Biotechnology- Principles and Processes - Choices of Host and Vectors

Slide 1

Introduction to choices of host and vectors in biotechnology
Importance of host and vectors in recombinant DNA technology
Overview of the topics to be covered in this lecture
Examples of host organisms and vectors used in biotechnology

Slide 2

Host Organisms

Definition: organisms used to produce large quantities of recombinant DNA or desired proteins
Criteria for choosing a host organism:
- Compatible with the expression vector
- Capable of proper folding and post-translational modifications
- Easy to culture and manipulate
Examples of commonly used host organisms:
- Bacteria (Escherichia coli)
- Yeast (Saccharomyces cerevisiae)
- Mammalian cells (Chinese hamster ovary cells)

Slide 3

Bacterial Hosts

Advantages of using bacteria as host organisms:
- Rapid growth rate
- Well-characterized genetics
- Easy to manipulate and culture
Example: Escherichia coli (E. coli)
- Widely used bacterial host
- Strong promoter sequences available
- Efficient protein expression
- Simple transformation protocols

Slide 4

Yeast Hosts

Advantages of using yeast as host organisms:
- Eukaryotic expression system
- Capable of performing post-translational modifications
- Suitable for protein secretion
Example: Saccharomyces cerevisiae (S. cerevisiae)
- Commonly used yeast host
- Efficient protein secretion
- Easy genetic manipulation
- Well-characterized genetics

Slide 5

Mammalian Cell Hosts

Advantages of using mammalian cells as host organisms:
- Ability to carry out complex post-translational modifications
- Close resemblance to human cells
- Suitable for expression of complex proteins
Example: Chinese hamster ovary cells (CHO cells)
- Widely used mammalian cell host
- Capable of proper protein folding and assembly
- Efficient protein secretion
- Post-translational modification machinery similar to human cells

Slide 6

Vectors

Definition: DNA molecules used as carriers to transfer foreign DNA into host organisms
Criteria for choosing a vector:
- Capable of self-replication in the host organism
- Contains selectable markers for host cell identification
- Accommodates large insert DNA
Examples of commonly used vectors:
- Plasmids
- Bacterial artificial chromosomes (BACs)
- Viral vectors (retroviruses, adenoviruses)

Slide 7

Plasmids

Definition: small circular DNA molecules separate from the chromosomal DNA in bacteria
Advantages of using plasmids as vectors:
- Easy to manipulate and propagate
- Efficient transformation into host cells
- Can accommodate small to moderate-sized DNA inserts
Example: pUC19
- Widely used plasmid vector
- Contains selectable markers (ampicillin resistance)
- Multiple cloning sites for DNA insertion

Slide 8

Bacterial Artificial Chromosomes (BACs)

Definition: large DNA molecules capable of carrying large DNA inserts (100-300 kb)
Advantages of using BACs as vectors:
- Able to accommodate large DNA inserts
- Stable replication in bacterial cells
- Compatible with high-throughput automation
Example: pBeloBAC11
- Commonly used BAC vector
- Contains selectable markers (chloramphenicol resistance)
- Large DNA insert capacity

Slide 9

Viral Vectors

Definition: viruses used as vectors to transfer foreign DNA into cells
Advantages of using viral vectors:
- Efficient gene delivery into host cells
- Can target specific cell types
- Suitable for in vivo gene therapy applications
Examples of viral vectors:
- Retroviruses
- Adenoviruses
- Lentiviruses

Slide 10

Retroviral Vectors

Derived from retroviruses (e.g., HIV)
Advantages of using retroviral vectors:
- Can integrate the foreign DNA into the host genome
- Suitable for long-term gene expression
Example: pLenti6/V5-DEST
- Commonly used retroviral vector
- Efficient gene transfer into a wide range of cell types
- Compatible with large DNA inserts

======

Slide 11

Factors Affecting Choice of Host and Vectors

Purpose of the experiment (recombinant protein production, gene therapy, etc.)
Compatibility between host organism and expression vector
Ease of genetic manipulation and culture conditions
Protein folding and post-translational modification capabilities
Type of application (in vitro or in vivo)
Safety considerations (use of viral vectors)
Cost and scalability

Slide 12

Basics of Recombinant DNA Technology

Definition: Technology that involves the manipulation of DNA sequences from different sources to create new combinations
Steps involved in recombinant DNA technology:
1. Isolation of DNA from a donor organism
2. Cloning of the DNA fragment into a vector
3. Introduction of the recombinant DNA into a host organism
4. Selection and amplification of the transformed host cells
5. Expression of the desired gene or protein in the host organism

