Question

1. Explain briefly (and within the context of the plasmid you just created) the role of each of the 10 elements that are in bold

   it needs a “ColE1 origin” (E. coli origin of replication)

   · it needs a “2μ ori” (yeast origin of replication)

   · a AmpR gene (coding for the bacterial resistance against the antibiotic ampicillin) and its promoter.

   · a yeast gene coding for the synthesis of uracil and its promoter.

   · a yeast gene coding for the resistance against the antibiotic Nourseothricin, and that uses a GAL1 promoter and a TEF terminator.

   · two restriction enzyme cut sites that will allow the subcloning of a sequence of interest somewhere in the plasmid.

   
   

Answer 1

A plasmid is a small extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate indepnedently.Artificially created plasmids are used as vectors in Genetic engineering.plasmids are the most commonly bacterial cloning vectors.

1)An origin of replication is the place where the process of DNA replication begins.it is a critical component of a DNA plasmid because it ensures, the plasmid is passed from mother to daughter cells during cell division.

ColE1 is a plasmid found in bacteria,got its name as it carries a gene for colicin E1(the cea gene).Its size and the presence of a single EcoRI recognition site caused it to be considered as a vector candidate.

2)Plasmids have been extensively studied inn yeast and deveeloped into yeast cloning vectors.One interesting yeast plasmid is called the 2u circle.The 2u circle is a circular extrachromosomal element found in the nucleus of most saccharomyces cerevisiae strains.,the properties require the integrity of four plasmid loci.

REP1 andREP2 active in trans and corresponds to two open coding regions of 2u.The other two loci are active in cis and correspond to origin of replication and to a region,REP3 located away from the origin.

3)The AmpR gene ,also known as Ampicillin resistance gene confers resistance to the antibiotic Ampicillin.ThesE genes can be introduced into bacterial cells with thE help of a plasmid that acts like a vector in transforming the bacterial cells and making them ampicillin resistant.E.coli cells containing this plasmid can survive and form colonies on lactobacillus agar supplimented with ampicillin.

The AmpR gene codes for an enzyme(b-lactamase)that is secreted into the periplasmic space of the bacterium where it catalyses hydrolysis of the b-lactam ring of the ampicillin.thus,the gene product of the AmpR gene destroys the antibiotic

4) URA3-Uracil biosynthesis gene;LaC4 as promoter.

5)TEF gene of the filamentous fungus ashbya gossypi,dual gene expression cassette vectors.=-resistance against nourseothricin

6)Restriction sites are located on a DNA molecule containing specific nucleotide sequence,recognised by restriction enzymes.If the plasmids were cut at the replication,it would not be able to reproduce transfer genetic information to its host cell.It would become fragments if dna and no more the plasmid will be in circular form.restriction enzymes cut dna at specific 4- to 8 bp sequence,leaving self complimentary single stranded tails,.