Cloning vectors

Question 1

Once different pieces of DNA with complementary ends could be ligated scientists developed a means of copying or replicating this recombinant DNA.

Answer 1

= Cloning Vectors

Question 2

What are cloning vectors?

Answer 2

DNA molecules that are able to “house” other DNA fragments and acts as a ”vehicle” to get DNA into host cell (transformation)

Question 3

When these vectors are introduced into host cells (e.g. E. coli) they can then

Answer 3

replicate themselves and the DNA fragment they house.

Question 4

DNA cloning vectors share a number of key features/ prerequisites:

Answer 4

1. They have restriction sites that allow insertion of the DNA fragments to be cloned.
   [Contain various unique RE sites]
2. An origin of replication allows them to replicate in host cells.
   [Must replicate autonomously]
3. Antibiotic selection markers allow one to distinguish only those host cells which have the vector.
   [Selectable marker (AbR)]
4. Many DNA cloning vectors have specific sequences that allow for sequencing inserted DNA,
   these are termed primers
   [Should be easy to recover from host cell]

Question 5

Types of vectors

Answer 5

• plasmids
• phages
• cosmids
• shuttles
• BACs
• YACs

Question 6

lacZ gene. What is it doing in a cloning vector?

Answer 6

used in a process called “blue-white” screening

Question 7

What is blue-white screening and how does it work?

Answer 7

1. In alpha-complementation, the vector molecule contains the regulatory and coding regions for the first 146 amino acids of the ß-galactosidase (lacZ) gene.
2. A multiple cloning site (MCS) is engineered into the vector sequence without disrupting the activity of this piece of the lacZ gene.
3. During ligation, vector molecules are cut at a particular sequence in the MCS and incubated with pieces of insert DNA cut with the same restriction enzyme (using DNA ligase).

Question 8

Two experimental outcomes of blue-white screening are possible;

Answer 8

1. insert DNA is successfully ligated into the vector disrupting the lacZ coding sequence on the vector.
2. vectors will not be cut by the restriction endonuclease or, their ends will re-ligate without an insert.

After ligation, the resulting rDNA is used to transform a competent E. coli strain where the genome contains the rest of the lacZ gene.

Question 9

Slide 7 and 8 - Chapter 20 - Part 1

Question 10

recombinant DNA experiment steps:
To clone a piece of foreign DNA into a vector

Answer 10

1. Plasmid vector is cut with a restriction enzyme
   - produces sticky overhangs
2. DNA to be cloned is cut - using the same restriction enzyme
   = Generates a series of fragments with compatible sticky ends
3. The 2 DNA molecules are brought together and ligated to form a recombinant molecule
4. This new recombinant vector is introduced into bacterial host cells using transformation methods
5. Cells carrying recombinant plasmids are selected and plated on agar gel containing antibiotics and colour indicators

Question 11

Blue/white selection

Answer 11

ß-galactosidase catalyses the conversion of the X-GAL substrate to form a bright blue precipitate
No insertion means the ß-galactosidase enzyme is functional, thus a blue precipitate forms
Insertion of a cloned DNA fragment disrupt the lac Z ORF, thus ß-galactosidase is inactivated, and no blue colour visible

Question 12

What limits the bacterial plasmid vector

Answer 12

The size of the insert they are able to house
- max size of 20 Kb

Question 13

Phage vectors

Answer 13

• Can hold larger inserts of DNA than bacterial vectors

Question 14

Phage vector characteristics

Answer 14

1. Genome of Gamma phage has been sequenced and modified to incorporate key features of bacterial plasmids
2. Phage vectors can accept up to about 45 kb inserts (almost twice that of a bacterial plasmid).
3. DNA is ligated and “packaged” into phages which, are then used to infect E. coli cells.
4. The phage then replicates inside the host cells, producing many copies of the DNA insert.
5. As they reproduce they lyse the E.coli cell forming zones of clearing on a plate (called plaques).
6. From these plaques one can recover more phage and recombinant DNA.

Summary:
Viruses that infect bacteria
Genetically modified to have importantcharacteristics of plasmids (e.g. an MCS)
Up to 45kb inserts
Ligated vector packaged into phage heads
Infect plated-out bacteria and can be identified as plaques (lysis of cells)

Question 15

Unlike what we have seen for blue-white screening, the circles on the plate of phage vectors represent zones of

Answer 15

clearing where the phage has killed E.coli cells forming plaques.

Question 16

bacterial and yeast artificial chromosomes (BACs and YACs)

Answer 16

Examples of vectors which can house very large DNA inserts.

Question 17

When are bacterial and yeast artificial chromosomes (BACs and YACs) useful?

Answer 17

when analysing Eukaryotic genomes (e.g. the human genome).

Question 18

BACs

Answer 18

can accept 100 – 300 kb DNA inserts but, occur in very low copies in the bacterial cell.

Question 19

YACs

Answer 19

• have telomeres at each end
• origins of replication
• and a centromere just like normal chromosomes.
• Unlike normal chromosomes, YACs also have a selectable marker and an MCS with restriction enzyme sites.
• YACs can accept 100 to 1000 kb DNA inserts and they were critical to the Human Genome Project.

Question 20

Question 21

Artificial chromosomes are promising tools for gene therapy applications. Similar to endogenous chromosomes, artificial chromosomes can replicate and segregate autonomously without integration into the host chromosomes. Human artificial chromosomes (HACs), can carry DNA inserts larger than 1Mb.

Question 22

When are expression vectors used?

Answer 22

When DNA inserts need to be transcribed (converted to mRNA) and translated (converted to protein).

Question 23

Expression vector used for _______________ cells and, are able to initiate ___________________ of the DNA insert (cloned gene). it is found adjacent to a _______ and ______

Answer 23

bacterial and eukaryotic host
both transcription and translation
T7 promoter and lac operator

Question 24

The biotechnology industry relies heavily on _____________ to produce commercially valuable ____________

Answer 24

expression vectors
protein products from cloned genes.

Question 25

Bacterial expression vectors are used to

Answer 25

introduce “foreign-DNA” into a host in order to replicate and amplify the foreign DNA sequences as a recombinant molecule (protein).

Question 26

The pET vectors

Answer 26

allow one to clone a gene and express recombinant protein in bacterial cells. For example recombinant human interferon-β (rhIFN-β), expressed in E. coli, is used to treat multiple sclerosis in humans.

Question 27

Plasmids

Answer 27

Natural, self replicating, extra-chromosomal
Small circular DNA molecule that functions independently from the host cell (autonomous replication)
Modified to contain an MCS/polylinker
Contains AbR selectable marker gene
High copy number (+/- 1000)
Proper MCS in lac Z (insertional inactivation)
+/- 20 kb inserts

Question 28

Cloning in plants

Answer 28

Soil bacterium Agrobacterium tumefaciens infects plants
Ti-plasmid (tumour inducing)- modified
Integration into host-cell chromosome
Direct root & shoot development (transgenic plant)
Not all plant hosts can be infected