Molecular Diagnostics | Molecular Cloning | Vaccines

Molecular Diagnostics, Therapeutic Agents, Synthesis of Commercial Products by Recombinant Microorganisms.

by Farah Sabrin
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Molecular Diagnostics
The success of modern medicine depends on the detection of specific molecules e.g. Ø Viruses Ø Bacteria Ø Fungi Ø Parasites Ø Proteins In water, plants, soil and humans.

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Characteristics of a Detection System
A good detection system should have 3 qualities:
♣ ♣ ♣

Sensitivity Specificity Simplicity

Sensitivity means that the test must be able to detect very small amounts of target even in the presence of other molecules. Specificity: the test yields a positive result for the target molecule only. Simplicity: the test must be able to run efficiently and inexpensively on a routine basis. 33

ELISA involves: Binding of the test molecule or organism to a solid support e. micro titer plate. 44 .Immunological Diagnostic Procedures Immunological diagnostic procedures are often used to: ♠ ♠ ♠ Test drugs Monitor cancers Detect pathogens ELISA (Enzyme Linked Immunosorbent Assay) This involves the reaction of an antibody with an antigen and a detection system to determine if a reaction has occurred.g.

Addition of a colourless substrate which will react with the secondary antibody to give a colour reaction which indicates a positive result. The secondary antibody usually has attached to it an enzyme e.g. Wash to remove unbound antibody. 55 .ELISA Addition of a specific antibody (primary antibody) which will bind to the test molecule if it is present. Washing to remove unbound molecules. Addition of secondary antibody which will bind to the primary antibody. alkaline phosphatase.

ELISA 66 .

ELISA Animation Click to edit Master text styles Second level ● Third level ● Fourth level ● Fifth level 77 .

ELISA Lab/Detection of HIV Click to edit Master text styles Second level ● Third level ● Fourth level ● Fifth level 88 .

Detection of HIV 99 .

DNA Diagnostic Systems DNA Diagnostic Systems include: Ø Ø Ø Ø Ø DNA Hybridization PCR Restriction endonuclease analysis RAPD (random amplified polymorphic DNA) DNA fingerprinting 1010 .

This involves using a DNA probe during DNA hybridization. Genetic diseases often are due to mutations or absence of particular gene or genes. These genes (DNA) can be used as diagnostic tools. What is a DNA probe? How does DNA hybridization work? 1111 .DNA Hybridization Bacterial and viral pathogens may be pathogenic because of the presence of specific genes or sets of genes.

Attach the target to a solid matrix e. If there is sequence homology between the target and the probe. Denaturation of both the probe and target. 1212 . Detection of the hybridized probe e.g.g. chemiluminsence or colorimetric. the probe will hybridize or anneal to the target. by autoradiography.DNA Hybridization For DNA hybridization: A probe is needed which will anneal to the target nucleic acid. membrane. Add the denatured probe in a solution to the target.

DNA hybridization movie Click to edit Master text styles Second level ● Third level ● Fourth level ● Fifth level 1313 .

g ELISA does not differentiate between past and present infection. kidney and other organs. which is labour intensive. Other methods e. What kind of organism is P. Why? 1414 .Detection of Malaria Malaria is caused by the parasite Plasmodium falciparum. Current treatment involves microscopic observations of blood smears. Symptoms include fever. falciparum? The parasite infects and destroys red blood cells. rashes and damage to brain.

The probes which hybridized strongly were tested further. The probes were tested for their ability to hybridize to other Plasmodium species which do not cause malaria and to human DNA.Detection of Malaria A DNA diagnostic system would only measure current infection. (Why?) The procedure involves: A genomic library of the parasite was screened with probes for parasitic DNA. 1515 .

The probe was able to detect 10 pg of purified DNA or 1 ng of DNA in blood smear. falciparum only could be used as a diagnostic tool. Other DNA probes were developed for the following diseases: Salmonella typhi (food poisoning) E.Detection of Malaria Probes which hybridized to P. coli (gastroenteritis) Trypanosoma cruzi (chagas’ disease) 1616 .

coli.Polymerase Chain Reaction PCR uses 2 sequence specific oligionucleotide primers to amplify the target DNA. tuberculosis). The presence of the appropriate amplified size fragment confirms the presence of the target. viruses (HIV) and fungi. M. Specific primers are now available for the detection of many pathogens including bacteria (E. 1717 .

