Molecular Biology - Vectors PDF

GENE CLONING  Gene cloning in recombinant DNA (rDNA) technology is the process of creating multiple identical co of a specific gene of interest by inserting it into a suitable vector and propagating it within a host organism (e.g., bacteria, yeast). Dr Ala Abuhammad, PhD...

GENE CLONING  Gene cloning in recombinant DNA (rDNA) technology is the process of creating multiple identical co of a specific gene of interest by inserting it into a suitable vector and propagating it within a host organism (e.g., bacteria, yeast). Dr Ala Abuhammad, PhD Nov- 4  GENETIC CLONING  Steps in Gene Cloning in rDNA Technology: 1. Isolation of Gene of Interest: The gene to be cloned is extracted from the source DNA (e.g., human, plant, or microbial DNA) using restr enzymes or PCR. 2. Insertion into aVector : The isolated gene is inserted into a vector (e.g., plasmid or phage DNA) that can carry the gene into the h - Ligation is performed using DNA ligase to attach the gene into the vector at a cloning site. 3. Introduction/ insertion into Host Cell ( ): Transformation The recombinant vector is introduced into a host cell (usually E. coli for its fast replication and ease of use 4. Selection of Clones: Only the host cells that successfully incorporated the recombinant DNA are selected, typically using select markers like antibiotic resistance genes. 5. Replication and Expression: Inside the host, the recombinant DNA replicates, producing many copies of the gene. Under specific condi the cloned gene can also be expressed to produce the corresponding protein. Dr Ala Abuhammad, PhD Nov- 5  VECTOR TYPES FOR CLONING  Vector -it carries recombinant DNA into cells.  Small DNA molecule capable of self replication that are used as cloning vehicle or carrier o fragment.  Selection of the vector type depends on host used in the cloning and size of insert  Plasmids Virosome Bacteriophage  Phages (e.g. Bacteriophages)  Hybrid vectors (e.g. virosome)  Artificial chromosomes e.g.( YAC, BAC, HAC ) Dr Ala Abuhammad, PhD Nov- 6 VECTOR TYPES FOR CLONING Pharmaceu Vector Descript Insert Size tical Example/ Type ion Capacity Circular, double- Application Production of Plasm stranded DNA found in bacteria; ~1-10 growth insulin or ids used for small insert kb hormones in E. recombinant cloning. Bacteriophages coli. Generation of DNA Phag (viruses infecting bacteria) ~10-20 for libraries identifying es vectors used as for kb target therapeutic cloning. features of Combine s. Gene therapy: and phages (e.g., plasmids Hybrid cosmid, virosome), to enhance ~35-45 delivering genes to correct Vectors kb disorde genetic functionali their rs. ty. Synthetic chromosomes Humanized Up to 300 kb (BAC), Artificial BACs, (e.g., YACs) used for 1 (YA Mb antibodies using monoclonal Chromosomes scale large- transgenic anima C) Dr Ala Abuhammad, PhD cloning. ls. Nov- 7 VECTOR CHARACTERISTICS  Self-Replication  Vectors must have an origin of replication (Ori), enabling them to replicate independently within the host cell.  Allows production of multiple copies of the gene of interest.  Cloning Site  Vectors contain multiple cloning sites (MCS) or restriction sites, which are unique sequences recognized by restrict enzymes.  These sites allow precise insertion of the gene of interest without disrupting essential vector functions.  Selectable Marker Gene  Vectors carry genes like antibiotic resistance (e.g., ampicillin or kanamycin resistance) to help identify and select h that successfully took up the vector.  Selectable markers are critical for screening recombinant cells.  Small Size  Smaller vectors are easier to manipulate and transfer into host cells.  Reduces metabolic burden on the host, enhancing replication efficiency. Dr Ala Abuhammad, PhD Nov- 8 VECTOR CHARACTERISTICS  Stability  The vector should be stable within the host and not undergo structural alterations or degradation.  Prevents loss of the gene of interest during replication.  Replication in Host Cell  The vector's origin of replication determines compatibility with specific hosts (e.g., plasmids for bacteria, ye artificial chromosomes for yeast).  