of Interest using PCR

o The Polymerase Chain Reaction (PCR) results in the selective amplification of a chosen region of a DNA molecule.

Requirements for PCR

o Thermostable enzyme: Taq Polymerase

▪ Source: Bacterium - Thermus aquaticus

▪ Property:The enzyme remains active during the high temperature.

o Sets of Primers:

▪ Primers are small chemically synthesized oligonucleotides that are complementary to the regions of DNA.

▪ They delimit the region of DNA to be amplified.

Step involved in PCR

o PCR is carried out in a single test tube simply by mixing DNA with a set of reagents and placing the tube in a thermal cycler.

1. Denaturation

o The mixture is heated to 94°C, at which temperature the hydrogen bonds that hold together the two strands of the double-stranded DNA molecule are broken, causing the molecule to denature.

o It forms single stranded DNA (ssDNA).

2. Annealing

o The mixture is cooled down to 50󈞨°C.

o The primers anneal (joins) with the ssDNA molecules at specific positions.

3. Extension

o The temperature is raised to 74°C.

o The Taq DNA polymerase works best at this temperature.

o It attaches to one end of each primer and synthesizes new strands of DNA, complementary to the template DNA molecules.

o This results in four stands of DNA instead of the two that there were to start with.

Insertion of Recombinant DNA into the Host Cell/Organism




o Recipient cells after making them ‘competent’ to receive, take up DNA present in its surrounding.

o So, if a recombinant DNA bearing gene for resistance to an antibiotic (e.g., ampicillin) is transferred into E. coli. cells, the host cells become transformed into ampicillin-resistant cells.

o If we spread the transformed cells on agar plates containing ampicillin, only transformants will grow, untransformed recipient cells will die.

o Since, due to ampicillin resistance gene, one is able to select a transformed cell in the presence of ampicillin. The ampicillin resistance gene in this case is called a selectable marker.

Obtaining the Foreign Gene Product

o In most of the recombinant technologies, the ultimate aim is to produce a desirable protein.

o The foreign gene gets expressed and produces the maximum protein in appropriate conditions.

o Recombinant protein : If any protein encoding gene is expressed in a heterologous host.

Culturing of Host cell

o The host cells with the rDNA is grown on an appropriate condition (proper nutrients, temperature, pH, etc.)

o Small scale culture: Cells are grown in the laboratory in cultures. The protein are then extracted and purifies by using different separation techniques.

o Large scale culture: Cells are grown in continuous culture system wherein the used medium is drained out from one side while fresh medium is added from the other to maintain the cells in their physiologically most active log/exponential phase.

o These are large vessels of large volumes (100-1000 litres), in which raw materials are biologically converted into specific products, individual enzymes, etc., using microbial, plant, animal or human cells.

o It provides the optimal conditions for achieving the desired product by providing optimum growth conditions (temperature, pH, substrate, salts, vitamins, oxygen).

Components of a bioreactor:




▪ an oxygen delivery system

▪ a temperature control system

▪ sampling ports so that small volumes of the culture can be withdrawn periodically

Stirred Tank Bioreactor

o A stirred-tank reactor is usually cylindrical or with a curved base to facilitate the mixing of the reactor contents.

o The stirrer facilitates even mixing and oxygen availability throughout the bioreactor.

Sparged Stirred Tank Bioreactor

o It is a stirred-tank reactor type bioreactor where the air is bubbled.

Downstream processing

o It involves all the stages after the expression of the gene product in the culture system by the host cells (biosynthetic stage).

o The processes include separation and purification, which are collectively referred to as downstream processing.

o Suitable preservatives if required are added.

o If the product is a drug, then it undergoes clinical trials.

o The downstream processing and quality control testing vary from product to product.

Download PDF Notes for Biotechnology : Principles and Processes - Class-XII.