The RNA is precipitated using isoproponal. 1818 . Reverse transciptase is used to make a cDNA copy of the RNA of the virus. This cDNA is used as a template to make dsDNA. Lyse plasma cells from the potentially infected person to release HIV RNA genome.Using PCR to Detect for HIV RT-PCR (reverse transcriptase PCR). HIV has a ssRNA genome.

RT-PCR Diagnosis of HIV 1919 .

PCR can also be used as a prognostic tool to determine viral load.Using PCR to Detect for HIV Specific primers are used to amplify a 156 bp portion of the HIV gag gene. Using standards the amount of PCR product can be used to determine the viral load. (Brock Biology of Microorganisms 9th ed. 2020 . pg 883-886). This method can also be used to determine the effectiveness antiviral therapy.

Paternity testing 2121 Ë .g. Ë In legal proceedings to identify suspects and clear others. DNA fingerprinting (DNA typing) is used to characterize biological samples e. DNA fingerprinting analyses genotypic traits.DNA Fingerprinting (RFLP) RFLP = Restriction Fragment Length Polymorphism Regular fingerprinting analyses phenotypic traits.

Membrane is hybridized with 4-5 different probes. and skin. The DNA is digested with restriction enzymes. blood. Digested DNA is separated by agarose gel electrophoresis. semen. Detection of hybridization. Examination of sample to determine if there is enough DNA for the test. DNA is transferred by Southern blotting to a membrane.DNA Fingerprinting (RFLP) The procedure involves: Collection of sample e. hair.g. 2222 .

Microsatellite DNA After hybridization the membranes are stripped and reprobed. 2323 .ATTAG….g ATTAG…. The microsatellites have different length and numbers in different individuals. These sequences occur in the human genome as repeated sequences. E. The variability is due to either a gain or lost of repeats during replication. The length of the repeat is 9-40 bases occurring 10-30 times.ATTAG…. The probes used are human microsatellite DNA.

2424 . An individual inherit one microsatellite from each parent. The chance of finding two individuals within the same population with the same DNA fingerprint is one in 105 . In other words an individuals DNA fingerprint is almost as unique as his or her fingerprint.Microsatellite DNA These changes do not have any biological effect because the sequences do not code for any protein.108.

DNA Fingerprinting 2525 .

RAPD is often used to show relatedness among DNA populations. 2626 .Random Amplified Polymorphic DNA (RAPD) Another method widely used in characterization of DNA is RAPD. In this procedure arbitrary (random) primers are used during PCR to produce a fingerprint of the DNA. A single primer is used which must anneal in 2 places on the DNA template and region between the primers will be amplified.

This procedure is widely used to differentiate between different cultivars/varieties of the same plant. If the samples have similar genetic make up then the pattern of bands on the gel will be similar and vice versa. quality of DNA. Amplified products are separated by agarose gel electrophoresis and visualized. Issues to consider when using this procedure include reproducibility. and several primers may have to be used. 2727 .RAPD The primers are likely to anneal in many places on the template DNA and will produce a variety of sizes of amplified products.

RAPD 2828 .

Bacteria with bioluminescent are good candidates for pollutant sensors. The structural genes (luxCDABD) encodes the enzyme for bioluminescent was cloned into the soil bacteria Pseudomonas fluorescens.Bacterial Biosensors Bacterial sensors can be used to test for environmental pollutants. The cells that luminescence to the greatest extent and grew as well as the wild type were tested as pollutant sensors. In the presence of pollutants the bioluminescent decreases. 2929 .

The procedure is rapid. cheap and a good screen for pollutants. 3030 .Bacterial Biosensors To screen water samples for pollutants (metal or organic) a suspension of P. simple. When the solution contained low to moderate levels of pollutants the bioluminescence was inhibited. After a 15 min incubation the luminescence of the suspension was measured. fluorescens was mixed with the solution to be tested.

Click to edit Master text styles Second level ● Third level ● Fourth level ● Fifth level Bacterial Bisensor 3131 .

