lab 7

Question 1

what is competency?

Answer 1

Bacterial cells must be in a particular physiological state before they can be transformed. This state is referred to as competency. Competency can occur naturally in some species under certain conditions.

Question 2

In nature, what is the source of external DNA for bacteria?

Answer 2

In nature, the source of the external DNA is from other cells that have died and lysed.

Question 3

what do Bacillus subtilis do to acquire foreign DNA?

Answer 3

take up DNA molecules present in the soil surroundings as a result of the death and breakdown of other bacteria, and they pull the DNA through their cell membrane to the inside of the cell

Question 4

what is the difference between bacteria acquiring foreign DNA in nature and in lab

Answer 4

nature: source of external DNA is from other cells that have died and lysed

lab: source of DNA is plasmid

Question 5

why cant all eukaryotic proteins be expressed in bacteria? what is a solution to this?

Answer 5

due to the lack of post-transcription and translation modification in prokaryotic cells, not all eukaryotic proteins can be expressed in bacteria.

solution: many eukaryotic proteins are made in bacteria, which have been transformed with recombinant plasmids carrying eukaryotic genes.

Question 6

true or false, e coli enters a state of competency naturally.

Answer 6

false, it must be induced

Question 7

how can we induce competency for e-coli?

Answer 7

when treated with chloride salts of the metal cations calcium, magnesium, and rubidium. Calcium chloride is the most commonly used salt. In addition, sudden cycles of heat and cold help to bring about competency.

Question 8

what is the most commonly used salt to induce competency for e-coli?

Answer 8

calcium chloride

Question 9

how does treating e-coli with chloride salts and making it go through hot-cold cycles induce competency?

Answer 9

affect the structure and permeability of the cell wall and membrane so that DNA molecules can pass through.

Question 10

why is the method by which e-coli uptakes DNA unknown?

Answer 10

-Unlike salts and small organic molecules such as glucose, DNA molecules are too large to diffuse or be easily transported through the cell membrane.

• Some bacteria possess membrane proteins that recognise DNA and facilitate the uptake of small pieces, but E. coli does not have this property.

Question 11

how do we suspect that e-coli takes in DNA?

Answer 11

It is suspected that in E. coli DNA molecules pass through any of several hundred of adhesion zones where the outer and inner cell membranes of E. coli are fused to pores in the bacterial cell wall

Question 12

what are adhesion zones?

Answer 12

where the outer and inner cell membranes of E. coli are fused to pores in the bacterial cell wall

Question 13

what is the main factor preventing DNA from diffusing through the cell wall of e-coli?

Answer 13

presence of negatively charged phospholipids in the cell membranes, since DNA is also negatively charged

Question 14

how can we solve the problem of the DNA and the cell wall having the same charge, which prevents DNA from entering?

Answer 14

It is suspected that treatment of the cells at 0 degrees C crystallises the fluid cell membrane, stabilising the distribution of charged phosphates.

Cations form complexes with exposed phosphate groups, shielding the negative charges, making passage of DNA possible.

Heat shock appears to facilitate the movement by creating a thermal imbalance on either side of the membrane, physically helping to move the DNA through the adhesion zone

Question 15

what is transformation efficiency?

Answer 15

The amount of cells transformed per 1 μg of DNA

Question 16

what is the procedure of selection?

Answer 16

plated on agar media that would allow only the cells that acquired the DNA to grow

Question 17

what’s a colony?

Answer 17

a colony is a group of identical cells derived from a single cell which underwent many divisions

each colony originally grew from ONE transformed cell

Question 18

true or false. transformation is ways 100 percent effective

Answer 18

false

Question 19

in this lab, which plasmid is getting introduced into e-coli?

Answer 19

pGLO plasmid.

Question 20

what strain of the pGLO plasmid is used?

Answer 20

HB101

Question 21

what are the interesting features of the pGLO plasmid?

Answer 21

-origin of replication

-the Bla or ampicillin resistance gene which codes for β-lactamase. This is a
secreted enzyme that inactivates ampicillin and allows transformed bacteria, carrying the plasmid, to grow into colonies on ampicillin containing medium.

-the gene coding for the Green Fluorescent Protein (GFP) from the jelly fish Aequoria victoria. GFP is a protein that emits green light in the range of 500nm (509nm) when excited in the ultraviolet range (395nm). Emission is fluorescent because it has a different (higher) wavelength than excitation.

-an AraC gene which codes for the AraC arabinose repressor.

-an AraC promoter, pBAD, controlling transcription of the GFP gene. this promoter functions in the L-arabinose bacterial operon for arabinose sugar catabolism

-arabinose breakdown operon

Question 22

what is pBAD?

Answer 22

araC promoter, which is the promoter for the L-arabinose bacterial operon for arabinose sugar catabolism.

Question 23

what does the expression of the GFP gene depend on? how?

Answer 23

on the amount of arabinose. if L-arabinose is present, it binds to the araC arabinose repressor and inactivates it, which allows for transcription of the metabolic pathway to occur. since GFP is a part of this pathway, if arabinose is present, GFP will be transcribed

Question 24

why is the coding for antibacterial resistance useful for bacteria?

Answer 24

-In nature: can give them a selective advantage in an environment containing antibiotics
-in experiments: provides an easy screening technique to distinguish between transformed bacteria (carrying plasmids) and non-transformed bacteria

Question 25

how can we use antibiotic resistance in our favour in this lab?

