Molecular Biology Vectors Quiz

Genetic Engineering (GE-1) - Methodology of Gene Manipulation

• Lecture 7 covered methodology of gene manipulation.
• The lecture was given by Dr. Mohamed Hazman on November 13, 2024.

Promoters: Screening and Gene Expression (APB1303, Lecture 7)

• The lecture discussed promoters and gene expression.
• Diagrams showed constitutive promoters, teto expression cassette of interest, and the hypertrophic heart (with diseased and normal versions).
• Various IPTG concentrations (0.05 mM, 0.5 mM, 1 mM) were tested, illustrating how IPTG affects gene expression.
• The diagrams show RNA polymerase and TetR.

Reading Vectors: Basic Elements

• The lecture explained basic elements of reading vectors.
• Elements included origin of replication (Ori), multiple cloning site (MCS), selectable marker, promoter, and sequence elements of translation.
• There was a visual representation of a plasmid map, showing various restriction sites (e.g., EcoRI, HindIII, BamHI).
• The diagram of the plasmid included pBluescript II SK(+).

Vectors

• The lecture described vectors for cloning and expression.
• Vectors are used to create copies of genes, and to express proteins.
• A diagram of different vectors was provided, and different parts of the vectors were highlighted.
• Parts of the vector included the origin of replication (ORI), multiple cloning site (MCS), selectable marker, promoter, and elements of translation.

Cloning and Expression

• Cloning involves making copies of a gene, and expression involves producing a protein from that gene.
• Vectors are crucial to achieve both of these tasks in molecular biology.
• The flow chart linked cloning with the production of proteins, with special elements for protein expression and purification highlighted.
• The diagrams illustrate the various stages of cloning and expression, including gene of interest, amplification, and protein expression.

Blue/White Colony for Cloning

• Blue/white colony screening is used to identify recombinant vectors, and differentiating them from empty vectors.
• The optimal amounts of IPTG and X-Gal are crucial for efficient blue/white colony screening.

Rosetta Stone

• The Rosetta Stone was mentioned but not discussed in detail.

To Express a Gene

• A special E. coli strain (like Rosetta) and a specialized expression vector are required to express a gene.
• The process of using a vector for expression is linked to the presence of chloramphenicol resistance gene within the vector.
• Production of a toxic protein due to the lack of the insert needs an expression vector.

IPTG Induces Protein Expression

• IPTG, isopropyl-β-D-1-thiogalactopyranoside, induces protein expression.
• IPTG is added in controlled quantities (0.05 mM, 0.5 mM, 1 mM) to illustrate the resulting effect on protein expression.
• The lac repressor is explained and its role in controlling expression.

Recombinant Protein Tags

• Maltose Binding Protein (MBP) is used as a tag for better protein solubility and purification.
• MBP fusion proteins are purified using amylose resin.
• 6xHis tag is used for rapid purification, detection and specific quantification.
• 6xHis tag is useful for use in assays, without using specific antibodies.

Purification Strategy

• The lecture presented a diagram illustrating protein purification strategies, including sample loading, washing and elution steps.
• The procedure clarifies protein purification using MBP (Maltose Binding Protein) affinity tags.
• There are different stages leading to the purification of the target protein, which is shown in an image format.

Mammalian Inducible Promoters (Ex2)

• The expression system uses the Tn10 tetracycline resistance operon.
• Three configurations were presented: repression-based, Tet-off and Tet-on.
• The role of IPTG induction of gene expression was included.

Tetracycline

• Tetracycline eliminates sensitive cells by binding to ribosomes, mis-reading mRNA and thus disrupting protein synthesis.
• Tetracycline-resistant E. coli strains/types were presented.
• Drug efflux mechanism was also mentioned

Repression-based Configuration

• A diagram illustrating a repression-based configuration with a constitutive promoter, TetR, Teto, and the gene of interest.
• The mechanism showed how Dox (Doxycycline) dissociates TetR-teto and initiates RNA transcription.

Tet-off Configuration

• Diagram showing Tet-off configuration, with Tet-Response Element (TRE), tTA, and the regulatory mechanisms.
• The expression is ON in the absence of tetracycline.
• Adding Tetracycline dissociates the protein and stops the expression.

Tet-on Configuration

• Diagram showing Tet-on regulatory mechanisms.
• The expression is OFF until the presence of tetracycline, which will lead to expression.

Tissue-Specific Promoters

• Tissue-specific promoters ensure gene expression is localized to specific tissues or cell types.
• Cis-acting elements and trans-acting proteins are highlighted and how they interact to control expression.
• Enhancer elements play an important role in fine-tuning gene expression.
• The importance of these elements in tissue-specific genetic therapeutic strategies is noted.

Focused vs Dispersed Transcription

• The differences between TATA-containing and TATA-less gene promoters were explained with diagrams to illustrate the concepts.
• Housekeeping and tissue-specific promoters are distinguished.

CpG Islands and TATA Box

• CpG islands are regions devoid of methylation, with higher G+C content.
• They are associated with generic transcription initiation sites (without TATA box).
• TATA box is associated with basic expression in tissue-specific promoters and less chromatin accessibility.

Colorectal Cancer-Specific Promoters (Ex1)

• The prevalence of colorectal cancer was noted, with the benefit of gene suicide and cytotoxic effects highlighted.
• Therapeutic genes targeting tumor cells, not normal cells, were discussed.

Specificity and Targeting

• Diagrams illustrate the mechanisms of targeting therapeutic genes to colorectal cancer cells, avoiding their expression in normal cells.

Endosperm-Specific Glutelin Promoter

• The need for tissue-specific promoters, particularly in endsoperm was highlighted.
• How tissue-specific promoters like glutelin promoters can help to focus transgene expression in the endosperm of seeds, thus minimizing any negative consequences, without unnecessary metabolic penalties, is discussed.

Thanks and Good Luck

• A general closing message.