Recombinant DNA technology Flashcards
What is Biotechnology?
technology based on biology; the harnessing of biological processes to develop technologies and products geared at improving life on this planet.
What are the colours of Biotechnology?
- Red Biotechnology
- Green Biotech
- White Biotech
- Yellow Biotech
- Brown Biotech
- Gold Biotech
- Blue Biotech
- Violet Biotech
- Grey Biotech
- Dark Biotech
Bioinformatics and Nanotechnology fall under which colour of biotechnology?
Gold Biotechnology
Describe Blue Biotechnology.
The use of sea resources to make products. Deals with refining and combustion, and the production of bio-oils with photosynthetic micro-algae.
I am the branch of biotechnology that uses agricultural processes such as micropropagation, transgenic plants and industrial applications.
Green Biotechnology
Red Biotechnology is…
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.
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.
White Biotechnology
Yellow Biotechnology is…
- 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.
Environmental usage and emphasis on maintaining biodiversity and removing pollutants is which colour of biotechnology?
Grey Biotechnology
What are brown biotechnology and violet biotechnology associated with?
- Brown biotechnology is associated with management arid lands and deserts
-violet biotechnology is associated with law, ethical, philosophical issues relating to biotechnology
What are the four applications of biotechnology in industrial areas?
- Healthcare
- Crop Production
- Non-Food Industrial Uses of crops and other products
- Environmental Uses
What are the ways biotechnology is used in the following sections:a)Medicine b) Agriculture c)Industrial and D)Environmental?
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
Define Genetic Engineering.
Genetic engineering is a technique used to transfer the desired gene to an organism to manipulate its genome.
What are some applications of genetic engineering?
- Production of pharmaceutical compounds such as insulin and growth hormone
- production of transgenic plants and animals
- Production of pathogens and insect-resistant plants
- Understanding biological events in biological courses
What is recombinant DNA technology?
Recombinant DNA technology is the incorporation/transfer of desired gene from one organism to another.
What are the steps in r-DNA technology?
- Extraction and Isolation
- Gene Splicing
- Transfer of r-DNA into the host cell (transformation)
- Generation of r-DNA molecules when the vector self replicates in host cell
- Selection and Screening
- Replication of cells carrying r-DNA molecules
- Culture and DNA purification
Explain Extraction and Isolation.
- 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.
What is a R plasmid?
a plasmid that contains the antibiotic resistance gene
What are some common plasmids used recombinant DNA technology?
- pBR322
- pUC18
- Ti plasmid
- Ri plasmid
What are some characteristics of the pBR322 plasmid?
*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
What other vectors can be used instead of plasmids?
- Phages
- Plant virus
- Animal virus
- Artificial Chromosomes
Explain Splicing
- 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
For the restriction enzyme ECORI, what sequence does it cut at?
GAATTC…cuts between the G and the A
Explain Transformation
- 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
Why is E coli used as a common host cell?
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
Generation of rDNA molecules when the vector self-replicates in the host cell refers to…
- A single recombinant molecule will enter any individual bacterial cell and replicate like a normal cell subsequently producing more copies /clones
Why are plasmids used to replicate recombinant DNA?
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
Expound on Selection and Screening of Recombinant DNA?
- 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
What are the types of selectable markers?
- Positive - selectable markers that confer a selective advantage to the host cell.
- Negative - selectable markers that eliminate or inhibit growth of the host organism upon selection.
- Positive or Negative - can serve as both positive or negative to the host organism depending on the condition.
What is replica plating?
transfer of colonies from one plate to another using an absorbent pad.
What happens to the rDNA after Screening and Selection take place?
- 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
Explain rDNA purification/protein extraction
- 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
What is a bioreactor? (see slide for more information)
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)
