lab 2: bacterial transformation

Question 1

bacterial transformation

Answer 1

-an important technique in molecular biology
-changing the genotype & phenotype of a bacteria by uptake of foreign/new DNA

Question 2

DNA

Answer 2

-contains genetic instructions specifying the biological development
-instructions are carried out by proteins

Question 3

proteins

Answer 3

-made up of amino acids joined by peptide bonds
-proteins are synthesized in rough ER
-all amino acids have: acidic group (-COOH) = carboxyl group; amino group (H2N); R group that varies (determines the identity of amino acid

Question 4

bacteria

Answer 4

-prokaryotes
-no nucleus
-bacterial DNA is organized in 1 double stranded circular molecule
-DNA is located in nucleoid
-

Question 5

Escherichia coli (E. Coli)

Answer 5

-host organism we use for the lab
-one of the most studied organisms
-grows rapidly
-bacteria competence (the chances for successful transformation)

Question 6

genetic transformations

Answer 6

-inserting new DNA so the bacteria contains whatever products we want to mass produce
-ex: proteins like insulin (made from bacteria & yeast), genetically modified foods (GMOs)

Question 7

bioremediation

Answer 7

the use of either naturally occurring or deliberately introduced microorganisms or other forms of life to consume & break down environmental pollutants, in order to clean up a polluted site

Question 8

plasmids

Answer 8

-small circular pieces of DNA that replicate independently from the host’s chromosomal DNA
-can be engineered to carry genes from one organism into a bacterial cell
-contain a gene that confers some selective advantage (AMP & Beta-galactosidase)
-can be used to carry genes into a cell

Question 9

AMP

Answer 9

-ampicillin
-resistance to an antibiotic

Question 10

Beta-galactosidase

Answer 10

produces an enzyme that breaks down lactose & a chemical like lactose, X-Gal -> Indo Blu

Question 11

bacterial conjugation

Answer 11

-bacteria can pass their plasmid on to other bacteria by bacterial conjugation

Question 12

plasmid conditions for successful transformation

Answer 12

1. a host into which DNA can be inserted
2. a means of carrying the DNA into the host
3. a method for selecting & isolating the successfully transformed organisms

Question 13

what’s carried on the plasmid?

Answer 13

-amp= ampicillin resistance gene
-betagalactosidase gene= turns bacteria blue

Question 14

result 1: no change = control

Answer 14

bacteria on the control plate (no antibiotic & no X-gal) will grow beige “lawn”

Question 15

result 2: antibiotic resistance

Answer 15

-amp
-this gene codes for a protein that confers antibiotic resistance
-it will survive if exposed to antibiotics

Question 16

result 3: turning bacteria blue

Answer 16

-betagalactosidase gene
-this gene codes for an enzyme that breaks down the X-Gal substance to make Indo Blu pigment

Question 17

calcium chloride

Answer 17

neutralizes negative charge of DNA

Question 18

ampicillin

Answer 18

antibiotic designed to kill any bacteria that lack the plasmid

Question 19

X-gal

Answer 19

the sugar that is present in order for the bacteria to express the blue color

Question 20

luria broth

Answer 20

-a nutritionally rich medium given to E.Coli after they have been stressed with temperature changes
-used as a food source

Question 21

transformation

Answer 21

bacteria take up plasmid DNA from their environment

Question 22

LB/AMP/X+ plate

Answer 22

-the purpose is to grow only transformed colonies which express the blue color (protein)
-colony count should be greatly reduced since only a few colonies contain the plasmid
-plasmid in presence of XGAL turned transformed (AMP resistance) bacteria blue

Question 23

LB+ plate

Answer 23

-the purpose is to show that the E.Coli are viable & healthy after the transformed procedures
-all bacteria should grow & express white color
-this is a positive control for LB/A/X+ plate
-the bacteria will contain the plasmid
-have a lawn of bacteria; this proves that the bacteria are capable of growing on the agar & there was nothing preventing growth besides the ampicilllin

Question 24

LB- plate

Answer 24

-the purpose is to show that the E.Coli are viable & healthy after the transformation procedures
-all bacteria should grow & express white color
-this is the positive control for the LB/A/X- plate
-the bacteria will NOT contain plasmid
-also proves that bacteria is capable of growing on agar & nothing preventing growth beside ampicillin

Question 25

LB/AMP/X- plate

Answer 25

-the purpose is to show that without the plasmid, the bacteria can not grow in the presence of ampicillin
-bacteria killed by AMP so no transformed bacteria to turn blue

Question 26

LB/AMP-

Answer 26

-no growth

Question 27

LB/AMP+

Answer 27

-small growth
-this shows that the bacteria was transformed & developed a resistance to ampicillin