The disease results in progressive anemia and damage to heart.Restriction Digest Analysis Diagnosis of sickle cell anemia. 3232 . brain. A (normal) glutamic acid and S (sickle) valine. In the homozygous state SS the red blood cells are irregularly shaped. Sickle cell anemia is a genetic disease which is caused by a single nucleotide change in the 6th aa of the  chain of hemoglobin. This occurs because the mutant hemoglobin is unable to carry enough oxygen to supply these systems. joints and other organ systems. lung.

3333 .Diagnosis of Sickle Cell Anemia The single mutation in hemoglobin cause a change in the restriction pattern of the  globin gene abolishing a CvnI site. CvnI site CCTNAGG (N = any nt) Normal DNA sequence CCTGAGG (A) Mutant DNA sequence CCTGTGG (S) Two primers which flank the mutant region of the  globin gene is used during PCR to amplify this region of the gene. The PCR products is digested with CvnI and separated by agarose gel electrophoresis.

Detection of Sickle cell anemia by PCR 3434 .

E.g. 3535 . 2 short oligonucleotides (oligo) are synthesized Oligo 1 (probe x) is complementary to the wild type has A at 106 (3’ end). A single nt change can be detected by PCR/OLA ( oligonucleotide ligation assay).? Many diseases are caused by a single nucleotide (nt) change in the wild type gene.PCR/OLA Like sickle cell anemia many genetic diseases are caused by mutant genes. E.g. The normal gene has A at nt position 106 and mutant has a G.

PCR/OLA Oligo 2 ( probe y) has G at 107 (5’ end). 3636 . For the mutant gene the nt at the 3’ end of probe x is a mismatch and does not anneal. The two probes are incubated with the PCR amplified target DNA. For the wild type the two probes anneal so that the 3’end of probe x is next to the 5’end of probe y.

PCR/OLA Click to edit Master text styles Second level ● Third level ● Fourth level ● Fifth level 3737 .

PCR/OLA
DNA ligase is added. The two probes will only ligate if the two probes are perfectly aligned (as in the wild type). To determine if the mutant or wild type gene is present it is necessary to detect for ligation. Probe x is labeled at 5’ end with biotin Probe x is labeled at 5’ end with digoxygenin.
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PCR/OLA
Digoxygenin serves as an antibody binding indicator. After washing a colourless substrate is added. If a coloured substrate appears this is indicative that the biotin probe (x) ligated to the dioxygenin probe (Y) and that the wild type gene is present.
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PCR/OLA

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4141 . expressed in the host cells and are being tested as therapeutic agents (drugs) in humans. Today hundreds of genes for human proteins have been cloned. sequenced.Therapeutic Agents Before the advent of molecular biotechnology most human proteins were available in only small (limited) quantities.

Enzymes as Therapeutic Agents/ DNase1 Cystic fibrosis (CF) is one of the most fatal heredity diseases among European and their descendants with ~30. Individuals with CF are highly susceptible to bacterial infection and antibiotic treatment often results in resistant strains.000 cases in the US and 23.500 live birth and 1 in 25 are carriers.000 in Canada. Furthermore among European descendants it occurs in 1 in 2. It is caused by more than 500 different mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. 4242 .

4343 . Scientist at Genentech isolated the gene for DNase1 The purified enzyme was delivered as an aerosol to the lung where it hydrolysed the DNA into short oligionucleotides. This decrease the viscosity in the lungs and made breathing easier.DNase 1 A thick mucus which is a results of: Alignate produced by bacteria DNA from lysed cells Leucocytes which accumulate due to the infection Makes breathing difficult.

Alginate Lyase Alginate is a polysaccharide polymer that is produced by seaweed and some soil and marine bacteria. The excretion of alginate by Pseudomonas aeruginosa of patients with CF contributes to the viscosity in the lung. Alginate lyase was isolate from Flavobacterium sp. coli. 4444 . and cloned into E. The enzyme alginate lyase can liquefy bacteria alginate.

4545 .000 Da protein was cleaved to produce a 43.000 Da.Aliginate Lyse The expressed gene produced a protein of 69.000 Da.000 Da which is able to liquefy bacterial alginate. alginate lyse is able to reduce the mucus in the lungs of patients with CF. Combined with DNase1. The 69.000 Da protein produced a proteolytic enzyme of 6. The remain 63.

Click to edit Master text styles Second level ● Third level ● Fourth level ● Fifth level DNAse 1 and Alginate lyase Click to edit Master text styles Second level ● Third level ● Fourth level ● Fifth level 4646 .

