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. Viruses Bacteria Fungi Parasites 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

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. ELISA involves: Binding of the test molecule or organism to a solid support e.g. micro titer plate.
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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. The secondary antibody usually has attached to it an enzyme e.g. alkaline phosphatase. Wash to remove unbound antibody. Addition of a colourless substrate which will react with the secondary antibody to give a colour reaction which indicates a positive result.
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ELISA

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ELISA Animation
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ELISA Lab/Detection of HIV

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Detection of HIV

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DNA Diagnostic Systems

DNA Diagnostic Systems include:

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

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

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Detection of Malaria
Malaria is caused by the parasite Plasmodium falciparum.
What kind of organism is P. falciparum?

The parasite infects and destroys red blood cells. Symptoms include fever, rashes and damage to brain, kidney and other organs. Current treatment involves microscopic observations of blood smears, which is labour intensive. Other methods e.g ELISA does not differentiate between past and present infection. Why?
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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. 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.

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

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Polymerase Chain Reaction

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

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Using PCR to Detect for HIV

RT-PCR (reverse transcriptase PCR). HIV has a ssRNA genome. Lyse plasma cells from the potentially infected person to release HIV RNA genome. The RNA is precipitated using isoproponal. 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.
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RT-PCR Diagnosis of HIV

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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. PCR can also be used as a prognostic tool to determine viral load. This method can also be used to determine the effectiveness antiviral therapy. (Brock Biology of Microorganisms 9th ed. pg 883-886). 2020

DNA Fingerprinting (RFLP)

RFLP = Restriction Fragment Length Polymorphism Regular fingerprinting analyses phenotypic traits. DNA fingerprinting analyses genotypic traits. DNA fingerprinting (DNA typing) is used to characterize biological samples e.g.

In legal proceedings to identify suspects and clear others. Paternity testing

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DNA Fingerprinting (RFLP)

The procedure involves: Collection of sample e.g. hair, blood, semen, and skin. Examination of sample to determine if there is enough DNA for the test. The DNA is digested with restriction enzymes. Digested DNA is separated by agarose gel electrophoresis. DNA is transferred by Southern blotting to a membrane. Membrane is hybridized with 4-5 different probes. Detection of hybridization.
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Microsatellite DNA
After hybridization the membranes are stripped and reprobed. The probes used are human microsatellite DNA. These sequences occur in the human genome as repeated sequences. E.g ATTAG.ATTAG.ATTAG. The length of the repeat is 9-40 bases occurring 10-30 times. The microsatellites have different length and numbers in different individuals. The variability is due to either a gain or lost of repeats during replication.
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Microsatellite DNA
These changes do not have any biological effect because the sequences do not code for any protein. 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 - 108. In other words an individuals DNA fingerprint is almost as unique as his or her fingerprint.
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DNA Fingerprinting

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Random Amplified Polymorphic DNA (RAPD)

Another method widely used in characterization of DNA is RAPD. RAPD is often used to show relatedness among DNA populations. 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.

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

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

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

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Restriction Digest Analysis

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

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

PCR/OLA
Oligo 2 ( probe y) has G at 107 (5 end). The two probes are incubated with the PCR amplified target DNA. For the wild type the two probes anneal so that the 3end of probe x is next to the 5end of probe y. For the mutant gene the nt at the 3 end of probe x is a mismatch and does not anneal.
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PCR/OLA
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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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Therapeutic Agents
Before the advent of molecular biotechnology most human proteins were available in only small (limited) quantities. Today hundreds of genes for human proteins have been cloned, sequenced, expressed in the host cells and are being tested as therapeutic agents (drugs) in humans.
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Enzymes as Therapeutic Agents/ DNase1

Cystic fibrosis (CF) is one of the most fatal heredity diseases among European and their descendants with ~30,000 cases in the US and 23,000 in Canada. Furthermore among European descendants it occurs in 1 in 2,500 live birth and 1 in 25 are carriers. It is caused by more than 500 different mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Individuals with CF are highly susceptible to bacterial infection and antibiotic treatment often results in resistant strains. 4242

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. 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.
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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. The enzyme alginate lyase can liquefy bacteria alginate. Alginate lyase was isolate from Flavobacterium sp. and cloned into E. coli.
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Aliginate Lyse
The expressed gene produced a protein of 69,000 Da. The 69,000 Da protein produced a proteolytic enzyme of 6,000 Da. The remain 63,000 Da protein was cleaved to produce a 43,000 Da which is able to liquefy bacterial alginate. Combined with DNase1, alginate lyse is able to reduce the mucus in the lungs of patients with CF.
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DNAse 1 and Alginate lyase

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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.

