Lab 24: pGLO (transformation)

Question 1

what is transformation?

Answer 1

uptake of naked DNA by competent cells

Question 2

what is competence?

Answer 2

the ability of cells to take up “naked” DNA

Question 3

what are two types of competence?

Answer 3

-natural: eg. Haemophilus influenzae, Neisseria gonorrhea

-artificial: via heat shock or electroporation

Question 4

what are plasmids?

Answer 4

extrachromosomal DNA elements

Question 5

what is the purpose of this lab? what are we trying to create?

Answer 5

-We will insert the pGLO plasmid with NEW GENES into E. coli cells and see if they express those new genes.
-We are trying to create recombinant organisms!

Question 6

what are the new genes that we are trying to insert into E. coli?

Answer 6

1. bla gene
2. GFP gene
3. araC gene

Question 7

what is the bla gene?

Answer 7

bla gene: confers ampicillin (an antibiotic). It is an antibiotic-resistant gene. (bla = beta-lactamase enzyme).

Question 8

what is the GFP gene?

Answer 8

GFP gene: codes for Green Fluorescent Protein (GFP - a jellyfish protein that glows green under UV light). It makes the cell that engulfs pGLO, glow.

Question 9

what is the araC gene?

Answer 9

araC gene: codes for a regulatory protein, AraC, that binds to the GFP promoter, called PBAD, and switches it ON/OFF in the presence/absence of arabinose sugar

Question 10

is e. coli naturally competent?

Answer 10

no

Question 11

what does pGLO stand for?

Answer 11

plasmid GLO (glo bc it has a fluorescent gene)

Question 12

pGLO gene regulation:
what happens when arabinose is available?

Answer 12

when arabinose is available, it binds to the araC gene. Then, PBAD turns the GFP gene on. This then makes GFP, since transcription can now occur. Now, E. coli (or any cell that uptakes plasmid) will glow.

Question 13

is PBAD inducible or repressible?

Answer 13

inducible

Question 14

in the first step of pGLO gene regulation, transcription is usually turned off by what?

Answer 14

transcription is usually turned off by the AraC protein

Question 15

when arabinose is present, the AraC protein does what?

Answer 15

when arabinose is present, the AraC protein changes shape and allows RNA polymerase to transcribe the 3 genes.

Question 16

in the pGLO experiment, the PBAD promoter has been attached to the ___ gene, so that it controls the expression of the GFP protein

Answer 16

GFP

Question 17

what will we use in the lab to introduce the pGLO plasmid into the host E. coli cells?

Answer 17

artificial transformation - heat shock

Question 18

what will we do in the lab after we heat shock the E. coli cells?

Answer 18

-We will then spread the E. coli cells evenly on some agar plates and incubate 24-48 hours at 37 degrees Celsius.
-We then assess if E. coli took in the pGLO plasmid or not.

Question 19

do all E. coli cells take up the plasmid?

Answer 19

no, not all E. coli cells take up the plasmid - transformation is not 100% efficient

Question 20

how can we tell, by looking at the plates, if E. coli cells took up the plasmids?

Answer 20

if the plate has growth and glows

Question 21

transformed bacteria that take up the pGLO plasmid express what gene, and how can it be “selected for”?

Answer 21

transformed bacteria that take up the pGLO plasmid express the ampicillin resistance bla gene and therefore can be “Selected for” by growing on an agar plate with ampicillin

Question 22

ampicillin in the agar does what to bacteria that do not take up the pGLO plasmid?

Answer 22

Ampicillin in the agar kills any bacteria that did not take up the pGLO plasmid?

Question 23

transformed bacteria that take up the pGLO plasmid also express what gene?

Answer 23

transformed bacteria that take up the pGLO plasmid also express the GFP gene, making a green fluorescent protein that glows green under UV light.

Question 24

how do we look for green glowing when the GFP gene is expressed?

Answer 24

by shining a UV light on the colonies and look for green glowing

Question 25

when is glowing only see??

Answer 25

Glowing is only seen if the arabinose sugar is present in the agar because the GFP gene has been attached to promoter, PBAD, and only turns on IF arabinose is in the agar.

Question 26

what two plates did we use in lab?

Answer 26

-Control plates (without pGLO plasmids)
-Transformation plates (with pGLO plasmids)

Question 27

what are the 3 different agar options we used in lab?

Answer 27

-LB: Luria-Bertani nutrient agar (a medium on which E. coli grows well).
-LB + amp: LB agar with ampicillin in the agar.
-LB + amp + ara: LB agar with ampicillin and arabinose sugar in the agar

Question 28

If there is no arabinose in the agar, is the PBAD on or off? Is GFP made?

Answer 28

Arabinose in agar: NO
PBAD: OFF
GFP made? NO

Question 29

If there is arabinose in the agar, is the PBAD on or off? Is GFP made?

Answer 29

Arabinose in agar: YES
PBAD: ON
GFP MADE? YES

Question 30

In the LB plate: what will grow
E. coli (-pGLO)
E. coli (+pGLO)

Answer 30

In the LB plate:
E. coli (-pGLO): YES, GROWTH
E. coli (+pGLO): YES, GROWTH

Question 31

In the LB + AMP plate: what will grow
E. coli (-pGLO)
E. coli (+pGLO)

Answer 31

In the LB + AMP plate:
E. coli (-pGLO): NO GROWTH
E. coli (+pGLO): YES, GROWTH - NO GLOW

Question 32

In the LB + AMP + Ara plate: what will grow
E. coli (-pGLO)
E. coli (+pGLO)

Answer 32

In the LB + AMP + Ara plate:
E. coli (-pGLO): NO GROWTH
E. coli (+pGLO): YES, GROWTH - YES, GLOW

Question 33

In the LB plate, why does E. coli grow both without (-pGLO) and with (+pGLO)?

Answer 33

Because the LB, Luria-Bertani nutrient agar, is a medium that allows E. coli to grow well on. There is solid growth (a lawn), since there is nothing in the agar to inhibit growth.

Question 34

In the LB + Amp, why does E. coli not grow without (-pGLO)?

Answer 34

There is no growth because ampicillin in agar kills bacteria without pGLO plasmid.

Question 35

In LB + Amp, why does E. coli grow but not glow?

Answer 35

Colonies grew, however, it was not a lawn because transformation is not 100% efficient.
There is no glow, since there is no arabinose in the agar. When there is arabinose in the agar, the PBAD turns on, making GFP, which makes the glow.

Question 36

In LB + Amp + Ara, why does E. coli not grow without (-pGLO)?

Answer 36

There is no growth because the ampicillin in the agar kills the bacteria without pGLO plasmid.

Question 37

In LB + Amp + Ara, why does E. coli grow AND glow with (+pGLO)?

Answer 37

Colonies grow, but there is not a full lawn since transformation is not 100% efficient.
They DO GLOW green since arabinose is in the agar.

Question 38

what is -pGLO in the experiment?

Answer 38

without the plasmid glow, this is our control

Question 39

In our results, what is the only plate that glew?

Answer 39

LB + Amp + Ara