4747 .Production of Monoclonal Antibodies Monoclonal antibodies results from a clone of a B lymphocyte producing a single antibody which will bind to a specific epitope of an antigen. What is a polyclonal antibody? Monoclonal antibodies are produced: Fusion of a myeloma (B cell which has become cancerous) with a spleen cell that is immunized with a specific antigen. The resulting hybridomas are tested for the production of a monoclonal antibodies.

Production of Monoclonal Antibodies 4848 .

4949 . coli. The amplified products are cut with restriction enzymes and cloned into Lambda vector.Production of Human Monoclonal Antibodies by E. Both heavy and light chains are amplified separately from the cDNA using PCR. coli Hybridoma cells grow relatively slow and require expensive media. cDNA is synthesized from the mRNA by the enzyme reverse transciptase. The produce involves: mRNA is isolated from the B cell. To circumvent this problem human monoclonal antibodies are grown in E.

Production of Human Monoclonal Antibodies by E. coli 5050 .

The DNA of one heavy and one light chain are cloned into the same vector. 5151 . coli. coli During cloning different light and heavy chains are cloned. The L and H chains are excised from Lambda and cloned into an E. Lambda is not useful for producing large amounts of proteins.Production of Human Monoclonal Antibodies by E. coli plasmid and the recombinant plasmid transformed into E. Many different combinations of H and L chains are cloned together in the same vector.

coli 5252 .Production of Human Monoclonal Antibodies by E.

coli will produce large amounts of monoclonal antibodies which are harvested. 5353 . Therapeutically for the treatment of infection.g detection of HIV by ELISA.Production of Human Monoclonal Antibodies by E. These monoclonal antibodies can be used for: ¶ ¶ Diagnostic purposes e. coli E.

Theoretically a small ss nucleic acid can hybridize to a specific gene or mRNA and diminish transcription or translation. parasites) are often caused by overproduction of a normal protein. Ribozyme (catalytic RNA) and interfering RNA ( RNAi) can target specific mRNA for degradation.g. 5454 . cancer and inflammatory conditions (virus. An oligo that binds to mRNA and blocks translation is called an antisense oligo. An oligonucleotide (oligo) that binds to a gene and blocks transcription is an antigene.Nucleic Acids as Therapeutic agents (e 3rd) Many human disorders e.

Antisense RNA Click to edit Master text styles Second level ● Third level ● Fourth level ● Fifth level 5555 .

Antisense Oligonucleotide Click to edit Master text styles Second level ● Third level ● Fourth level ● Fifth level 5656 .

A similar treatment of mice which were injected with prostate carcinoma cells caused small or no tumor to develop. 5757 .Antisense RNA Episomally based expression vectors with cDNA for insulin-like growth factor 1 (ILGF-1) receptors were constructed in the antisense version. Culture of glioma cells when transfected with the antisense version of ILGF-1 in ZnSO4 lost its tumurous properties. ILGF-1 is prevalent in malignant glioma a common form of brain cancer and prostate carcinoma.

5858 . However when injected into the body is deoxynucleotides are susceptible to degradation. Free oligos are usually introduced into to the body encapsulated in a liposome.Antisense Oligonucleotides Antisense deoxynucleotides can also be used as therapeutic agents. phosphoramidate and polyamide. To prevent this modified deoxynucleotides are used including phosphorothioate.

Modified Deoxynucleotides Phosphodiester linkage Phosphorothioate linkage 5959 .

Modified Deoxynucleotides Phosphoramidite linkage Polyamide linkage 6060 .

Click to edit Master text styles Second level ● Third level ● Fourth level ● Fifth level Liposome 6161 .

Narrowing of the coronary and carotid arties can lead to heart attacks and strokes.Therapeutic Nucleic Acids Several preclinical trials have been conducted with antisense oligos. This involved inserting a balloon into the blocked artery and inflating it. 6262 . This often results in injury of the artery and subsequent healing can result in blockage in 40% of patients within 6 months. This condition is alleviated by angioplasty.

