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Introduction to Biotechnology 1 Introduction to Biotechnology The term biotechnologist will be used to describe a person who deals with fermentation and microbial systems. This term was coined because a biotechnologist is a professional who integrates and applies a number of disciplines such as microbiology, genetics, biochemistry, chemistry, pharmacy, food technology, mathematics and engineering in his or her work. When designing or working with fermentations, the biotechnologist’s understanding of the above disciplines needs to be sufficient enough to allow the manipulation of process conditions in a rational manner. For instance, when biotechnologists are modelling fermentation systems, they integrate their knowledge of biological concepts (microbiology, biochemistry and genetics) with their mathematical and engineering skills. The idea of integrating biodisciplines and engineering is summarised by Figure 1. Chemistry Biochemistry Biotechnology Mathematics Engineering Microbiology Genetics Pharmacy Food Technology Figure 1: The discipline of biotechnology, an integration of other disciplines. This study is an ideal example that shows the integration between disciplines and in particular, it looks at the production of a recombinant protein hormone via fermentation while relying on the integration of the engineering, microbiology and biochemistry disciplines. Details of the fermentation are explained in the following sections. 1.1 The Biotechnological Process A typical biotechnological process is similar to a chemical process in many ways. For instance, both processes generally have a reaction step, followed by a separation step. Yet the biotechnological process exhibits clear distinctions in that it relies on live cells or organisms to carry out the reaction. These may be thought of as “bio-catalysts”. The steps required in a typical biotechnological process are shown in Figure 2.

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Page 1: 3804 Intro Summary

Introduction to Biotechnology

1 Introduction to Biotechnology

The term biotechnologist will be used to describe a person who deals with fermentation and microbial systems. This term was coined because a biotechnologist is a professional who integrates and applies a number of disciplines such as microbiology, genetics, biochemistry, chemistry, pharmacy, food technology, mathematics and engineering in his or her work. When designing or working with fermentations, the biotechnologist’s understanding of the above disciplines needs to be sufficient enough to allow the manipulation of process conditions in a rational manner. For instance, when biotechnologists are modelling fermentation systems, they integrate their knowledge of biological concepts (microbiology, biochemistry and genetics) with their mathematical and engineering skills. The idea of integrating biodisciplines and engineering is summarised by Figure 1.

Chemistry Biochemistry

Biotechnology

MathematicsEngineering

Microbiology

Genetics

Pharmacy

Food Technology

Figure 1: The discipline of biotechnology, an integration of other disciplines.

This study is an ideal example that shows the integration between disciplines and in particular, it looks at the production of a recombinant protein hormone via fermentation while relying on the integration of the engineering, microbiology and biochemistry disciplines. Details of the fermentation are explained in the following sections.

1.1 The Biotechnological Process A typical biotechnological process is similar to a chemical process in many ways. For instance, both processes generally have a reaction step, followed by a separation step. Yet the biotechnological process exhibits clear distinctions in that it relies on live cells or organisms to carry out the reaction. These may be thought of as “bio-catalysts”. The steps required in a typical biotechnological process are shown in Figure 2.

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ProductformulationRaw materials Bio-reactor

Process Control

Bio-catalystSelection andManipulation

ProductSeparation and

Purification

Figure 2: Schematic showing the typical steps in a biotechnological process.

The bio-reactor is at the heart of the process and its performance relies on the material input, the bio-catalyst performance and the control strategy implemented. A typical biotechnological process runs at low temperatures and consumes little energy. It also often utilises inexpensive raw materials for feed26. However antibiotics and inductants can have considerable costs. 1.1.1 Bio-catalyst Selection and Manipulation

The initial step in any biotechnological process is the selection of a suitable bio-catalyst. In the selection process, the biotechnologist aims to find a microorganism that exhibits the following characteristics26:

• It should be a pure culture,

• Be genetically stable,

• Be easily propagated,

• Exhibit rapid growth characteristics,

• Have good rate of product formation,

• Be free of toxic byproducts and

• Be amenable to genetic manipulation. Natural selection and mutation selection, are two ways of obtaining the desired bio-catalyst. Recent developments in the field of “recombinant DNA technology” are becoming more commonplace and are widely used in the selection and preparation of suitable bio-catalysts. 1.1.2 Natural Selection

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Most microorganisms exhibit a great capacity for natural variation. Microorganism cultures isolated from nature generally lead, once grown, to variant cultures, some of which may be superior in their capacity to synthesize a product6. This capacity is exploited in biotechnology where this improved variant is preserved and utilised. 1.1.3 Mutation Selection Once a bio-catalyst is found, it may be improved by genetic modification. The most effective method previously used for improving the yield of product is by way of induced mutation followed by selection of an improved variant7. Genetic and microbiology texts21,22 offer detailed discussions of mutation and procedures used for selection. Although mutation selection is very effective, it has been superseded by the new techniques employed in genetic engineering because it is not as directed and relies heavily on chance.

1.2 Recombinant DNA Technology

Recent developments in the genetic engineering techniques have presented tools that enable the handling and manipulation of DNA, allowing more flexibility in the bio-catalyst selection process. DNA is a molecule found in living cells and is responsible for several complex functions. In humans for instance, DNA determines the physical features such as height and eye colour. Bacterial DNA, as a second example, has specialised functions that allow the bacteria to metabolise nutrients and regulate this metabolism. DNA is also involved in development and reproduction of an organism. DNA is a polymeric molecule made of four different monomeric units called nucleotides.

Each nucleotide consists of a 5-carbon sugar to which is attached a phosphate group and

one of four nitrogenous (nitrogen containing) bases - adenine, guanine, cytosine and

thymine. The structure of DNA is shown in Figure 3.

A series of nucleotides is called a gene. Different genes are responsible for different functions. A complete collection of genes is called a genome and this would fully represent an organism’s complete DNA sequence. Genes are organised inside the cells in coiled structures known as chromosomes. Alteration, addition or deletion of genes may from natural or human effect, reconstruct the chromosomes. A more important reconstruction method is by way of recombination.

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Figure 3: A space filling model showing the structure of the DNA double helix [21].

Recombination of DNA allows the variation and diversity of living organisms. The joining of the male’s sperm cell with the female’s egg cell is a natural recombination process. The DNA of the male joins and mixes with the female’s DNA resulting in an offspring cell with a combination of both parents. This is evident in the offspring’s physical appearance, which exhibits features that are similar to both parents. Natural recombination is now mimicked by biotechnologists allowing the reconstruction of DNA molecules in test tubes. In this process, the construction of a new DNA molecule takes place from two different sources. The techniques and procedures associated with this are known as recombinant DNA technology and are the specialty of the geneticist. Today, this technology is commonly known as “gene cloning”. 1.2.1 Gene Cloning

Genes are cloned by isolating a piece of DNA from an organism and inserting it into a cloning vector to make a recombinant DNA molecule. This molecule is then introduced into the target organism carrying with it the cloned gene(s) which allow(s) the organism to carry out new functions or improve existing ones. An organism carrying foreign genes is known as recombinant organism. The steps required for cloning a gene are illustrated in Figure 4. Here the gene of interest is integrated into a vector DNA molecule using cut-and-paste techniques facilitated by special enzymes. The combination of the vector and the gene is called a recombinant molecule. This molecule is introduced into the desired host organism by transformation, where the effect of the gene is amplified and the host acquires the new characteristics brought about by the new gene.

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Figure 4: Recombinant plasmid construction and cloning [21].

1.2.2 DNA Isolation

Before cloning a particular gene, the source DNA must be isolated and the gene identified. Isolation usually is a process consisting of three steps9: 1) cell disruption, 2) removal of cell debris, proteins and enzymes and 3) purification and concentration of the DNA. Once the DNA is isolated, the gene to be cloned is cut out of the DNA by the use of restriction endonuclease enzymes. These are special enzymes that cleave DNA at certain base sequences called restriction sites. They merely act like special scissors that cut out the desired gene. Since their discovery in the 1960’s, hundreds have been identified. The next step is to paste the excised gene into a cloning vector before transferring to host cell.