Insertional inactivation of the Streptococcus mutans dexA (dextranase) gene results in altered adherence and dextran catabolism

Streptococcus mutans is able to synthesize extracellular glucans from sucrose which contribute to adherence of these bacteria. Extracellular dextranase can partially degrade the glucans, and may therefore affect virulence of S. mutans. In order to isolate mutants unable to produce dextranase, a DNA library was constructed by inserting random Sau3AI-digested fragments of chromosomal DNA from S. mutans into the BamHI site of the streptococcal integration vector pVA891, which is able to replicate in Escherichia coli but does not possess a streptococcal origin of replication. The resultant plasmids were introduced into S. mutans LT11, allowing insertional inactivation through homologous recombination. Two transformants were identified which did not possess dextranase activity. Integration of a single copy of the plasmid into the chromosome of these transformants was confirmed by Southern hybridization analysis. Chromosomal DNA fragments flanking the plasmid were recovered using a marker rescue technique, and sequenced. Comparison with known sequences using the BLASTX program showed 56% homology at the amino acid level between the sequenced gene fragment and dextranase from Streptococcus sobrinus, strongly suggesting that the S. mutans dextranase gene (dexA) had been inactivated. The colony morphology of the dextranase mutants when grown on Todd-Hewitt agar containing sucrose was altered compared to the parent strain, with an apparent build-up of extracellular polymer. The mutants were also more adherent to a smooth surface than LT11 but there was no apparent difference in sucrose-dependent cell-cell aggregation.(ABSTRACT TRUNCATED AT 250 WORDS)


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Abstract

Methionine (Met) S-methyltransferase (MMT) catalyzes the synthesis of S-methyl-Met (SMM) from Met andS-adenosyl-Met (Ado-Met). SMM can be reconverted to Met by donating a methyl group to homocysteine (homo-Cys), and concurrent operation of this reaction and that mediated by MMT sets up the SMM cycle. SMM has been hypothesized to be essential as a methyl donor or as a transport form of sulfur, and the SMM cycle has been hypothesized to guard against depletion of the free Met pool by excess Ado-Met synthesis or to regulate Ado-Met level and hence the Ado-Met toS-adenosylhomo-Cys ratio (the methylation ratio). To test these hypotheses, we isolated insertional mmtmutants of Arabidopsis and maize (Zea mays). Both mutants lacked the capacity to produce SMM and thus had no SMM cycle. They nevertheless grew and reproduced normally, and the seeds of the Arabidopsis mutant had normal sulfur contents. These findings rule out an indispensable role for SMM as a methyl donor or in sulfur transport. The Arabidopsis mutant had significantly higher Ado-Met and lowerS-adenosylhomo-Cys levels than the wild type and consequently had a higher methylation ratio (13.8 versus 9.5). Free Met and thiol pools were unaltered in this mutant, although there were moderate decreases (of 30%–60%) in free serine, threonine, proline, and other amino acids. These data indicate that the SMM cycle contributes to regulation of Ado-Met levels rather than preventing depletion of free Met.

The SMM cycle and its relationship to the activated methyl cycle. The reactions catalyzed by MMT and HMT are bolded. CH3-THF, 5-Methyltetrahydrofolate THF, tetrahydrofolate.

The SMM cycle and its relationship to the activated methyl cycle. The reactions catalyzed by MMT and HMT are bolded. CH3-THF, 5-Methyltetrahydrofolate THF, tetrahydrofolate.

The functions of SMM and its seemingly wasteful cycle are for the most part unknown. The only established role of SMM is in transporting reduced sulfur in the phloem, for which there is qualitative evidence in a range of plants including Arabidopsis ( Bourgis et al., 1999). The importance of SMM relative to other translocated forms of sulfur has been quantified only in wheat (Triticum aestivum), where it accounts for one-half the sulfur moving to developing grains ( Bourgis et al., 1999). However, the contribution of SMM to sulfur transport may be less in other species ( Bourgis et al., 1999) and may depend on developmental stage and sulfur nutrition ( Fitzgerald et al., 2001). A hypothetical role for SMM is as methyl donor for a plant-specific reaction ( Giovanelli et al., 1980). This role has not been tested but is attractive because it would obviously explain why plants alone have SMM.