6363 .Therapeutic Nucleic Acids When antisense oligo that target the mRNA for a protein essential for the cell cycle was applied to rat carotid arteries after angioplasty the reoccurring blockage was reduced by 90%. Furthermore smooth muscle cell proliferation is implicated in other disease such as: Ω Ω Ω Ω Atherosclerosis Hypertension Diabetics mellitus Failing of coronary bypass graft Similar antisense therapeutics could be used to help alleviate these conditions.

ILGF-1 receptors are implicated in the pathogenesis of psoriasis. 15 nt antisense oligo were transferred into keratinocytes using liposome and the amount of ILGF-1 protein was decreased by 45-65%. When mouse with human psoriasis lesions were injected with anitsense oligi complementary to ILGF-1 receptor mRNA there was significant reduction (58-69%) in epidermal thickness.Antisense Oligos and Psoriasis Antisense oligos have also been tested in the treatment of psoriasis. 6464 . Psoriasis is uncontrollable epidermal growth.

Gene silencing has been shown to be a natural mechanism which plant and animals use to protect against viruses. 6565 . These short oligos complex with RISC ( RNA inference inducing silencing complex) which degrade the mRNA complimentary to the oligos. This process is called gene silencing or RNA inference (RNAi). This process can be used to target specific mRNA. The dsRNA that is introduced is cleaved by dicer-like dsRNAse into ssRNA of 21-23 nt.Interfering RNA The addition of dsRNA to an animal cell causes the degradation of the mRNA from which it is derived.

6666 .RNA1 as Therapeutic Agents A viral vector was used to deliver a small fragment of RNA to brain cells of mice with SCA1 (human neurodegenerative disease spinocerebellar ataxia 1). Scientists are optimistic about using RNAi to treat other neurological diseases such as Alzheimer’s and Hunting’s disease. This suppress the SCA1 gene and the mice has normal coordination and movement.

slowly shutting down the immune system.HIV Therapeutic Agents (e 2nd) Acquired immune deficiency syndrome (AIDS) is caused by the human immunodeficiency virus (HIV). 6767 . The target of HIV are the T helper cells (TH). The TH cells become infected with the virus and are destroyed. TH cells play a pivotal role in the immune system by the release of cytokines which stimulate other immune cells. The gp120 glycoprotein of HIV binds to CD4 receptors of TH cells.

Interaction of HIV with CD4 Click to edit Master text styles Second level ● Third level ● Fourth level ● Fifth level 6868 .

Both strategies do not destroy HIV but only block infection. To stop HIV infection we need to develop strategies which will destroy HIV.HIV Therapeutic Agents HIV antiviral strategies may include: Production of antibodies to CD4 (will block CD4 receptors on TH cells and prevent infection by HIV). 6969 . Production excess CD4 protein (react with gp120 protein therefore HIV cannot infect TH cells).

HIV Therapeutic Agents
One strategy which will protect TH cells and destroy HIV include the production of a fusion protein. The fusion protein will have 2 parts CD4 protein attached to the Fc portion of an immunoglobulin (CD4 immunoadhesion). The CD4 portion will attach to the gp120 protein of HIV or virus infected cells. The immunoglobulin portion will initiate a cytotoxic response to destroy the virus or virus infected cell.
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CD4 Immunoadehesion Fusion Protein

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HIV Therapeutic Agents
Another strategy involves making a second fusion protein. The CD4 sequence is ligated to the sequence of Pseudomonas exotoxin A to form a fusion protein. HIV infected cells have gp120 proteins on their surfaces. The CD4 portion of the fusion protein will attach to the infected cells. The fusion protein will enter the cells and initiate the killing of the infected cell. Pseudomonas exotoxin A inactivates the protein synthesis by affecting elongation factor EF-2. This prevents further protein synthesis and eventually causes death of the infected cell.
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CD4-Pseudomonas Exotoxin Fusion Protein 7373 .

During vaccination a vaccine is injected or given orally.Principles of Vaccination A vaccine renders the recipient resistant to infection. 7474 . The host produces antibodies for a particular pathogen. Generally to produce a vaccine the pathogen is grown in culture and inactivated or nonvirulent forms are used for vaccination. Upon further exposure the pathogen is inactivated by the antibodies and disease state prevented.

© The procedure is expensive and sometimes unsafe. HIV vaccination is not appropriate.g. why? 7575 . © For some pathogens e.Principles of Vaccination There are many disadvantages and they include: © Not all organisms can be cultured. © New pathogens keep occurring.

