Eukaryotic Vectors in Biotechnology

Eukaryotic Vectors

• Vectors are DNA molecules acting as destinations for Gene of Interest (GOI).
• Vectors transport the gene into a host cell, which acts like a biofactory.
• Vectors include plasmids, bacteriophages, cosmids/phagemid, transposons, viruses, and artificial chromosomes.

Vectors for Yeast and Fungi

• 2 µm plasmid is found in eukaryotic organisms (EK!).
• 2 µm plasmid size is 6kb.
• Copy number ranges from 70-200.
• Rep1 and Rep2 facilitate plasmid replication.
• FLP protein converts one plasmid form (A) to another (B), altering gene order through intramolecular recombination.
• Selectable markers include methtrexate/Cu tolerance.
• Auxotrophic hosts like leu- can be converted into Leu+.

Vectors Based on 2 µm Plasmid (YEp)

• YEp plasmid size is 10.7kb.
• Selectable marker is Leu gene.
• Shuttle vector: replicates in both E. coli and yeast.
• Can exist as both plasmid and episome, which can be problematic during DNA isolation and sequencing.
• Selection is done in bacteria then introduced into yeast.

Other Types of Yeast Cloning Vectors

• Yeast integrative plasmid (Ylps): Ura3 selectable marker, replicates only when integrated.
• Yeast Replicative plasmid (YRps): Can multiply independently, with Trp1 as the selectable marker.

Transformation Frequency of Vectors

• YEp has a high transformation frequency of 10,000 to 100,000 per μg.
• YRps have a transformation frequency of 1,000 to 10,000 per μg.
• Ylp has a transformation frequency of 1000 per μg, only 1 to 10 without special measures.

Copy Number of Vectors

• YEp has a copy number of 20-50.
• YRp has a copy number of 5-100.
• Ylp typically has only one copy.

Artificial Chromosomes

• Yeast Artificial Chromosome (YAC)
  • Contains a centromere for accurate chromosome distribution during cell division.
  • Has telomeres to ensure proper replication of chromosome ends and prevent degradation.
  • Includes origins of replication to initiate DNA replication.

Applications of YAC

• Stable during segregation.
• Accommodates more DNA than other vectors (up to 1 million base pairs!).
• Used in gene libraries.

Vectors for Plants

• Ti plasmid, carried by Agrobacterium tumefaciens, is a tool for plant transformation.
• It has oncogenic genes, and it regulates auxin and cytokinin production.
• The T-DNA region of the Ti plasmid is transferred to the plant's genome.
• Different regions and traits of Ti plasmid are useful in plant transformation like Opine synthesis and its catabolism.

Vectors for Expression

• Vectors are used for cloning (mapping and sequencing) or for gene expression.

Expression Optimization

• Optimizing protein expression involves factors such as:
  • Transcription initiation and termination.
  • Promoter strength.
  • Plasmid copy number.
  • Plasmid stability.
  • Host-cell physiology.
  • Translation initiation sequences.
  • Codon choice.
  • mRNA structure.
  • Toxicity and metabolic drain/load.

Metabolic Load

• Overexpression of unnatural proteins has detrimental effects on bacterial growth.
• Using regulatory/inducible promoters helps to control protein expression levels. -Promoters like AP, T7, trc, tac, or BAD.

Translation Optimization

• mRNA must be effectively translated into protein.
• Ribosome interaction with regions upstream of the initiation codon is crucial.
  • Bacteria use the Shine-Dalgarno sequence(S-D).

Characteristics of the Translation Initiation Sequence

• The degree of similarity to the consensus sequence, the distance between the S-D sequence and the initiation AUG affects translation efficiency greatly (most optimal length is 8 bases).
• Presence of certain bases following the S-D site affect translation efficiency.
• Composition of the triplet immediately preceding the AUG codon affects translation efficiency greatly, especially for important or needed genes.

Codon Choice and Biasness

• Codon choice and biasness are correlated with tRNA availability in the cell and non-random choices exist between pyrimidine-ending codons.
• The translation stoppage is also dependent on codon biasness (e.g., UAA is used for high-expression genes, whereas UAG and UGA are more common in low-expression genes).

Protein Purification and Expression Vector

• Use of Tags: Tags facilitate protein purification through affinity chromatography, determine the presence of the cloned protein, and identify their size. Common tags include GST, MBP, and His tags.
  • Tag removal is necessary after purification, if purification is stringent, otherwise it can interfere with the final protein products. Inteins can be used.
• Use of Antibody/Immuno-assay: Antibodies can be generated against proteins of interest and then used for detection and purification via affinity chromatography or immuno-assay.
• Inclusion Bodies: Proteins of interest can form inclusion bodies (aggregates) due to improper folding. factors that encourage inclusion body formation include factors like temperature, pH, and media composition.
  • Modifications in the vector can help prevent inclusion body formation; examples include engineering the host cell to overexpress chaperones or making minor changes to the amino acid sequence of the cloned gene. Adding signal peptide or fusion partner may also be important for producing soluble protein and exporting them into the periplasm of the host cell.

Vector to Promote Protein Export

• Secreted proteins need transport to outer membranes or outside cells.
• Signal sequences (peptides) have distinct N-terminal domains: a positively charged region, a hydrophobic core, and a cleavage site for leader peptidase.
• Signal peptides are usually derived from naturally-occurring proteins.

Explore the fascinating world of eukaryotic vectors used in biotechnology, focusing on their role in transporting genes into host cells. Learn about various types of vectors such as plasmids, bacteriophages, and artificial chromosomes, particularly in yeast and fungi. This quiz will challenge your knowledge on the specifics of these genetic tools and their applications.