Ensures high copy numbers or controlled replication as needed.  Control Elements for Gene Expression  Includes promoters, operators, ribosome binding sites, and terminators for regulated expression of the gene interest.  Allows flexibility for induced or constitutive gene expression.  Easily Isolated and Purified  Vectors should be simple to extract and purify from host cells using standard molecular biology techniques. Dr Ala Abuhammad, PhD Nov- 9  PROMOTERS AND CONTROL ELEMENTS  Promoters regulate gene transcription in host cells.  Types of Promoters:  Strong bacterial promoters (e.g., T7) for recombinant protein overexpression.  Recognized by the T7 RNA polymerase (not the host’s RNA polymerase).  Extremely strong, leading to high levels of transcription.  Typically used for overexpression of recombinant proteins in bacterial systems (e.g., ). E. coli  Produces large amounts of therapeutic proteins, such as insulin or monoclonal antibodies.  Used for industrial production of enzymes, vaccines, and research proteins.  Mammalian promoters (e.g., CMV) for eukaryotic applications. Dr Ala Abuhammad, PhD Nov- 10  PROMOTERS AND CONTROL ELEMENTS  Other Control Elements:  Operators are incorporated into expression vectors to regulate the transcription of the gene of intere  They allow conditional expression, meaning the gene is expressed only under specific conditions (e.g., presenc of an inducer or repressor).  Repressors: Proteins that bind to the operator and prevent transcription.  Activators: Proteins that enhance transcription by binding to the operator or adjacent sequences, increasing RN polymerase activity.  By using a controlled operator system, therapeutic proteins can be produced in bacteria or mammalian cells on needed, preventing wastage of cellular resources or toxic protein accumulation.  Ribosome binding sites (RBS): Ensure efficient translation.  Adjusting Sequence: Altering the RBS sequence can fine-tune translation efficiency, controlling the rate of prot  An optimized RBS can increase protein production by improving ribosome recruitment and alignment with the s Dr Ala Abuhammad, PhD Nov- 11 HOST SPECIFICITY AND COMPATIBILITY  Vectors must be compatible with the host for successful gene expression.  Common hosts and suitable vectors:  Bacteria: Plasmids, phages (e.g., E. coli).  Yeast: YACs for larger inserts.  Mammalian Cells: Viral vectors for therapeutic applications.  Transformation Methods:  Heat shock and electroporation for plasmids.  Viral delivery for mammalian cells. Dr Ala Abuhammad, PhD Nov- 12 SELECTABLE MARKER GENES  Selectable markers help differentiate transformed from non- transformed cells.  Common Types:  Antibiotic Resistance: E.g., ampicillin resistance gene.  Reporter Genes: E.g., GFP (green fluorescent protein) for visual confirmation.  Selection Methods:  Antibiotic plates for bacterial colonies.  Fluorescence or colorimetric assays. Dr Ala Abuhammad, PhD Nov- 13 COLONY SCREENING  X-gal is a substrate of beta- galactosidase and turns blue in the presence of functional beta- galactosidase is added to the medium. Dr Ala Abuhammad, PhD Nov- 16 PRACTICAL CONSIDERATIONS IN VECTOR SELECTION  Insert Size: Plasmids (small inserts), BACs/YACs (large inserts).  Host Compatibility: Optimize for bacterial, yeast, or mammalian systems.  Scalability: Plasmids for high-yield production; viral vectors for therapeutic doses.  Cost: Balancing vector production costs with efficiency. Dr Ala Abuhammad, PhD Nov- 19 PHARMACEUTICAL APPLICATIONS OF VECTORS  Recombinant Protein Production:  Human insulin using plasmids in E. coli.  Monoclonal antibody production using mammalian expression vectors.  Gene Therapy:  Viral vectors delivering therapeutic genes (e.g., for cystic fibrosis).  Vaccine Development:  Plasmids encoding antigens (e.g., mRNA COVID-19 vaccines).  The starting material for mRNA vaccines is often a DNA plasmid encoding the gene for the antigen, such as SARS-CoV-2 spike protein.  This plasmid is used in a lab or production facility to synthesize mRNA via in vitro transcription. Dr Ala Abuhammad, PhD Nov- 20 THE END … Any questions?  Dr Ala Abuhammad, PhD Nov- 21