ª Virulence genes are deleted and organism is still able to stimulate an immune response.New Generation of Vaccines Recombinant DNA technology is being used to produce a new generation of vaccines. coli.g. genes of proteins for antigenic determinants can be cloned and expressed in an alternative host e. If the agent cannot be maintained in culture. Live nonpathogenic strains can carry antigenic determinants from pathogenic strains. 7676 ª ª . E.

Binding of antibodies to these proteins will stimulate an immune response. 7777 . Therefore proteins can be use to stimulate an immune response.New Generation of Vaccines There are three types of vaccines we will be discussing: v v v Subunit (protein) vaccines Attenuated vaccines Vector vaccines Subunit Vaccines Antibodies usually bind to surface proteins of the pathogen or proteins generated after the disruption of the pathogen.

7878 ¥ .g: ¥ ¥ Herpes simplex virus envelop glycoprotein O. E.Principles of Vaccination It has been showed that the capsid or envelope proteins are enough to illicit an immune response. Foot and mouth disease virus capsid protein (VP1) Extracellular proteins produced by Mycobacterium tuberculosis.

bovis often responds to diagnostic test for M. About 2 billion people are infected and there are 3 million deaths/year. tuberculosis (e 2nd) Tuberculosis is caused by Mycobacterium tuberculosis. tuberculosis. Currently tuberculosis is controlled by a vaccine called BCG (Bacillus Calmette-Guerin) which is a strain of M. 7979 .A Subunit Vaccine for M. bovis. The bacterium form lesions in the tissues and organs causing cell death. Often the lung is affected. M.

These animals were then challenged with M. tuberculosis were purified. After 9-10 weeks examination showed that some combinations of the purified proteins provided the same level of protection as the BCG vaccine. Separately and in combinations these proteins were used to immunized guinea pigs. tuberculosis. tuberculosis Six extracellular proteins of M.A Subunit Vaccine for M. 8080 .

The Development of a Live Cholera Vaccine. With this in mind a live vaccine for cholera was developed. dehydration abdominal pain and diarrhea. Cholera is characterized by fever. Live vaccines are more effective than a killed or subunit (protein) vaccines. 8181 .Attenuated Vaccines Attenuated vaccines often consists of a pathogenic strains in which the virulent genes are deleted or modified.

8282 . Presently the cholera vaccine consist of a phenolkilled V. A live vaccine would be more effective. cholerae and it only last 3-6 months. In the sequence of the A peptide a tetracycline resistance gene is inserted. V. cholerae produces a enterotoxin with an A subunit and 5 B subunits.A Live Cholera Vaccine The causal agent of cholera is Vibrio cholerae and is transmitted through contaminated water.

This removes 550 bases of A peptide. This plasmid was mixed with V. 8383 . By conjugation the plasmid was transferred to the strain with the tetR gene inserted into it’s chromosomal DNA.A Live Cholera Vaccine A plasmid with A peptide was digested with 2 restriction enzymes Cla1 and Xba1. cholerae with tetracycline resistant gene. A Xba1 linker was added and T4 ligase used to ligate the DNA.

Production of a Live Cholera Vaccine 8484 .

cholerae with a 550 bp of the A peptide deleted. If this can be used as a vaccine is being tested. The final result is V.A Live Cholera Vaccine By recombination the A peptide with the tetR gene was replaced by the deleted A peptide. 8585 .

Production of a Live Cholera Vaccine 8686 .

The vaccinia virus is generally nonpathogenic.Vector Vaccine A vector vaccine is a vaccine which is introduced by a vector e. vaccinia virus. The virus replicates in the cytoplasm rather than in the nucleus. The genome of the vaccinia virus has been completely sequenced. The vaccinia virus as a live vaccine led to the globally eradication of the smallpox virus. 8787 .g.

The procedure involves: The DNA sequence for the specific antigen is inserted into a plasmid beside the vaccinia virus promoter in the middle of a non-essential gene e. 8888 .g.Vector Vaccine These characteristics makes the vaccinia virus a good candidate for a virus vector to carry gene for antigenic determinants form other pathogens. thymidine kinase.

