Recombinant DNA technology

Question 1

What is Biotechnology?

Answer 1

technology based on biology; the harnessing of biological processes to develop technologies and products geared at improving life on this planet.

Question 2

What are the colours of Biotechnology?

Answer 2

1. Red Biotechnology
2. Green Biotech
3. White Biotech
4. Yellow Biotech
5. Brown Biotech
6. Gold Biotech
7. Blue Biotech
8. Violet Biotech
9. Grey Biotech
10. Dark Biotech

Question 3

Bioinformatics and Nanotechnology fall under which colour of biotechnology?

Answer 3

Gold Biotechnology

Question 4

Describe Blue Biotechnology.

Answer 4

The use of sea resources to make products. Deals with refining and combustion, and the production of bio-oils with photosynthetic micro-algae.

Question 5

I am the branch of biotechnology that uses agricultural processes such as micropropagation, transgenic plants and industrial applications.

Answer 5

Green Biotechnology

Question 6

Red Biotechnology is…

Answer 6

The branch of biotechnology uses techniques and procedures in the field of medicine and the health sector. Deals with the production of vaccines, antibiotics etc.

Question 7

Also called Industrial Biotechnology, I deal with the processes and resources functional to industries to produce industrial goods such as enzymes as a chemical catalyst.

Answer 7

White Biotechnology

Question 8

Yellow Biotechnology is…

Answer 8

• Biotechnology which is used in food production such as making wine, cheese and beer by fermentation.
  -Also applies to the biotechnology of insects; the control of harmful insects and the use of genes of insects for research and utilization in agricultural and medicinal sectors.

Question 9

Environmental usage and emphasis on maintaining biodiversity and removing pollutants is which colour of biotechnology?

Answer 9

Grey Biotechnology

Question 10

What are brown biotechnology and violet biotechnology associated with?

Answer 10

• Brown biotechnology is associated with management arid lands and deserts
  -violet biotechnology is associated with law, ethical, philosophical issues relating to biotechnology

Question 11

What are the four applications of biotechnology in industrial areas?

Answer 11

1. Healthcare
2. Crop Production
3. Non-Food Industrial Uses of crops and other products
4. Environmental Uses

Question 12

What are the ways biotechnology is used in the following sections:a)Medicine b) Agriculture c)Industrial and D)Environmental?

Answer 12

a)Medicine: Pharmaceutical drug discoveries, Pharmacogenomics, Genetic Testing

b) Agriculture: Genetically modified crops, Food crop resistance, Bioremediation

c) Industrial: Industrial fermentation, Usage of microorganisms to generate industrially useful products

D) Environmental: Bioremediation

Question 13

Define Genetic Engineering.

Answer 13

Genetic engineering is a technique used to transfer the desired gene to an organism to manipulate its genome.

Question 14

What are some applications of genetic engineering?

Answer 14

1. Production of pharmaceutical compounds such as insulin and growth hormone
2. production of transgenic plants and animals
3. Production of pathogens and insect-resistant plants
4. Understanding biological events in biological courses

Question 15

What is recombinant DNA technology?

Answer 15

Recombinant DNA technology is the incorporation/transfer of desired gene from one organism to another.

Question 16

What are the steps in r-DNA technology?

Answer 16

1. Extraction and Isolation
2. Gene Splicing
3. Transfer of r-DNA into the host cell (transformation)
4. Generation of r-DNA molecules when the vector self replicates in host cell
5. Selection and Screening
6. Replication of cells carrying r-DNA molecules
7. Culture and DNA purification

Question 17

Explain Extraction and Isolation.

Answer 17

• This is the step where the desired DNA and the vector of choice are isolated.
• DNA can be isolated by lysing cells (using enzymes), purification ( precipitation and seperation), agarose gel electrophoresis or PCR method
• Vector (plasmids) can be isolated using ultracentrifugation or alkaline lysis.

Question 18

What is a R plasmid?

Answer 18

a plasmid that contains the antibiotic resistance gene

Question 19

What are some common plasmids used recombinant DNA technology?

Answer 19

1. pBR322
2. pUC18
3. Ti plasmid
4. Ri plasmid

Question 20

What are some characteristics of the pBR322 plasmid?

Answer 20

*It is the
naturally occurring E. coli plasmid
*It has 4.3 Kbs. (kilo base pair size)
*It contains one Ori site ( origin of replication site)
*It contains two antibiotic resistance genes Amp + and
Tet
* It contains specific restriction endonuclease
recognizing site

Question 21

What other vectors can be used instead of plasmids?

