Aquaculture & Marine Biotechnology

• Mechanism of $\alpha$-Complementation:
• The host E. coli strain (e.g., DH5$\alpha$, JM109) carries a mutant $\beta$-galactosidase gene (lacZ$\Delta M15$) encoding an inactive enzyme missing its N-terminal segment (the $\alpha$-peptide).
• The plasmid vector (e.g., pUC19, pBluescript) carries the coding sequence for this missing $\alpha$-peptide within its multiple cloning site (MCS).
• In transformed cells, the plasmid-encoded $\alpha$-peptide physically complexes with the host's inactive dimer to reconstitute functional $\beta$-galactosidase holoenzyme by providing the missing N-terminal domain.
• Functional enzyme cleaves chromogenic X-gal to produce blue colonies; insertion of foreign DNA disrupts the $\alpha$-peptide, yielding white recombinant colonies.
• nalysis:
• (alpha; beta-galactosidase; N-terminal): Correct.

Correct option: (2) alpha; beta-galactosidase; N-terminal

• Definition of a Phagemid:
• phagemid is a hybrid cloning vector that contains both a standard plasmid origin of replication (e.g., ColE1) and an origin of replication derived from a filamentous bacteriophage (e.g., f1 ori from M13 or fd phage).
• When helper phage (e.g., M13K07) is co-infected, the phagemid replicates via rolling-circle replication and packages single-stranded DNA into phage virions.
• pBluescript (pBC, pBS) is the classic prototype phagemid vector widely used in cDNA libraries, sequencing, and mutagenesis.
• $\lambda$ZAP is a lambda replacement/insertion vector; $\lambda$gt11 and EMBL4 are bacteriophage lambda vectors.
• nalysis:
• (pBluescript): Correct phagemid.

Correct option: (2) pBluescript

• Preparation of Chemically Competent Cells (Mandel & Higa, 1970):
• Bacterial cells (E. coli) possess net negative surface charges due to lipopolysaccharides and phospholipids, repelling negatively charged plasmid DNA.
• Incubating cells in ice-cold calcium chloride ($CaCl_2$):
• Divalent calcium ions ($Ca^{2+}$) neutralize the negative electrostatic charges on both the outer membrane phosphate groups and the DNA backbone.
• This facilitates the binding of plasmid DNA to the bacterial cell surface.
• subsequent brief heat shock ($42^\circ\text{C}$ for 45–90 seconds) alters membrane fluidity and creates transient pores through which plasmid DNA enters.
• nalysis:
• (CaCl2): Correct.

Correct option: (4) CaCl2

• Blunt-End Cleavage by EcoRV:
• Restriction endonucleases cleave palindromic recognition sequences:
• EcoRV (from E. coli): Cleaves at $5'\text{-GAT}^\downarrow\text{ATC-3'}$, producing blunt (flush) ends:

• EcoRI ($5'\text{-G}^\downarrow\text{AATTC-3'}$), BamHI ($5'\text{-G}^\downarrow\text{GATCC-3'}$), and HindIII ($5'\text{-A}^\downarrow\text{AGCTT-3'}$) produce staggered sticky (cohesive) $5'$ overhangs.
• Correct Option: Option 4 (EcoRV).