Lecture 23: Biotechnology I

Question 1

Limitations of Selective Breeding

Answer 1

1. Must have to wait a long time for the right gene to mutate into the new form
2. Be able to cross two closely related species with the right phenotypes

Question 2

Selective Breeding

Answer 2

-All animals/plants that are domesticated have been selectively bred
-Over years you select particular alleles of genes
-Chihuahuas to Great Dane (took approx. 100 years)
- If you want something new you must wait for a mutation

Question 3

Why use genetic engineering

Answer 3

1. To make an organism express a new phenotype
2. To understand how a specific gene works

Question 4

Gene cloning

Answer 4

Moving a single gene from one organism to another

Question 5

How does the pancreas work?

Answer 5

1. When glucose enters your blood stream some gets absorbed for metabolism and some gets stored in the liver as glycogen
2. The way your liver knows to take up glucose and make glycogen is due to a cell in your pancreas called the beta cell
3. The beta cell senses the glucose and then sends a signal, the signal is insulin. The insulin is released into the bloodstream and received by the liver
4. Then the liver starts taking up glucose

Question 6

What happens if you need glucose in the bloodstream?

Answer 6

If you are low on glucose the pancreas will release the hormone glucagon to the bloodstream which tells the liver to release glucose into the bloodstream for cells to eat

Question 7

What happens to people with diabetes?

Answer 7

-The beta cells are destroyed in people with diabetes
-Then you have no cells in the pancreas to tell the liver to take up glucose

Question 8

What did Frederick Banting and Charles Best do?

Answer 8

Purified insulin from dogs and showed that it could be used to treat diabetes.

Question 9

What was the problem with pig insulin?

Answer 9

-Expensive
-People often formed immune reactions to it because it is not exactly human insulin

Question 10

Solution to pig insulin

Answer 10

Make a gene put it into a plasmid and transform it into bacteria

Question 11

Bacteria don’t know how to splice, how do you make a gene with no introns?

Answer 11

You make a DNA copy of the mRNA from which the introns have already been spliced out

Question 12

How can you get a DNA copy from mRNA?

Answer 12

Reverse transcriptase, takes the mRNA and makes DNA copy called cDNA

Question 13

How do you find the insulin mRNA which is mixed with every other mRNA in a cell?

Answer 13

You make every single mRNA and convert them into cDNAs and clone them all into bacteria

Question 14

How do you get the cDNAs into the bacteria?

Answer 14

Put the cDNAs into a plasmid, the plasmid with the cDNA is called the vector. These plasmids must then replicate

Question 15

How do you get the cDNAs into the plasmid?

Answer 15

1. Cut the plasmid with the restriction endonuclease(ragged ends)
2. Next cut the cDNA with the restriction endonuclease(ragged ends)
3. Both being ragged ends means they are more likely to stick together now

Question 16

Covalently attaching the cDNA to the plasmid vector

Answer 16

-Requires the enzyme DNA ligase
-Forms phosphodiester bonds between the cDNA and the plasmid vector

Question 17

cDNA library

Answer 17

-Growing the bacteria on an ampicilin plates creates a cDNA library because the bacteria represent every mRNA in cDNA form

Question 17

How do we know which bacteria have been transformed
and which haven’t?

Answer 17

-we use an antibiotic
resistance gene on the plasmid (usually ampicillin resistance
-That way if we grow the bacteria on
ampicillin, only those that have a plasmid will grow
-Transformation marker

Question 18

How do
we find the colony that has the plasmid that has the insulin gene?

Answer 18

-We know the amino acid sequence of the insulin gene
-We will create a DNA probe that is the complementary strand to the strand
-Complementary strands bind to each other (anneal) more tightly than non-complementary strands -maximizes number of hydrogen bonds

Question 19

Expression vector

Answer 19

Vector that is good for expressing proteins

Question 20

How to get insulin gene to express?

Answer 20

-Cut the promoter with restrictive enzymes
-Cut the cDNAs with restrictive enzymes
-Combine them together, they will stick due to sticky ends, add ligase to form phosphodiester bonds

Question 21

How to know if the plasmid has the proper number of inserts?

Answer 21

Agarose gel electrophoresis.
-Bigger pieces will get stuck and move more slowly
-Negatively charged DNA will move toward the positive cathode
-You will be able to find the one with the proper amount of insulin

Question 22

Can you turn the gene off and on with lactose?

Answer 22

Yes