RDT (Vectors) By – Shweta Singh

Question-Answers -I
Q1. Write a short note on pBR322.

Ans . pBR322 is the earliest constructed vectors to be used for cloning. It was
constructed by Boliver and Rodriguez in 1974. It is a double stranded DNA
molecule of size 4361 bp and is the most commonly used plasmid cloning vector
in E.coli. This plasmid contains two different antibiotic resistance genes (tet R and
ampR) and recognition sites for several restriction enzymes (such as BamH1,
EcoRI, HindIII, PstI, PuvI etc).

Q2. What are expression vectors? State their importance.

Ans. An expression vector, otherwise known as an expression construct, is

generally a plasmid that is used to introduce a specific gene into a target cell. Once the expression vector is
inside the cell, the protein that is encoded by the gene is produced by the cellular-transcription and translation
machinery ribosomal complexes. The goal of a well-designed expression vector is the production of large
amounts of stable messenger RNA, and in extension, proteins. Expression vectors are basic tools for
biotechnology and the production of proteins such as insulin, which is important for the treatment of diabetes.

Q3. What are shuttle vectors?

Ans. A shuttle vector is a vector that can propagate in two different host species, hence, inserted DNA can be
tested or manipulated in two different cell types. Since these can be grown in one host and moved to another
without any extra manipulation, hence are called shuttle vectors.Shuttle vectors can be used in both
eukaryotes and prokaryotes. Shuttle vectors are frequently used to quickly make multiple copies of the gene in
E. coli (amplification). These vectors contain two types of origin of replication and selectable markers, one set
which functions in eukaryotic cells (eg. Yeast) and another in E.coli. One of the most common types of shuttle
vectors is the yeast shuttle vector (Yep).

Q4. Write a brief note on pUC vectors.

Ans. pUC are a large family of cloning vectors which have been constructed by
modification of pBR322 vector in the University of California (thus its name,
pUC). pUC vectors are smaller than pBR322 (size approx. 2.7kb) and have a
higher copy number (500-600 copies per cell). It has two selectable markers –
one ampicillin resistance gene and the other lac Z gene that codes for the
enzyme β-galactosidase. Its also contains multiple cloning sites (polylinker)
and thus offers great variability in insertion of foreign gene. However, pUC
vectors cannot accommodate a gene insert larger than 15kb.

Q5. What is the significance of LEU2 gene in Yep vectors?

Ans. The LEU2 gene serves as a selectable marker. It codes for an enzyme which is needed for the synthesis of
the amino acid leucine. Yeast cells having this plasmid can grow on a medium lacking leucine and hence can be
selected over cells not containing the plasmid.

Q6. What is Ti plasmid? Why is it useful in RDT?

Ans. Ti (tumor-inducing) plasmid is a circular plasmid of pathogenic bacteria Agrobacterium tumefaciens that
enables the bacterium to infect plant cells and produce a tumour (crown gall tumour). For inducing infection,
the bacteria sends a part of it’s Ti plamid (T-DNA) into the host cell. These plasmids are specially useful in
genetic engineering of the plants as they facilitate easy transfer of a foreign gene into a plant cell.