Lab 24: pGLO (transformation) Flashcards
what is transformation?
uptake of naked DNA by competent cells
what is competence?
the ability of cells to take up “naked” DNA
what are two types of competence?
-natural: eg. Haemophilus influenzae, Neisseria gonorrhea
-artificial: via heat shock or electroporation
what are plasmids?
extrachromosomal DNA elements
what is the purpose of this lab? what are we trying to create?
-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!
what are the new genes that we are trying to insert into E. coli?
- bla gene
- GFP gene
- araC gene
what is the bla gene?
bla gene: confers ampicillin (an antibiotic). It is an antibiotic-resistant gene. (bla = beta-lactamase enzyme).
what is the GFP gene?
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.
what is the araC gene?
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
is e. coli naturally competent?
no
what does pGLO stand for?
plasmid GLO (glo bc it has a fluorescent gene)
pGLO gene regulation:
what happens when arabinose is available?
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.
is PBAD inducible or repressible?
inducible
in the first step of pGLO gene regulation, transcription is usually turned off by what?
transcription is usually turned off by the AraC protein
when arabinose is present, the AraC protein does what?
when arabinose is present, the AraC protein changes shape and allows RNA polymerase to transcribe the 3 genes.
in the pGLO experiment, the PBAD promoter has been attached to the ___ gene, so that it controls the expression of the GFP protein
GFP
what will we use in the lab to introduce the pGLO plasmid into the host E. coli cells?
artificial transformation - heat shock
what will we do in the lab after we heat shock the E. coli cells?
-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.
do all E. coli cells take up the plasmid?
no, not all E. coli cells take up the plasmid - transformation is not 100% efficient
how can we tell, by looking at the plates, if E. coli cells took up the plasmids?
if the plate has growth and glows
transformed bacteria that take up the pGLO plasmid express what gene, and how can it be “selected for”?
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
ampicillin in the agar does what to bacteria that do not take up the pGLO plasmid?
Ampicillin in the agar kills any bacteria that did not take up the pGLO plasmid?
transformed bacteria that take up the pGLO plasmid also express what gene?
transformed bacteria that take up the pGLO plasmid also express the GFP gene, making a green fluorescent protein that glows green under UV light.
how do we look for green glowing when the GFP gene is expressed?
by shining a UV light on the colonies and look for green glowing
when is glowing only see??
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.
what two plates did we use in lab?
-Control plates (without pGLO plasmids)
-Transformation plates (with pGLO plasmids)
what are the 3 different agar options we used in lab?
-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
If there is no arabinose in the agar, is the PBAD on or off? Is GFP made?
Arabinose in agar: NO
PBAD: OFF
GFP made? NO
If there is arabinose in the agar, is the PBAD on or off? Is GFP made?
Arabinose in agar: YES
PBAD: ON
GFP MADE? YES
In the LB plate: what will grow
E. coli (-pGLO)
E. coli (+pGLO)
In the LB plate:
E. coli (-pGLO): YES, GROWTH
E. coli (+pGLO): YES, GROWTH
In the LB + AMP plate: what will grow
E. coli (-pGLO)
E. coli (+pGLO)
In the LB + AMP plate:
E. coli (-pGLO): NO GROWTH
E. coli (+pGLO): YES, GROWTH - NO GLOW
In the LB + AMP + Ara plate: what will grow
E. coli (-pGLO)
E. coli (+pGLO)
In the LB + AMP + Ara plate:
E. coli (-pGLO): NO GROWTH
E. coli (+pGLO): YES, GROWTH - YES, GLOW
In the LB plate, why does E. coli grow both without (-pGLO) and with (+pGLO)?
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.
In the LB + Amp, why does E. coli not grow without (-pGLO)?
There is no growth because ampicillin in agar kills bacteria without pGLO plasmid.
In LB + Amp, why does E. coli grow but not glow?
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.
In LB + Amp + Ara, why does E. coli not grow without (-pGLO)?
There is no growth because the ampicillin in the agar kills the bacteria without pGLO plasmid.
In LB + Amp + Ara, why does E. coli grow AND glow with (+pGLO)?
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.
what is -pGLO in the experiment?
without the plasmid glow, this is our control
In our results, what is the only plate that glew?
LB + Amp + Ara