Thus virus can now be used as a vaccine for the specific antigen.Vector Vaccine The plasmid is used to transform thymdine kinase negative cells which were previously infected with the vaccinia virus. 8989 . Recombination between the plasmid and vaccinia virus chromosomal DNA results in transfer of antigen gene from the recombinant plasmid to the vaccinia virus.

Insertion of antigen gene into vaccinia virus genome 9090 .

A recombinant vaccinia virus vaccine for rabies is able to elicit neutralizing antibodies in foxes which is a major carrier of the disease.Vector Vaccine A number of antigen genes have been inserted into the vaccinia virus genome e. 9191 . v v v Rabies virus G protein Hepatitis B surface antigen Influenza virus NP and HA proteins.g.

Y Y Y Vitamins Amino acids Antibiotics We will be investigating the use of recombinant organisms to improve or enhance the production of : z z z z Restriction enzymes Ascorbic acid Microbial synthesis of the dye indigo Production of xanthan gum 9292 .g.The Production of Commercial Products by Recombinants Microorganisms Molecular biotechnology can be used to enhance the production of many commercially important compounds e.

To avoid maintaining a large # of microorganisms with differing growth requirements investigators usually clone the RE genes into E. These enzymes are produced by a number of bacteria with different specific growth requirements e.g. This allows for the standardized production of RE in E. coli. 9393 . coli.Restriction Endonucleases (REs) Currently there are more than 400 RE available. temperature. pH and O2 tension.

Advantages of Using E. coli has been genetically characterized extensively. coli to Produce REs Ò Ò Ò Ò Ò Using E. coli up to 40% of the protein produced can be from the cloned gene. coli has certain advantages: E. coli cells will grow rapidly to high density. What are restriction enzymes? How do they work? How does a cell that produces restriction enzymes protect its DNA from the RE it produces? 9494 . The target RE can be over expressed for increased production. In E. E.

In some Gram –ve bacteria the RE is localized in the periplasmic space. What is the periplasmic space? One way to avoid the problem of degradation of host DNA by a cloned RE is to clone and express both the restriction enzyme and the modification enzyme in the same host.Protection of DNA from REs The microorganism that produces the RE protects its DNA from degradation by methylation of the recognition sequence. 9595 .

Periplasmic Space 9696 .

coli. coli cells are infected with Lambda phage. The chromosomal DNA of P. 9797 . The transformed E. coli PstI is produced by Providencia stuartii. coli then the cells will be resistant to the lytic action of lambda because the RE will degrade the DNA of the infecting phage. The clone bank is transformed into E. stuartii is digested with HindIII and ligated into the HindIII site of pBR322.Cloning of Pst1 into E. If the RE is expressed in E.

Cloning of Restriction Enzyme Gene 9898 .

Cloning of Pst1 into E. 9999 . coli The lambda resistant transformants are grown and assayed for Pst1 activity. stuartii. The level of Pst1 expression in E. coli was 10 times compared to P. The positive clones are assayed for Pst1 methylase activity.

Ascorbic acid (vitamin C) is currently synthesized by a very expensive process which includes: ♦ A microbial fermentation step ♦ Number of chemical steps ♦ Which converts glucose to ascorbic acid 100100 .Synthesis of L-Ascorbic Acid With recombinant DNA technology it is possible to introduce new genes which will modify metabolic pathways.

Commercial Synthesis of Ascorbic Acid 101101 .

5-DKG  2-KLG (2-keto-L-gluconic acid).5-DKG).g. Many organisms can synthesize 2.Synthesis of L-Ascorbic Acid Glucose  2.5-DKG e.: û û û Acetobacter Glucanobacter Erwinia The enzyme 2.5-DKG reductase will convert 2.5-diketo-D-gluconic acid (2. Organisms which can carry out this reaction include: è è è Corynebacteria Brevibacterium Arthrobacter 102102 .

The best way to convert glucose to 2-KLG would be to engineer an organism which could carry out both reactions. 103103 .Synthesis of L-Ascorbic Acid The last step in the process involves the conversion of: 2-keto-L-gulonic acid (2-KLG)  L-ascorbic acid.

Production of 2-KLG 104104 .

herbicola. This recombinant organisms would be able to convert glucose to 2KLG. Which involves a single reaction.5DKG reductase from Corynebacterium into E. 105105 .5-DKG is done by Erwinia herbicola and involves several reactions.Microbial Synthesis of L-Ascorbic Acid The conversion of glucose to 2. The simplest thing to do would be to clone 2. The conversion of 2.5-DKG to 2-KLG is possible by Corynebacterium sp.