Slide 13

Expression Systems in Biotechnology

Definition: Systems used to express and produce desired proteins from recombinant DNA
Prokaryotic expression systems:
- Utilize bacterial hosts (e.g., E. coli)
- High growth rate and cost-effective
- Suitable for small proteins and enzymes
Eukaryotic expression systems:
- Utilize yeast or mammalian cells
- Capable of performing post-translational modifications
- Suitable for complex proteins and therapeutic antibodies

Slide 14

Methods of Vector Introduction into Host Organisms

Transformation:
- Direct introduction of DNA into host cells (e.g., electroporation in bacteria)
- Commonly used in bacterial hosts
Transfection:
- Introduction of DNA into eukaryotic cells using lipid-based or electroporation methods
- Used for both transient and stable gene expression
Infection:
- Introduction of DNA into host cells using viral vectors (e.g., lentiviral transduction)
- Efficient gene delivery into a wide range of cell types

Slide 15

Selectable Markers

Definition: Genes incorporated into vectors to identify and select transformed host cells
Commonly used selectable markers:
- Antibiotic resistance genes (e.g., ampicillin, kanamycin)
- Fluorescent proteins (e.g., green fluorescent protein - GFP)
- Enzyme activity markers (e.g., β-galactosidase, luciferase)
Selectable markers help distinguish transformed cells from non-transformed cells in the selection process

Slide 16

Examples of Applications of Biotechnology

Production of recombinant proteins
Development of genetically modified crops
Gene therapy for treating genetic disorders
Bioremediation to clean up environmental pollutants
Production of vaccines and therapeutic antibodies
Forensic DNA analysis and paternity testing

Slide 17

Challenges and Ethical Considerations

Regulatory and ethical concerns in the use of genetically modified organisms (GMOs)
Potential environmental impacts of GMOs
Safety considerations in the use of viral vectors in gene therapy
Protection of intellectual property and patent rights
Public perception and acceptance of biotechnology
Biosecurity measures to prevent misuse of biotechnology

Slide 18

Conclusion

Choice of host organisms and vectors is crucial in biotechnology applications
Different host organisms have their advantages and limitations
Vectors play a key role in the successful expression of recombinant DNA
Consideration of factors such as purpose, compatibility, and scalability is necessary
Ethical and safety considerations need to be addressed in biotechnology research and applications

Slide 19

References

Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., & Walter, P. (2002). Molecular Biology of the Cell. Garland Science.
Griffiths, A. J., Miller, J. H., Suzuki, D. T., Lewontin, R. C., & Gelbart, W. M. (2000). Introduction to Genetic Analysis. W.H. Freeman and Company.
Nelson, D. L., Cox, M. M. (2017). Lehninger Principles of Biochemistry. W. H. Freeman and Company.