Answer 25

we can see which bacteria took up the plasmid, cause the ones that took up the plasmid will be the only ones to grow

Question 26

what’s the antibiotic used in this lab?

Answer 26

ampicillin

Question 27

what should the colonies look like if arabinose if present on the plate?

Answer 27

After transformation, bacteria should be forming ampicillin resistant colonies that emit green fluorescent light when exposed to UV light

Question 28

What are the steps to transformation?

Answer 28

Question 29

which substance specifically inactivates ampicillin?

Answer 29

β-lactamase

Question 30

what is the bla gene?

Answer 30

ampicillin resistant gene

Question 31

what are our two controls in the experiment?

Answer 31

positive control: just our normal cells that grow to make sure our cells are viable (no plasmid, no ampicillin)

negative control: our cells, but no plasmid so when its put into ampicillin, it doesn’t grow

Question 32

for the lac operon of E-coli, what are the three genes?

Answer 32

lacZ, lacY and lacA

Question 33

which gene codes for ß-galactosidase

Answer 33

lac z

Question 34

what is ß-galactosidase

Answer 34

enzyme involved in lactose breakdown. cleaves lactose into the monosaccharides glucose and galactose

Question 35

is the lac operon by default on or off?

Answer 35

off (allolactose turns it on)

Question 36

what is IPTG?

Answer 36

ß-galactosidase can also be induced by the addition of IPTG (isopropyl- B-D-thiogalactoside) to a growing culture. IPTG is a non-metabolizable analog of the normal substrate, lactose

Question 37

what is the inducer of ß-galactosidase?

Answer 37

allolactose, or IPTG

Question 38

What are the three bacterial strains we are using (mutants). what do they each do?

Answer 38

-Lac Z+ (wild type)
-Lac Z- (cannot make the enzyme ß-galactosidase, the protein that
cleaves lactose into the monosaccharides glucose and galactose)
-Lac Zoc (has an altered operator region that does not bind lac repressor very well, thus the lac operon is active even in the absence of lactose)

Question 39

what are the 2 indicator plates used?

Answer 39

-Maconkey lactose plate
-xgal plate

Question 40

why does the gal plate have 2 subcategories of plates? what are they called?

Answer 40

-X-gal / glucose plates
-X-gal / IPTG / glycerol plates

-we need these 2 extra plates cause
Xgal plates are extremely sensitive. One molecule of ß-galactosidase is all
that is needed to cleave enough Xgal to turn the colony blue. For this reason you will use two types of Xgal plates when you assay the strains.

Question 41

what do u know ab maconkey plates?

Answer 41

 These plates contain lactose and a pH indicator.
 Colonies able to metabolize lactose (lac Z+) generate lactic acid, acidifying
the medium and maintaining a red color.
 Colonies that are unable to metabolize lactose (lac Z-) turn yellowish-white.
 MacConkey plates are not very sensitive. Approximately 1000 molecules
must be metabolized to generate enough lactic acid to turn the colony red.

Question 42

what do u know ab x-gal plates?

Answer 42

The compound Xgal (5-bromo-4-chloro-3-indoyl- ß-D-galactoside) is cleaved by the enzyme ß-galactosidase (product of the lacZ + gene) to give galactose and a blue dye, figure 6.
 Therefore lacZ + colonies turn blue, while lacZ - colonies remain white.
 Xgal plates are extremely sensitive. One molecule of ß-galactosidase is all
that is needed to cleave enough Xgal to turn the colony blue. For this reason you will use two types of Xgal plates when you assay the strains.

Question 43

what is the Xgal/glucose repressor plates?

Answer 43

By adding glucose, cAMP levels will be low and the lactose operon should not be expressed provided that the operator region is also functional.

Question 45

What is LB?

Answer 45

Lysogeny broth. It’s all the stuff that the bacteria needs to ensure proper growth (sugar, nutrients, etc.)

Question 46

Wha type of Operon is the arabinose bacterial operon?

Answer 46

Inducible

Question 47

What are the 4 plates used after our transformation experiment?

Answer 47

Question 48

Why is IPTG useful?

Answer 48

Because we have have an unlimited supply of it, since b-galactisodase does not cleave it

Question 49

How do Xgal/glycerol IPTG inducer plates lead to maximal transcription?

Answer 49

-Glycerol makes CAMP levels very high
-IPTG will never be cleaved by b-galactosidase so we have an excess

Question 50

What two questions does the x-gal plate answer?

Answer 50

Question 1:Does the strain make ß-galactosidase?

Question 2 - If the strain does make ß-galactosidase, is it constitutively expressed, or is it inducible via IPTG induction? Careful analysis of the color of the colonies on Xgal/glucose repressor plates versus Xgal/glycerol IPTG inducer plates will tell you whether the lac operon is constitutive or inducible in that strain.

Question 51

What plate is this

Answer 51

MacConkey plate

Question 52

What plate is this

Answer 52

X gal

Question 53

True or false, Lac Z- doesn’t show up in any of the plates

Answer 53

True

Question 54

Identify

Answer 54

Question 55

how many micrograms in 1 gram?

Answer 55

1000000

Question 56

which plates have LB loaded onto them?

Answer 56

all of them