5-DKG Reductase The enzyme was purified and the sequence of the first 40 aa were determined. The clone with 2. coli.5-DKG reductase).5-DKG reductase was sequenced. Any clone that reacted with both probes was assumed to be the target DNA. The Corynebacterium DNA was probed for the target DNA (gene for 2. Regulatory sequences were added to the 2. Based on the aa sequence two 43 nt probes were made from different portions of the protein.5-DKG reductase gene and cloned into E. 106106 .Cloning of 2.

5-DKG reductase was subcloned into E. herbicola cells were able to convert glucose to 2-KLG. Site-directed mutagenesis was used to increase (double) the activity of the 2.5-DKG reductase by changing Glu (192)  Arg.5-DKG Reductase The 2. The recombinant E. Gly at 55 and 57  Ala increased the thermal stability of the enzyme.Cloning of 2. 107107 . hericola.

It is the colouring agent in blue jeans and is the largest selling dye in the world. Approximately 13 million kg is produced each year worth $200 million USD. 108108 .Indigo Dye Indigo is an important blue pigment used to dye both cotton and wool. It is isolated from plants or made chemically.

g. xylene and phenol as their sole carbon source. coli. The enzymes for the degradation of these compounds are usually located on a large plasmid (~50-200 kb). 109109 . NAH7 is one such plasmid.Microbial Synthesis of Indigo Many Pseudomonas species have the ability to use a variety of organic carbon compounds e. During the characterization of NAH7 the DNA was digested with HindIII and ligated into pBR322 and transformed into E. toluene. napthalene.

Microbial Synthesis of Indigo One transformant with a 10.5 kb insert when grown on minimal media with tryptophan turned blue. Therefore the combination of enzymes from 2 different organisms resulted in the synthesis of a commercially important product. formaldehyde and cyanide which are used in the chemical synthesis of indigo. The microbial production of indigo avoids the use of hazardous chemicals such as aniline. Analysis of the blue colour revealed that the E. 110110 . coli cells were synthesizing the dye indigo.

Biosynthesis of Tryptophan E.coli NAH7 111111 .

Æ In industrial it produces xanthan gum which is used in the food. cosmetics and oil industries. Æ In agriculture it is a pathogen that causes black rot on a number of crops.Production of Xanthan Gum Xanthomonas campestris pv. campestris is an agricultural and industrial important bacterium. 112112 .

Xanthan Gum Xanthan gum is a high molecular weight exopolysaccharides with: Ø Ø Cellulose backbone Trisaccharides side chain 113113 .

Xanthan Gum Due to its: Ö Ö Ö High viscosity Stable properties in extreme conditions Pseudoplastic behaviour It has applications as: Stabilizing Viscosifying Emulsifying Thickening Suspending agent 114114 .

campestris X. and starch as carbon source but not lactose. This enzyme converts lactose to galactose and glucose. campestris is able to use glucose. sucrose.Growth of X. This is due to the low level of enzyme  galactosidase. 115115 .

It contains 95% water.Production of Xanthan Gum Whey is a nutrient rich by product of cheese making. 4-5% lactose. and 0. xanthan gum.81. It is produced by the diary industry in large quantities and its disposal is a major problem for the cheese industry.0% glucose. One useful way to dispose of the whey would be to use it as a substrate for the production of a useful product e.g. 116116 .

campestris which produce xanthan gum was not able to grow on whey because of the low level of  galactosidase.Production of Xanthan Gum X. The E. With this in mind X. coli LacZY gene which codes for  galactosidase and lactose permease was cloned into a plasmid under the control of bacteriophage ( LO) promoter. campestris. One way to resolve this problem would be to increase the level of  galactosidase in X. 117117 . campestris was engineered to grow on whey. Lactose permease transports lactose into the cell.

campestris. campestris were able to grow on lactose and produce as much xanthan gum as wild type X. campestris was able to express  galactosidase and lactose permease at high levels. coli to X. The recombinant X. The transformed X. 118118 . campestris.Production of Xanthan Gum The LacZY genes were transferred from E.

THE END 119119 .

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