Ingeniería Genética: Vectores y Bibliotecas Genómicas

• These are study notes that cover genetic engineering, specifically focusing on vectors, cloning, and constructing genomic libraries.

Cloning Vectors and Construction of Genomic Libraries

• Genomic and cDNA Libraries: Explores methods for constructing both genomic and cDNA libraries.
• Vectors: Highlights expression vectors used in biotechnology.
• Library Screening: Discusses screening for specific sequences in genomic libraries.
• Probing: Covers the use of nucleic acids and antibodies as probes.

Recombinant DNA Experiment

• DNA from a donor organism is extracted and cut with restriction endonucleases.
• It is then ligated into a cloning vector to make recombinant DNA, also known as DNA construction.
• The cloning vector-DNA insert is transferred into a host cell via transformation.
• Cells containing the construct are identified and selected.
• The construct can be engineered to ensure protein expression by the host cell.

Cloning Vectors

• Requires a bacterial plasmid or cloning vector to introduce mainly eukaryotic DNA into a prokaryotic organism for protein expression.
• The plasmid's promoter enables expression of the inserted DNA.
• This leads to the production of the protein the gene encodes, thus becoming an expression vector.
• The process involves extracting DNA, cutting DNA and the vector with restriction enzymes for ligation, inserting into bacteria, and selecting bacteria with the plasmid.
• Various molecules such as plasmids (up to 10kb inserts), bacteriophages (20kb), cosmids (50kb), and YACs (200-800kb) can be used for cloning vectors.

Plasmids as Cloning Vectors

• Focuses on the pBR322 plasmid
• The nomenclature includes:
• "p" for plasmid
• "BR" for the initials of the creators (F. Bolivar and R. Rodriguez)
• "322" distinguishes it from others developed in the same lab.
• pBR322 plasmid has genes for ampicillin (Ampr) and tetracycline (Tetr) resistance, allowing selection of bacteria incorporating these genes.
• It features unique restriction sites for BamHI, HindIII, and Sall within the Tetr gene, Pst I in the Ampr gene, and EcoRI outside of coding regions.
• The plasmid contains:
• pMB1 replication origin, functional in E. coli
• high copy number
• is non-transferable to other bacteria.
• Synthetic plasmids are made from DNA, assembled to order.
• These include a polylinker (multiple cloning site) for easy insertion of any insert near the replication origin (OriC).

Ways to Select Bacteria that Include the Plasmid

• Selectable markers such as genes that break down a substrate into a colored product can be used to identify bacteria with the plasmid.
• For example, the LacZ gene encodes β-galactosidase, which breaks down X-gal to produce a blue-colored product.
• Bacteria with β-galactosidase will turn blue in the presence of X-gal.
• B-galactosidase is activated by IPTG in the culture medium with the plasmid containing the LacZ gene for identification.

Selecting Through Hybridization

• Bacteria are grown on a plate then transferred to a nitrocellulose filter to generate a copy for analysis.
• The DNA is exposed by lysing the cells and then denatured, allowing hybridization with a fluorescent or radioactive probe.
• Only bacteria containing DNA complementary to the probe will be marked.

Selection by Immunodetection

• Utilizes an antibody specific to the protein of interest.
• It is associated with a secondary antibody that produces a colorimetric reaction for protein marking.
• A replica of the bacterial plate is made on a nitrocellulose filter.
• Adding a primary antibody and then a secondary antibody linked to an enzyme, generates a colored product to indicate the protein of interest's presence.
• For example, detecting insulin involves using an anti-insulin antibody (primary) and then a secondary antibody linked to a fluorescent or radioactive particle for visualization.

Negative Selection of Clones in pBR322

• pBR322 confers resistance to ampicillin and tetracycline
• The bacteria that incorporate the plasmid will be resistant to both antibiotics.
• To select the bacteria, a media containing both antibiotics is used to kill tetracycline-sensitive bacteria.
• A replica without tetracycline is required in order to do this procedure.
• Replicas are made, and to avoid contaminations antibiotics are added
• amplicillin is added in this instance, not tetracycline

Selection by Nutritional Characteristics (Auxotrophic Markers):

• Selection is based on bacterial nutritional requirements with auxotrophic markers.
• Original bacterial plates transferred to a minimal medium lacking a nutrient
• e.g., glucose alongside a complete medium
• Depending on the introduced plasmid, bacteria either survive or die
• If they require glucose, they die
• The colonies bacteria that survive in medium gets picked for growing up.