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Production of Monoclonal Antibodies

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Production of Human Monoclonal Antibodies by E. coli

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

Production of Human Monoclonal Antibodies by E. coli

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Production of Human Monoclonal Antibodies by E. coli

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

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Production of Human Monoclonal Antibodies by E. coli E. coli will produce large amounts of monoclonal antibodies which are harvested. These monoclonal antibodies can be used for:

Diagnostic purposes e.g detection of HIV by ELISA. Therapeutically for the treatment of infection.

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Nucleic Acids as Therapeutic agents (e 3rd)

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

Antisense RNA
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Antisense Oligonucleotide
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Antisense RNA
Episomally based expression vectors with cDNA for insulin-like growth factor 1 (ILGF-1) receptors were constructed in the antisense version. ILGF-1 is prevalent in malignant glioma a common form of brain cancer and prostate carcinoma. Culture of glioma cells when transfected with the antisense version of ILGF-1 in ZnSO4 lost its tumurous properties. A similar treatment of mice which were injected with prostate carcinoma cells caused small or no tumor to develop.
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Antisense Oligonucleotides
Antisense deoxynucleotides can also be used as therapeutic agents. However when injected into the body is deoxynucleotides are susceptible to degradation. To prevent this modified deoxynucleotides are used including phosphorothioate, phosphoramidate and polyamide. Free oligos are usually introduced into to the body encapsulated in a liposome.
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Modified Deoxynucleotides

Phosphodiester linkage

Phosphorothioate linkage
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Modified Deoxynucleotides

Phosphoramidite linkage

Polyamide linkage

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Liposome

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Therapeutic Nucleic Acids

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

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:

Atherosclerosis Hypertension Diabetics mellitus Failing of coronary bypass graft

Similar antisense therapeutics could be used to help alleviate these conditions. 6363

Antisense Oligos and Psoriasis

Antisense oligos have also been tested in the treatment of psoriasis. Psoriasis is uncontrollable epidermal growth. 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. 6464

Interfering RNA
The addition of dsRNA to an animal cell causes the degradation of the mRNA from which it is derived. This process is called gene silencing or RNA inference (RNAi). Gene silencing has been shown to be a natural mechanism which plant and animals use to protect against viruses. The dsRNA that is introduced is cleaved by dicer-like dsRNAse into ssRNA of 21-23 nt. These short oligos complex with RISC ( RNA inference inducing silencing complex) which degrade the mRNA complimentary to the oligos. This process can be used to target specific mRNA.
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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). This suppress the SCA1 gene and the mice has normal coordination and movement. Scientists are optimistic about using RNAi to treat other neurological diseases such as Alzheimers and Huntings disease.
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HIV Therapeutic Agents (e 2nd)

Acquired immune deficiency syndrome (AIDS) is caused by the human immunodeficiency virus (HIV). The target of HIV are the T helper cells (TH). 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. The TH cells become infected with the virus and are destroyed, slowly shutting down the immune system.
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Interaction of HIV with CD4

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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). Production excess CD4 protein (react with gp120 protein therefore HIV cannot infect TH cells). Both strategies do not destroy HIV but only block infection. To stop HIV infection we need to develop strategies which will destroy HIV.
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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

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

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

Recombinant DNA technology is being used to produce a new generation of vaccines.

Virulence genes are deleted and organism is still able to stimulate an immune response. Live nonpathogenic strains can carry antigenic determinants from pathogenic strains. If the agent cannot be maintained in culture, genes of proteins for antigenic determinants can be cloned and expressed in an alternative host e.g. E. coli.
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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. Binding of antibodies to these proteins will stimulate an immune response. Therefore proteins can be use to stimulate an immune response.
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Principles of Vaccination
It has been showed that the capsid or envelope proteins are enough to illicit an immune response. E.g:

Herpes simplex virus envelop glycoprotein O. Foot and mouth disease virus capsid protein (VP1) Extracellular proteins produced by Mycobacterium tuberculosis.
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A Subunit Vaccine for M. tuberculosis (e 2nd)

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

Six extracellular proteins of M. tuberculosis were purified. Separately and in combinations these proteins were used to immunized guinea pigs. These animals were then challenged with M. tuberculosis. After 9-10 weeks examination showed that some combinations of the purified proteins provided the same level of protection as the BCG vaccine.
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Attenuated Vaccines
Attenuated vaccines often consists of a pathogenic strains in which the virulent genes are deleted or modified. The Development of a Live Cholera Vaccine. Live vaccines are more effective than a killed or subunit (protein) vaccines. With this in mind a live vaccine for cholera was developed. Cholera is characterized by fever, dehydration abdominal pain and diarrhea.
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A Live Cholera Vaccine

