Question

3. Below is a diagram of the pUC19 cloning vector (plasmid): Sall EcoRI Hinc Il Kpnl BamHI Accii Pst! Hind III T1 Sacl Xmal xbal BSDMI Sphi Smal Polylinker lacz -ori PUC19 amp Fig_18-08 Genetics, Second Edition 2005 WH Freeman and Company a. Describe briefly the purpose of the: ori: ampR; lacZt: Palxlinker b. When bacteria containing this plasmid are grown on media containing x-gal and ampicillin, why do sometimes you see blue colonies and sometimes white colonies? What does each type of colony tell us?

Answer 1

pUC19 vector is a commonly used cloning vector

Ori Purpose : Ori is the start point of DNA replication in Plasmids. Ori enables the plasmids to replicate and survive in the cells.

ampR Purpose : ampR means ampicillin resistance. Hosts like E.coli does not possess antibiotics resistance in nature. when Plasmids containing Ampicilin resistance is introduced into host via transformation, Only the Hosts containing Plasmids will show ampicillin resistance. Thus bacteria with plasmids in it can be easily screened using ampR   screening

lacZ+ : lacZ+ gene is used to confirm the presence of gene of interest in the plasmid.

lacZ+ gene of the plasmid (Without gene of insert inserted into plasmid) and lacZ deletion mutant of the E.coli combines to give blue color to the colonies. lacZ+ gene contains restriction site. When gene of interest gets inserted into the lacZ+ using restriction digestion , lacZ+ gets disturbed. So it cannot produce blue color. Thus white colonies are formed. From this Blue/ White screening we can conclude that plasmids with gene of interest will produce white colony and plasmids without gene of interest will produce Blue colony.

Note : acZ+ gene of the plasmid (Without gene of insert inserted into plasmid) and lacZ deletion mutant of the E.coli combines to produce enzyme called β-galactosidase. β-galactosidase act on X-gal, a colourless analog of lactose to produce bright blue.I.e Blue colored colonies

Polylinker : Polylinker is also called Multiple Cloning site.It is a very short sequence of DNA with many unique restriction sites. This is the site where any gene of interest can be inserted without much trouble

When bacteria containing this plasmid are grown on media containing X gal and ampicliin, why do sometimes you see blue colonies and sometimes you see white colonies?

What does each type of colony tell us?

Let's assume that we are using E.Coli . When Plasmids with ampR is inerted into E.Coli , E.Coli will exhibit ampplicillin resistance. This is the reason for survival of bacteria in the presence of ampicillin. Whereas why do they produc e Blue color and white color?. It is because of the presence of X-gal, colourless analog of lactose. The lacZ+ gene of the plasmid (Without gene of insert inserted into plasmid) and lacZ deletion mutant of the E.coli combines to give blue color to the colonies. lacZ+ gene contains restriction site. When gene of interest gets inserted into the lacZ+ using restriction digestion , lacZ+ gets disturbed. So it cannot produce blue color. Thus white colonies are formed. From this Blue/ White screening we can conclude that plasmids with gene of interest will produce white colony and plasmids without gene of interest will produce Blue colony.