## Slide 20</p>
<h3 id="questions">Questions</h3>
<ol>
<li>What are the factors that affect the choice of host organisms and vectors in biotechnology?</li>
<li>Compare and contrast the advantages of using bacteria, yeast, and mammalian cells as host organisms.</li>
<li>Explain the steps involved in recombinant DNA technology.</li>
<li>What are the different methods of introducing vectors into host organisms?</li>
<li>Why are selectable markers important in recombinant DNA technology?</li>
<li>Give examples of applications of biotechnology in various fields.</li>
<li>Discuss the challenges and ethical considerations in the use of biotechnology.</li>
<li>Summarize the key points in the lecture regarding host organisms and vectors in biotechnology.</li>
</ol>
<p>Please feel free to ask any questions related to the topic.</p>
<h2 id="slide-21">Slide 21</h2>
<h3 id="factors-affecting-choice-of-host-and-vectors-1">Factors Affecting Choice of Host and Vectors</h3>
<ul>
<li>Purpose of the experiment</li>
<li>Compatibility between host organism and expression vector</li>
<li>Ease of genetic manipulation and culture conditions</li>
<li>Protein folding and post-translational modification capabilities</li>
<li>Type of application</li>
<li>Safety considerations</li>
<li>Cost and scalability</li>
<li>Availability of suitable promoters and regulatory elements</li>
</ul>
<hr>
<h2 id="slide-22">Slide 22</h2>
<h3 id="advantages-of-bacteria-as-host-organisms">Advantages of Bacteria as Host Organisms</h3>
<ul>
<li>Rapid growth rate allows for high protein production</li>
<li>Well-characterized genetics enable easy manipulation</li>
<li>Easy to culture and scale up</li>
<li>Availability of strong promoters for efficient gene expression</li>
<li>Low cost and high yield production of recombinant proteins</li>
<li>Example: Escherichia coli (E. coli) used in insulin production</li>
</ul>
<hr>
<h2 id="slide-23">Slide 23</h2>
<h3 id="advantages-of-yeast-as-host-organisms">Advantages of Yeast as Host Organisms</h3>
<ul>
<li>Eukaryotic expression system allows for proper protein folding and post-translational modifications</li>
<li>Suitable for protein secretion and for targeting specific subcellular compartments</li>
<li>Capable of performing complex post-translational modifications such as glycosylation</li>
<li>Example: Saccharomyces cerevisiae used in production of hepatitis B surface antigen</li>
</ul>
<hr>
<h2 id="slide-24">Slide 24</h2>
<h3 id="advantages-of-mammalian-cells-as-host-organisms">Advantages of Mammalian Cells as Host Organisms</h3>
<ul>
<li>Capable of performing complex post-translational modifications similar to human cells</li>
<li>Able to produce properly folded and assembled proteins</li>
<li>Suitable for the production of complex proteins, therapeutic antibodies, and viral particles</li>
<li>Close resemblance to human cells allows for better compatibility in therapeutic applications</li>
<li>Example: Chinese hamster ovary (CHO) cells used in the production of monoclonal antibodies</li>
</ul>
<hr>
<h2 id="slide-25">Slide 25</h2>
<h3 id="types-of-vectors">Types of Vectors</h3>
<ul>
<li>Plasmids: Small circular DNA molecules capable of self-replication</li>
<li>Bacterial artificial chromosomes (BACs): Large DNA molecules capable of accommodating large DNA inserts</li>
<li>Viral vectors: Derived from viruses and used for gene delivery into host cells
<ul>
<li>Retroviral vectors: Efficient gene transfer and integration into the host genome</li>
<li>Adenoviral vectors: High gene delivery efficiency but transient expression</li>
<li>Lentiviral vectors: Efficient infection of both dividing and non-dividing cells</li>
</ul>
</li>
</ul>
<hr>
<h2 id="slide-26">Slide 26</h2>
<h3 id="advantages-of-plasmids-as-vectors">Advantages of Plasmids as Vectors</h3>
<ul>
<li>Easy to manipulate and propagate</li>
<li>Efficient transformation into host cells</li>
<li>Can accommodate small to moderate-sized DNA inserts</li>
<li>Multiple cloning sites allow the insertion of foreign DNA</li>
<li>Example: pUC19 plasmid containing ampicillin resistance gene as a selectable marker</li>
</ul>
<hr>
<h2 id="slide-27">Slide 27</h2>
<h3 id="advantages-of-bacterial-artificial-chromosomes-bacs-as-vectors">Advantages of Bacterial Artificial Chromosomes (BACs) as Vectors</h3>
<ul>
<li>Large DNA insert capacity (100-300 kb)</li>
<li>Stable replication in bacterial cells</li>
<li>Suitable for maintaining large DNA fragments</li>
<li>Compatible with high-throughput automation</li>
<li>Example: pBeloBAC11 containing the chloramphenicol resistance gene as a selectable marker</li>
</ul>
<hr>
<h2 id="slide-28">Slide 28</h2>
<h3 id="advantages-of-viral-vectors">Advantages of Viral Vectors</h3>
<ul>
<li>Efficient gene delivery into host cells</li>
<li>Can target specific cell types or tissues</li>
<li>Suitable for in vivo gene therapy applications</li>
<li>Example: Retroviral vectors derived from HIV used in treating genetic disorders</li>
</ul>
<hr>
<h2 id="slide-29">Slide 29</h2>
<h3 id="selectable-markers-1">Selectable Markers</h3>
<ul>
<li>Genes incorporated into vectors to identify and select transformed host cells</li>
<li>Antibiotic resistance genes (e.g., ampicillin, kanamycin) used in bacterial systems</li>
<li>Fluorescent proteins (e.g., GFP) used for visualization of transformed cells</li>
<li>Enzyme activity markers (e.g., β-galactosidase, luciferase) used to detect gene expression</li>
<li>Selectable markers help distinguish transformed cells from non-transformed cells during selection process</li>
</ul>
<hr>
<h2 id="slide-30">Slide 30</h2>
<h3 id="examples-of-applications-of-biotechnology-1">Examples of Applications of Biotechnology</h3>
<ul>
<li>Production of recombinant proteins, such as insulin, growth factors, and vaccines</li>
<li>Development of genetically modified crops with improved traits, such as pest resistance and increased yield</li>
<li>Gene therapy for treating genetic disorders, such as cystic fibrosis and muscular dystrophy</li>
<li>Bioremediation to clean up environmental pollutants, such as oil spills and toxic chemicals</li>
<li>Production of therapeutic antibodies for cancer treatment and autoimmune diseases</li>
<li>Forensic DNA analysis and paternity testing for identification purposes</li>
</ul>
<hr>

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