Roles in transport or methylation might explain why plants produce SMM, but not why there is futile cycling of SMM throughout the plant. Two hypotheses have been advanced to justify this cycling. The first is that the SMM cycle prevents overshoots in Ado-Met synthesis from depleting the free Met pool required for protein synthesis ( Mudd and Datko, 1990) by providing a way to convert Met moieties locked up in Ado-Met back to free Met. The second hypothesis is that the SMM cycle is a means whereby plants control Ado-Met level in the absence of the feedback loops between Ado-Met and the enzymes involved in its synthesis that occur in other eukaryotes ( Ranocha et al., 2001 Roje et al., 2002). Controlling the levels of Ado-Met and AdoHcy is considered crucial to the many methyl transfer reactions that take place in cells: AdoHcy is a potent competitive inhibitor of methyltransferases ( Cantoni et al., 1979) so that the Ado-Met:AdoHcy ratio (the methylation ratio) determines the activity of these enzymes ( Cantoni, 1977). Computer modeling of the SMM cycle in Arabidopsis leaves, based on data for wild-type plants, favored the hypothesis that the SMM cycle contributes to the control of Ado-Met level. Thus, when the SMM cycle was eliminated in silico, the Ado-Met level increased by up to 160%, but steady-state free Met levels did not change. Moreover, the free Met pool recovered from a simulated overshoot in Ado-Met synthesis almost as fast in the absence of the SMM cycle as in its presence ( Ranocha et al., 2001).

In the present study, we investigated the function of SMM and its cycle by isolating and characterizing insertional knockout mutants of MMT in Arabidopsis and maize (Zea mays), which both have singleMMT genes ( Bourgis et al., 1999). We found that SMM is dispensable but that eliminating it caused an increase in Ado-Met level and in the methylation ratio.


Analysis and nucleotide sequence of an origin of DNA replication in Acinetobacter calcoaceticus and its use for Escherichia coli shuttle plasmids

A shuttle plasmid for Acinetobacter calcoaceticus and Escherichia coli has been constructed from a cryptic A. calcoaceticus lwoffi plasmid and pBR322. It is transformed to A. calcoaceticus BD413 by natural competency, yielding about 10(6) transformants per microgram of plasmid DNA. The ApR and TcR genes of pBR322 are functional in A. calcoaceticus. A gene bank was constructed from chromosomal A. calcoaceticus DNA and the shuttle plasmid. Direct transformation to A. calcoaceticus yielded about 95% recombinants, indicating a sixfold enrichment of recombinant plasmids compared to E. coli. One clone complementing a trpE mutation carried a 20-kb insertion and transformed with a 30-fold higher efficiency when compared to the vector. A deletion analysis of the shuttle plasmid indicates that 2.2 kb is necessary for autonomous replication and stable maintenance in A. calcoaceticus. No rearrangements of the DNA or loss of plasmids are found in that organism, even in the absence of selective pressure, when this sequence is present. A further insertional inactivation analysis creating lacZ transcriptional fusions suggests that the origin of replication (ori) is contained within about 1350 bp. Analysis of beta-galactosidase production in A. calcoaceticus indicates that only a weak promoter activity is directed out of one end of this ori. Its sequence contains A + T-rich regions, an 18-bp element with nearly perfect palindromic symmetry and eleven repeats of the consensus sequence, AAAAAATAT, eight of which are clustered within 360 bp. However, no open reading frames or significant homologies to other ori were found.


DNA cloning in Bacillus subtilis. III. Efficiency of random-segment cloning and insertional inactivation vectors ☆

Random segments of Bacillus amyloliquefaciens and yeast Saccharomyces cerevisiae DNA were used to determine two parameters pertinent to cloning in Bacillus subtilis, the yield of hybrids and the mean size of cloned segments. 10 3 to 10 4 hybrids/μg of DNA segments were obtained. Hybrids represented 11–18% of transformants. Mean m. wt. of cloned DNA segments was about 1 × 10 6 , substantially lower than 3 × 10 6 found for donor DNAs after digestion with restriction endonucleases.

We have cloned a B. amyloliquefaciens DNA segment which complemented a deficiency in B. subtilis hisH and E. coli hisC genes, which encode imidazolylacetolphosphate aminotransferase. The cloning efficiency for this gene was 10 transformed hosts/μg of donor DNA.

Several B. subtilis insertional-inactivation cloning vectors were examined. One, pHV41, allows inactivation of the kanamycin-resistance (Km R ) gene by insertion into its unique BglII site. In two other vectors, pHV11 and pHV23, insertion in their unique Kpn site inactivates the tetracycline-resistance (Tc R ) gene. pHV23 replicates both in E. coli and B. subtilis, and carries unique sites for seven restriction endonucleases (BamHI, EcoRI, HpaI, KpnI, PstI, SalI, XbaI). This makes it one of the most versatile B. subtilis cloning vectors yet described.


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