Phages as Cloning Vectors

• The process starts with a phage infecting a bacterium, introducing its DNA, which then follows two growth cycles:
• Lysitic
• Lysogenic
• In the lytic cycle, the phage DNA does not integrate into the bacterial DNA.
• The viral DNA replicates and creates new phages, causing the bacterial cell to lyse and release the phages.
• In the lysogenic cycle, the phage DNA integrates into the bacterial genome.
• The bacterium contains viral DNA, dividing as if it were its own.
• In Bacteria the transformation is called infecting.

Amplification of the Clone via the Lytic Cycle

• Selection depends on the introduced material
• Usually more complex than in previous cases.
• Bacteria is cultivated in a petri dish and infeted with phages
• Phages have previously had DNA put into them.
• The end result is the formation of colonies according to the different phages that we had.

Phage Lambda (λ)

• Has a genome of about 45,000bp.
• It contains a non-essential, 15kbp central fragment for replication or infection.
• It can be eliminated by restriction enzymes and replaced with an insert
• About 15kbp after.
• "In vitro" packaging of DNA follows:
• Capsid formation
• Viral particle formation
• Transfection or infection of cells
• Propagation through the phages
• Human DNA may be introduced into the phages, and it is also possible to obtain two DNA fragments for DNA to be expressed
• If genes are marked with radiation this proves they are active.

Genomic Libraries

• Genomic libraries can be generated with the introduction of different genes into different phages.
• Another way to search for the bacteria that are needed:
• Make a replica on a nitrocellulose filter
• Lysing the bacteria
• Hybridize DNA to have a marked probe

Production and Purification of Recombinant Protein in E. coli

• Culture of bacteria with plasmids/phages containing the insert of interest
• Add IPTG to promote plasmid development.
• IPTG contains a polymerase, which expresses the insert
• Prepare cellular extracts from the bacteria producing the protein
• Introduce into nickel columns where the cellular debris goes.
• In the remaining extract is the protein that has been used in the ilusion with ididacol.
• The pure proteins, and the vectors of expressions can be identified by their vectors, and whether they have capacity to contain the protein if the expression is given.

Production of Polyclonal Antibodies

• Purified protein is injected into rabbits to develop polyclonal antibodies.
• Polyclonal antibodies:
• Divide and interact against many epitopes of a protein
• Monoclonal antibodies:
• Interact against one epitopes
• Extracted blood contains protein specific polyclonal antibodies that can be verified in affinity chromatography.

Coupled Transcription-Translation in Vitro (Cell-Free System)

• A tool for producing toxic proteins for E. coli.
• Requires DNA (circular or linear) for transcription in vitro by adding elements
• ribosomes, RNApoly, DNApoly etc.
• Translation then occurs to synthesize targeted protein from that DNA

Production of Recombinant Proteins

• To produce a recombinant protein, make to consider the aspects
• The gene must be cloned, and characterised
• It must be subcloned in a vector
• It must be transferred to cells
• Examples are somatostatin, and the insulin

Strategies for cloning and construction of genothics

• Genomics are produced by finding:
• the promoter
• the exons
• the intorns
• the restriction enzymes
• For construction of genothices
• Recombine the gene with DNA of the 2.
• It has the phago inject the culture of bacteria
• This will obtain the major DNA
• The bacteria freezes with the genothics
• Genomics, which are places where large quantities of genes are maintained.
• Genothics will be produced by synthesising cDNA which has retrotranscription.

Genomic DNA (λ y Cos) Genothics

• Are constructed from genthic DNA
• The cut out for enzymes of restriction
• As well it has a replesement vector where it will insert the ligate
• Digest
• ligatsation
• Amplication
• titration
• Bacteria which lisis of the fago litito

Consmids

• Re widely used vectors
• Used for genothics of great size.

Screening for Sequences in Genothics (Screening)

• The lasties of infection will have genes that have resistance to atipotics
• Making selection with blue, and whire
• Selection if there is a hybridization with raidicatica

Chromosomal Walking → Genothical DNA

• The mapping
• The representation
• Dna is cut where has not been inserted.
• The positination

D. Genothics of cDNA Expression

• For inserting the cDNA without introns

Trasngenic Seminar

Trasngenic Plants

• The gen modified
• The enhancement and the result of production.

Transgenic Animal

• Bacteria
• Restriction sight
• Plamist ti
• Recombinat ti
• Adnas

Mice Konk Out ( Trasnagenic)

• ES cells
• Vectorial Director
• Neo casetier
• Bloqued the replications of DNA