Answer 21

1. Phages
2. Plant virus
3. Animal virus
4. Artificial Chromosomes

Question 22

Explain Splicing

Answer 22

• Splicing refers to the cutting and joining of the DNA and the vector.
• This is achieved by using restriction enzymes (for cutting) and DNA ligase for joining.
• Restriction enzymes are enzymes that cut DNA at specific sites, There are two types-exo and endo-nucleases. The one used in r-DNA technology is endo (cuts inside)
• The fragments and the vector are joined by sticky ends

Question 23

For the restriction enzyme ECORI, what sequence does it cut at?

Answer 23

GAATTC…cuts between the G and the A

Question 24

Explain Transformation

Answer 24

• This is the transfer of the r-DNA to the host cell via a vector
• The recombinant DNA vector (plasmid) is added to a flask
  containing a culture of E.coli
• Calcium ions usually in the form of calcium chloride are added to
  the flask followed by a brief heat shock
• This allows holes to briefly appear in the cell surface membrane of
  the E.coli making it permeable to DNA and allowing the plasmid to
  enter

Question 25

Why is E coli used as a common host cell?

Answer 25

1.It is a simple prokaryotic cell 2.It is a non pathogenic bacteria 3.It can be cultured easily in laboratory condition 4.It has very short life span 5.It contains self replicating plasmid 6.The plasmid of E.coli can be easily handled as vector

Question 26

Generation of rDNA molecules when the vector self-replicates in the host cell refers to...

Answer 26

- A single recombinant molecule will enter any individual bacterial cell and replicate like a normal cell subsequently producing more copies /clones

Question 27

Why are plasmids used to replicate recombinant DNA?

Answer 27

Plasmids are small circles of DNA found in bacteria Plasmids replicate independently of the bacterial chromosome Replication often produces 50 to 100 copies of a recombinant plasmid in each cell Pieces of foreign DNA can be added within a plasmid to create a recombinant plasmid

Question 28

Expound on Selection and Screening of Recombinant DNA?

Answer 28

- Selection of the host cells carrying the rDNA molecule using a selectable marker - Bacteria that have been subjected to an rDNA technology to introduce foreign DNA are grown on a medium containing an antibiotic (ampicillin, tetracycline etc) - Those bacterial colonies that can grow have successfully taken up and expressed the introduced genetic material - Screening is an alternative to a selectable marker - The screenable marker helps to distinguish between wanted and unwanted cells, these wanted or unwanted cells are simply untransformed cells that were unable to take up the gene during the experiment

Question 29

What are the types of selectable markers?

Answer 29

1. Positive - selectable markers that confer a selective advantage to the host cell. 2. Negative - selectable markers that eliminate or inhibit growth of the host organism upon selection. 3. Positive or Negative - can serve as both positive or negative to the host organism depending on the condition.

Question 30

What is replica plating?

Answer 30

transfer of colonies from one plate to another using an absorbent pad.

Question 31

What happens to the rDNA after Screening and Selection take place?

Answer 31

- the cells carrying the rDNA are replicated to get genetically identical cells/clones -The original mixture of transformed bacterial cells is spread out on the surface of a growth medium in a flat dish (Petri dish) so that the cells are separated from one another *These individual cells are invisible to the naked eye, but as each cell undergoes successive rounds of cell division, visible colonies form *Each colony is a cell clone , but it is also a DNA clone because the recombinant vector has now been amplified by replication during every round of cell division *Thus, the Petri dish, which may contain many hundreds of distinct colonies, represents a large number of clones of different DNA fragments * This collection of clones is called a DNA library * The colony of interest can be transferred to a culture medium for further replication

Question 32

Explain rDNA purification/protein extraction

Answer 32

- rDNA expresses itself (target protein) under appropriate optimal conditions after cloning of rDNA to a particular number of copies *Target protein can be produced in large scale *Cells which harbour cloned genes of interest grown in laboratory in small scale *Culture of dividing host used to extract desired protein then purified by different separation techniques

Question 33

What is a bioreactor? (see slide for more information)

Answer 33

vessels in which raw materials (recombinant organisms like microbial plant, animal or human cell & culture medium with ambient condition) biologically converted into specific products (protein/ enzymes)