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

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

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A Live Cholera Vaccine

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

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Production of a Live Cholera Vaccine

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Vector Vaccine
A vector vaccine is a vaccine which is introduced by a vector e.g. vaccinia virus. The vaccinia virus as a live vaccine led to the globally eradication of the smallpox virus. The genome of the vaccinia virus has been completely sequenced. The virus replicates in the cytoplasm rather than in the nucleus. The vaccinia virus is generally nonpathogenic.
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Vector Vaccine
These characteristics makes the vaccinia virus a good candidate for a virus vector to carry gene for antigenic determinants form other pathogens. 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.g. thymidine kinase.
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Vector Vaccine
The plasmid is used to transform thymdine kinase negative cells which were previously infected with the vaccinia virus. Recombination between the plasmid and vaccinia virus chromosomal DNA results in transfer of antigen gene from the recombinant plasmid to the vaccinia virus. Thus virus can now be used as a vaccine for the specific antigen.
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Insertion of antigen gene into vaccinia virus genome

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Vector Vaccine
A number of antigen genes have been inserted into the vaccinia virus genome e.g.
v v v

Rabies virus G protein Hepatitis B surface antigen Influenza virus NP and HA proteins.

A recombinant vaccinia virus vaccine for rabies is able to elicit neutralizing antibodies in foxes which is a major carrier of the disease.
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The Production of Commercial Products by Recombinants Microorganisms

Molecular biotechnology can be used to enhance the production of many commercially important compounds e.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
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Restriction Endonucleases (REs)

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

Using E. coli has certain advantages: E. coli cells will grow rapidly to high density. E. coli has been genetically characterized extensively. In E. coli up to 40% of the protein produced can be from the cloned gene. The target RE can be over expressed for increased production.

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

Protection of DNA from REs

The microorganism that produces the RE protects its DNA from degradation by methylation of the recognition sequence. 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.
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Periplasmic Space

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Cloning of Pst1 into E. coli

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

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Cloning of Pst1 into E. coli

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

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Synthesis of L-Ascorbic Acid

With recombinant DNA technology it is possible to introduce new genes which will modify metabolic pathways. 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
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Commercial Synthesis of Ascorbic Acid

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Synthesis of L-Ascorbic Acid

Glucose 2,5-diketo-D-gluconic acid (2,5-DKG). Many organisms can synthesize 2,5-DKG e.g.:

Acetobacter Glucanobacter Erwinia

The enzyme 2,5-DKG reductase will convert 2,5-DKG 2-KLG (2-keto-L-gluconic acid). Organisms which can carry out this reaction include:

Corynebacteria Brevibacterium Arthrobacter

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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. The best way to convert glucose to 2-KLG would be to engineer an organism which could carry out both reactions.
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Production of 2-KLG

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Microbial Synthesis of L-Ascorbic Acid

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

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

Cloning of 2,5-DKG Reductase

The 2,5-DKG reductase was subcloned into E. hericola. The recombinant 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. Gly at 55 and 57 Ala increased the thermal stability of the enzyme.
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Indigo Dye
Indigo is an important blue pigment used to dye both cotton and wool. It is isolated from plants or made chemically. Approximately 13 million kg is produced each year worth $200 million USD. It is the colouring agent in blue jeans and is the largest selling dye in the world.
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Microbial Synthesis of Indigo

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

One transformant with a 10.5 kb insert when grown on minimal media with tryptophan turned blue. Analysis of the blue colour revealed that the E. coli cells were synthesizing the dye indigo. Therefore the combination of enzymes from 2 different organisms resulted in the synthesis of a commercially important product. The microbial production of indigo avoids the use of hazardous chemicals such as aniline, formaldehyde and cyanide which are used in the chemical synthesis of indigo.
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Biosynthesis of Tryptophan
E.coli
NAH7

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Production of Xanthan Gum

Xanthomonas campestris pv. campestris is an agricultural and industrial important bacterium. In agriculture it is a pathogen that causes black rot on a number of crops. In industrial it produces xanthan gum which is used in the food, cosmetics and oil industries.
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Xanthan Gum
Xanthan gum is a high molecular weight exopolysaccharides with:

Cellulose backbone Trisaccharides side chain

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Xanthan Gum
Due to its:

High viscosity Stable properties in extreme conditions Pseudoplastic behaviour

It has applications as: Stabilizing Viscosifying Emulsifying Thickening Suspending agent

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

Whey is a nutrient rich by product of cheese making. It contains 95% water, 4-5% lactose, and 0.81.0% glucose. It is produced by the diary industry in large quantities and its disposal is a major problem for the cheese industry. 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. xanthan gum.
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Production of Xanthan Gum

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

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