WEEK 7: Sub-cloning

Question 1

Restriction Endonucleases

Answer 1

An enzyme that recognizes particular base sequences in DNA and makes double-stranded cuts at those sequences; also called a restructure enzyme

Question 2

Endonuclease

Answer 2

A group of enzymes that break the phosphodiester bond present within the polynucleotide chain of a DNA molecule.

Question 3

DNA Ligase

Answer 3

An enzyme that catalyzes the formation of a phosphodiester bond between adjacent 3’-OH and 5’-phosphate groups in a DNA molecule without adding another nucleotide to the strand.

Question 4

Ligate

Answer 4

join (molecules or molecular fragments) together with a new chemical bond.
“We ligated these proteins to create three triangular nanostructures”

Question 5

Transformation

Answer 5

The mechanism by which DNA found in the environment is taken up by a cell. After transformation, recombination may take place between the introduced genes and those of the bacterial chromosome.

Question 6

Vector

Answer 6

In molecular cloning, a vector is any particle used as a vehicle to artificially carry a foreign nucleic sequence – usually DNA – into another cell, where it can be replicated and/or expressed. A vector containing foreign DNA is termed recombinant DNA

Question 7

Cloning Vector

Answer 7

Stable, replicating DNA molecule to which a foreign DNA fragment can be attached for introduction into a cell.

Question 8

Sub-cloning

Answer 8

Simply consists of moving a piece of DNA from one vector to another.

Question 9

lacZ

Answer 9

Originally isolated from the wild-type E.coli, is from the first gene in the lac operon.

Question 10

Beta-galactosidase

Answer 10

Is an enzyme that catalyzes the hydrolytic cleavage of lactose into glucose and galactose. Can be separated into two polypeptide parts:
- alpha-fragment (small part)
- w-fragment (larger part)

Question 11

X-gal

Answer 11

A chemical analogue of lactose, to form an insoluble blue-coloured product.

Question 12

Alpha-fragment

Answer 12

The alpha-fragment can be encoded by a small part of lacZ called lacZ’ that is neither the lac operon nor the entire lacZ gene - it is only the promoter and the first part of the coding region of lacZ

Question 13

w-fragment

Answer 13

w-fragment is encoded in a similar fashion to the alpha-fragment but instead only uses he last part of the lacZ as the coding region.
- the first part which corresponds to lacZ’ is deleted.

Question 14

lacZ’ (lac z prime)

Answer 14

Is neither the entier lac operon nor the entire lacZ gene; it is only the promoter and the first part of the coding region of lacZ

Question 15

alpha-complementation

Answer 15

The coming together of the alpha- and w- fragments of the Beta-galactosidase enzyme to make a functional enzyme is known as alpha-complementation.
- Used in DNA manipulations to determine if a piece of DNA was inserted into a plasmid vector.

Question 16

lux-operon

Answer 16

Is a sequence of genes contained within a DNA restriction fragment cloned from the MJ1 strain of V.fisheri, a bioluminescent bacterial species that live in the ‘light origins’ of the Japanese pinecone fish

All genetic information needed to confer the phenotype of bioluminescence is present within this restriction fragment.

Question 17

bioluminescence

Answer 17

The production and emission of light by a living organism; i.e., the ability to glow in the dark - a lux+ phenotype.

Question 18

luciferase

Answer 18

This enzyme is capable of catalyzing a reaction that produces a blue-green light. i.e., Luciferase is a light-producing enzyme

Question 19

Double Digests

Answer 19

Involves digesting plasmid vectors with two different restriction enzymes to generate restriction fragments that have different restriction cut sites at each end. i.e., Digesting a DNA substrate with two restriction endonucleases simultaneously

Question 20

Antibiotic Selection

Answer 20

Since the plasmid includes both the DNA of interest and a gene that confers resistance to a specific antibiotic, applying the antibiotic to the bacterial cells (i.e., antibiotic selection) can help determine which cells were genetically modified.

Question 21

Blue/white Screening

Answer 21

Blue-white screening is a rapid and efficient technique for the identification of recombinant bacteria. It relies on the activity of β-galactosidase, an enzyme occurring in E. coli, which cleaves lactose into glucose and galactose

Takes advantage of alpha-complementation to identify E.coli cells that have incorporated a piece of DNA into the target plasmid, which may or may not be your target restrction fragment or gene. These cells will be white between the inserted piece of DNA disrupts lacZ’ and prevents alpha-complementation.

Blue cells contain the plasmid vector we desire but do NOT have a piece of DNA incorporated into it.

When a plasmid containing the lacZ gene is introduced into E. coli, it can disrupt the lacZ gene and prevent the production of active β-galactosidase. Therefore, colonies containing plasmids with the cloned DNA fragment of interest will be white (i.e., no β-galactosidase activity), while colonies without the plasmid or with non-recombinant plasmids will be blue (i.e., β-galactosidase activity). This allows for easy visual identification and selection of colonies that contain the desired recombinant plasmid.

Question 22

Multiple cloning site (MCS)

Answer 22

Is located within the lacZ’ gene; the MCS contains a series of unique restriction enzyme sites that are useful for cloning restriction fragments.

Question 23

DH5alpha

Answer 23

Our DH5α competent E. coli is a versatile strain used for general cloning and sub-cloning applications and is available in a wide variety of transformation efficiencies.
- Recombinant deficient nad its restriction-modification system lacks the endonuclease but retains the methylase - both properties make the strain useful in cloning procedures
- it is sensitive to ampicillin as well as to chloramphenicol
- it has the necessary genetic information to encode for the w-fragment, but lacks the normal lac operon. This mutation is essential for blue/white screening
- DH5alpha is competent, meaning that it can take up DNA from its environment, such as the plasmids that are used in cloning. This allows for efficient transformation of the bacteria with the cloned plasmid.