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1.2.3 Cloning Vectors and Gene Cloning

Typical cloning vectors include plasmids, transposons, cosmids, phages, shuttle vectors and artificial chromosomes. Because they are most commonly used and because this present work uses one, features of plasmid vectors are discussed here. A plasmid is an extrachromosomal genetic element that replicates autonomously inside the host cell. It is circular and double-stranded in structure. To be useful, a plasmid ought to have the following qualities9: 1) single restriction site for a number of given enzymes, 2) High copy number of plasmids in a cell (presence of more than one plasmid in

the cell at any one time; approaching 100 copies), and 3) Selectable marker (this tells us that the cell has the plasmid, E.g. antibiotic

resistance marker).

The desired gene is joined to the appropriate vector forming a recombinant molecule, which is then introduced in a process called transformation into the host organism. Transformation is the process of introducing new DNA into a host cell. The recombinant DNA molecule is mixed with the appropriate host cell. The host cell is made permeable by addition of calcium chloride and incubated for a short time allowing the new DNA to be taken up into the cell. Once the DNA is inside, the cell begins to exhibit the features associated with the new DNA. The introduction of a DNA plasmid into a bacterial host is illustrated in Figure 5.

Figure 5: The transformation process. The recombinant plasmid molecule is introduced into the host cell [21].

1.2.4 Gene Expression

The rate of plasmid-encoded gene expression is important because of the direct influence on gene-product levels. Inserting a gene into a cloning vector does not ensure that expression will be successful. In order for a cloned gene to be expressed, the DNA must get transcribed and translated. A promoter site on the plasmid initiates transcription, while a terminator site induces cessation of transcription. Promoter and terminator sites merely act as signals functioning to turn the gene on or off. Cloned genes usually do not carry these DNA sites

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without which they can not be transcribed. To deal with this, the gene to be cloned is inserted in the correct location between these sites. To produce large amount of gene product, it is necessary to use a promoter that supports transcription at a high level. 1.2.5 Recombinant DNA Technology in Biotechnology

The overall efficiency of the biotechnology process is improved with the aid of the new recombinant techniques. The benefits of recombinant DNA technology to the biotechnology process lie in the bio-catalyst selection stage and include26: 1. Custom design of products with desirable characteristics, 2. Increase of a yield of a particular product, 3. Improve the efficiency of the production with respect to energy utilisation, 4. Transfer of a particular activity to a more desirable host, and 5. Minimise the purification steps required. Today it is possible to introduce mammalian genes into bacteria for the large-scale production of products encoded by those genes26. The impact of this will be felt by the pharmaceutical industry in particular with the production of insulin, growth hormone and vaccines. Clearly bio-catalyst selection is an area receiving a great deal of attention with optimism in the development of newer high-producing stable bio-catalysts.

1.3 Recombinant Protein Production

The emergence of recombinant DNA technology enabled the production of many important proteins that were previously extremely difficult to produce in large commercial scale quantities8. An example of a recombinant protein is porcine growth hormone (pGH), the production of which is studied in this thesis. Porcine growth hormone is a naturally occurring protein produced in pigs by their pituitary glands and is responsible for normal growth and muscle development. Advances in recombinant DNA technology now permit the large-scale production of synthetic analogues of pGH. pGH plays an important role in regulating skeletal growth, protein and fat metabolism of pigs. It is used in the pig farming industry to improve the efficiency of converting feed into muscle tissue. 1.3.1 Cloning the Porcine Growth Hormone

The DNA coding for pGH was cloned into the vector plasmid pET3a (Figure 6). The recombinant plasmid pET3a-pGH was transformed into E.coli strain BL21 DE3 for expression. pGH is produced intracellularly in the form of insoluble inclusion bodies. The formation of inclusion bodies inside E.coli cells is promoted by hydrophobic, ionic, and in some cases covalent interactions (i.e. disulfide bonds) between protein molecules8.

