module 6 - 21.4 genetic engineering

Question 1

what is genetic engineering?

Answer 1

manipulating an organisms genome

Question 2

what are the basic principles of genetic engineering?

Answer 2

isolating a gene for a desirable characteristic in one organism and placing it into another organism

Question 3

what is ‘transgenic’?

Answer 3

an organism that carries a gene from another organism (also referred to as a genetically modified organism - GMO)

Question 4

what is the first stage of genetic modification?

Answer 4

isolate desirable gene

Question 5

what is the process involved in isolating a gene using the enzyme restriction endonucleases?

Answer 5

• most common technique uses enzymes called restriction endonucleases, to cut required gene from the DNA of an organism
• these enzymes cut the DNA strands unevenly, resulting in exposed bases called sticky ends
• stick ends make it easier to indert desired gene into DNA of different organism

Question 6

what is the process involved in isolating a gene using the enzyme reverse transcriptase?

Answer 6

• used to produce a single strand of complementary DNA
• advantageous because makes it easier to identify desired gene as particular cell will make some very specific types of mRNA

Question 7

what is the formation of recombinant DNA?

Answer 7

DNA isolated by restriction endonucleases must be inserted into a vector that can carry it into a host cell

Question 8

what are the most commonly used vectors in genetic engineering?

Answer 8

bacterial plasmids - small circular molecules of DNA separate from chromosomal DNA that can replicate independently

Question 9

why are bacterial plasmids used in genetic engineering?

Answer 9

• once plasmid gets into new host cell it can combine with host DNA to form RECOMBINANT DNA
• plasmids contain marker gene

Question 10

what is a marker gene?

Answer 10

allows scientists to determine that the bacteria have taken up the plasmid by growing bacteria in media containing the antibiotic

Question 11

how do you insert DNA into a plasmid?

Answer 11

• DNA fragment must be cut open using same restriction endonuclease used to isolate the DNA
• results in plasmid having complementary sticky ends to sticky ends of DNA fragment
• once sticky ends are lined up, DNA ligase forms phosphodiester bonds between sugar and phosphate groups on the 2 strands of DNA, joining them together

Question 12

why might plasmids be given a second marker gene?

Answer 12

• used to show that the plasmid contains the recombinant gene
• it placed in plasmid by genetic engineering methods
• plasmid then cut by restriction enzyme within marker gene to insert desired gene
• if insertion is successful, marker gene will not function

Question 13

how do you transfer the vector?

Answer 13

plasmid with recombinant DNA must be transferred into host cell through process called transformation

Question 14

what are the 2 methods of transferring a vactor?

Answer 14

• culture bacterial cells and plasmids in calcium rich solution
• electroporation

Question 15

what is involved in the method of culturing bacterial cells and plasmids in calcium-rich solution?

Answer 15

• culture bacterial cells in calcium-rich solution and increase temperature
• this causes bacterial membranes to become permeable and plasmids can enter

Question 16

what is involved in electroporation?

Answer 16

• small electrical current is applied to bacteria
• this makes membranes very porous so plasmids move into cells
• can be used to get DNA fragments directly into eukaryotic cells, new DNA will pass through cell membrane and nuclear membrane to fuse with the nuclear DNA
• technique’s less useful in whole organisms

Question 17

what is electrofusion?

Answer 17

• tiny electric currents are applied to the membranes of 2 different cells
• this fuses the cell and nuclear membranes of 2 cells together to form a hybrid/polyploid cell, containing DNA from both
• important in production of nonclonal antibodies

Question 18

how are monoclonal antibodies produced?

Answer 18

produced by combination of cell producing one single type of antibody with a tumour cell, meaning it rapidly divides in culture

Question 19

What is one method of genetically modifying plants?

Answer 19

• Using agrobacterium tumefaciens (a bacterium that causes tumours in healthy plants)
• Desired gene is placed in the plasmid of A. tumefaciens along with maker gene
• this is then carried directly carried into me plant cell DNA
• transgenic plant cells form a callus due to bacteria plasmid

Question 20

What is a callus?

Answer 20

Mass of GM plant cells, each which can be grown into a new transgenic plant

Question 21

How can transgenic plants be produced by electrofusion (as well as using A. Tumefaciens)?

Answer 21

• The cells produced have chromosomes from both original cells so are polyploid
• The cells that are fused may be from similar species or very different ones
  Main stages in this process:
  1. Removal of cell wall by cellulases
  2. Electrofusion to form a new polyploid cell
  3. Use of plant names to stimulate growth of new cell wall
  4. Callus formation and production of many cloned, transgenic plants

Question 22

Why is it harder to engineer the DNA of eukaryotic animals, tran bacteria or plants?

Answer 22

Animal cell membranes are less easy to manipulate

Question 23

What is the summarised whole process of genetic engineering in plants?

Answer 23

1. Cut plant
2. Expose leat to bacteria carrying a resistant die and an antibiotic resistant gene. Allow bacteria to deliver the genes into leat cells
3. Expose leaf to antibiotic to kill plus hat lack new genes, wait for surviving (gene-altered) cells to multiply and form a clump (callus)
4. The plants are transferred to soil to develop into fully differentiated adult plants that are resistant