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Figure 7 shows electron micrographs of E.coli cells having inclusion bodies. The cells with inclusion bodies show bulging around where the inclusion bodies are found, while cells without inclusion bodies show a different morphology, with pointy ends in some cases. The genetic engineering designed the production of pGH by the bacteria to be induced by the addition of IPTG (inducer) and repressed by glucose.

Figure 6: Cloning of the pGH gene into the pET3a plasmid system. The restriction enzymes BamH I and Nde I were used to cut the pGH and the plasmid DNA.

Figure 7: Intracellular inclusion bodies in sectioned transmission electron micrographs of recombinant E.coli. Arrows point to inclusion bodies [3,8].

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1.3.2 Production of pGH

Production of pGH can takes place by fermentation of the E.coli culture growing on glucose as a substrate. The fermentations can be carried out in Chemap fermenter (Figure 8) This fermenter system is capable of automatic sterilisation and allows the on-line monitoring of temperature, pH and DO. Previous work has demonstrated success in the production of pGH. Kollaras15 performed nine fermentations and found optimum temperature and pH to be 34-36°C and 6.9 respectively. Downstream processing of the fermentation broth takes place in the bio-separation plant. This consists of a series of unit operations aimed at the separation and purification of the product. Figure 9 shows a view of the bio-separation plant. For optimisation purposes, the fermenter and bio-separation plant should be considered as one process as they interact heavily with each other to produce the final product.

Figure 8: The 14-litre Chemap Fermenter.

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Figure 9: The bio-separations plant for product separation and purification.

2 References

1. Aristidou AA., San KY., and Bennet GN., 1995, “Metabolic Engineering of Escherichia coli To

Enhance Recombinant Protein Production through Acetate reduction”, Journal of

Biotechnology Progress, Vol. 11, pp 475-478.

2. Bahri P,. and romagnoli J., 1997, “Advanced Process control”, University of Sydney, Australia.

3. Bailey JE., and Ollis DF., 1986, Design and Analysis of Biological Reactors, in:”Biochemical

Engineering Fundamentals”, McGraw-Hill Book Co., Singapore, pp 622-630.

4. Bastin G., and Dochain D., 1990, “On-Line Estimation and Adaptive Control of Bioreactors”,

Elsevier Science Publishing Company Inc., NY, USA.

5. Bech Jensen E., and Carlsen S., 1989, “Production of Recombinant Human Growth Hormone in

E.coli: Expression of Different Precursors and Physiological effects of Glucose, Acetate, and

Salts”, Journal of Biotechnology and bioengineering, Vol. 36, pp. 1-11.

6. Belter PA., 1979, General Procedures for Isolation of Fermentation Products, in: “Microbial

Technology – Fermentation Technology”, edited by Peppler HJ. and Perlman D., Academic

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7. Benson HJ., 1984, “Microbiological applications – a laboratory manual in general

microbiology”, C. Brown Company Publishers, USA.

8. Blanch HW., and Clark DS., 1996, “Biochemical Engineering”, Marcel Dekker, Inc., NY, USA.

9. Carter D., 1998, Lecture Series: Microbial Genetics, Regulation and Manipulation of the

Bacterial Genome in: “Molecular Microbiology of the Organism” course. Biochemistry

Department, University of Sydney.

10. Chen HC., Hwang CF., and Mou DG., 1992, “High-density Escherichia coli cultivation process

for hyperexpression of recombinant porcine growth hormone”, Journal of Enzyme

Microbiology and technology, Vol. 14, pp 321-326.

11. Cockshott AR., and Bogle IDL., 1992, “Modelling a Recombinant E.coli Fermentation

Producing Bovine Somatotropin”, IFAC Modeling and Control of Biotechnical Processes,

Colorado, USA, pp219-222.

12. Gaete-Muller D., 1997, “The fermentation of E.coli to Produce the Porcine Growth Hormone”,

University of Sydney, Australia.

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22. Russel PJ., 1996, Gene mutation, in: “Genetics”, Harper Collins College Publishers, New York,

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