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Genetic Transformation Using pGLO.

Groups 7-10
RESULTS:
1. Draw, write your observations, and rationale on each of the 4 plates.
Time of checking: 12nn, Feb. 11 (Wed)
+pGLO/LB/amp

OBSERVATIONS

RATIONALE

(+) growth: 2 small


colonies
(-) glow

Ideal: (+) growth (-) glow


(+) growth, because the binding of pGLO plasmid should
allow the bacteria to express the gene of amp resistance.
(-) glow, because despite the binding of pGLO, without ara
the expression of pGLO fluorescence gene via GFP cannot
be expressed.
Result: same with ideal
Ideal: (+) growth (+) glow
(+) growth, because the binding of pGLO plasmid should
allow the bacteria to express the gene of amp resistance.
(+) glow, because the binding of pGLO plasmid, activated by
the presence of ara, should allow the bacteria to glow under
UV light.

(-) growth, (-) glow

Result: (-) growth, (-) glow


(-) growth possibly due to:

Bacteria settled at the bottom of the bottom of the


test tube (inadequate mixing)

Failure to pipet adequate number of bacteria onto


corresponding plate (pipetting errors)

Plate was not sealed properly and moisture


affected growth

The group finished the experiment around 5PM,


and was asked to check the results at 12NN the next
day (19 hours). Given that it takes 18-24 hours for E.
coli to grow, this may explain why growth was not
observed.

(-) glow possibly due to:

Bacteria settled in the bottom of test tube (inadequate


mixing), therefore failure to pipet adequate number of
bacteria onto corresponding plate

(-) growth, (-) glow

(+) growth, (-) glow

no growth = no bacteria to express glow.


Ideal: (-) growth, (-) glow
(-) growth, because without pGLO, there was no gene to
express amp resistance in the bacteria. This causes the
bacteria to be killed by amp.
(-) glow, because the bacteria were killed, and also because
there was no pGLO present to express fluorescence via
GFP.
Result: same with ideal
Ideal: (+) growth, (-) glow
(+) growth, because although the bacteria is not amp
resistant due to absence of pGLO, there was also no amp
added to kill them. Thus, resulting in normal growth.
(-), because there was no pGLO present to express
fluorescence via GFP.
Result: same with ideal

+pGLO/LB/amp/ara

-pGLO/LB/amp

-pGLO/LB

= growth

= glow

2. Which of the traits that you originally observed for E. coli did not seem to become altered?
Which traits seem now to be significantly different after performing the transformation
procedure?
Before transformation, E. coli cultures that grew in the source plate are observed to be opaque, white and
in streaks. This means that there was (+) growth in the E. coli culture source without ampicillin. It is,
however, not amp resistant, and does not glow under UV light.
Ideally, in the presence of arabinose, E. coli should bind to the pGLO plasmid, causing it to express GFP
which allows the bacteria to fluoresce under UV light. It also allows E. coli to be resistant to ampicillin,
thus growing despite being exposed to ampicillin.
3. What evidence suggests that the changes were due to the transformation procedures?
There are two ways to check whether or not the transformation procedure led to any changes in the
bacteria.
First is to subject the bacteria to UV light. In the presence of arabinose, pGLO should allow the bacteria to
be able to glow green or fluoresce.
Second is to subject the bacteria to ampicillin. pGLO causes the bacteria to be resistant to ampicillin, thus
preventing it from dying in the presence of ampicillin.
4. Compare your observation when you shined the UV light onto the original pGLO plasmid
DNA vs the plate with pGLO plasmid. From this observation, what indicates the source of
fluorescence?
None of the groups were able to perform this step, because it was not stated in the procedure, and also
the UV light was not provided at the time.
However, ideally, if UV light were shined on the original pGLO plasmid DNA, nothing will happen (it wont
glow). The reason for this is that the DNA must first be encoded (transcribed then translated) into the
GFP, w/c causes the glow.
5. Was your experiment on performing a genetic transformation successful or not? Explain.
The groups E. coli genetic transformation was not successful for +pGLO and +ara, because results
showed that the E. coli did not grow or glow despite presence of both pGLO and arabinose.
(-) growth possibly due to:

Bacteria settled in the bottom of the bottom of the test tube (inadequate mixing)

Failure to pipet adequate number of bacteria onto corresponding plate (pipetting errors)

The group finished the experiment around 5PM, and was asked to check the results at 12NN the
next day (19 hours). Given that it takes 18-24 hours for E. coli to grow, this may explain why growth
was not observed
(-) glow possibly due to:
Bacteria settled at the bottom of test tube (inadequate mixing), therefore failure to pipet adequate
number of bacteria onto corresponding plate, thus no growth, no bacteria to express glow.
*Other conditions were standardized for all plates and therefore could not be attributed for the failure of
growth in +pGLO/LB/amp/ara.

*Failure to obtain a colony of bacteria from starter plate could not be a source of error because there are
2 +pGLO plates and +pGLO with transformation solution came from a single test tube, however,
+pGLO/LB/amp showed positive growth.
However, the +pGLO and ara plate showed successful genetic transformation, because there was
(+) growth in the presence of ampicillin.

GUIDE QUESTIONS:
1. What 2 factors must be present in the bacteria to see the green color/fluorescence?
Arabinose sugar in the agar plate is needed to turn on the expression of the gene for GFP (green
fluorescent protein) (Bio-Rad).
UV light is needed for the visualization of the expression of the GFP, causing the bacteria to
fluoresce (Bio-Rad).
2. How do these 2 factors cause genetically transformed bacteria to turn green?
The pGLO plasmid is composed of the genes for GFP and resistance to the antibiotic ampicillin. pGLO
also has a special gene, araC, which contributes as a gene regulation system by controlling the
expression of the green fluorescent protein in transformed bacterial cells. The addition of the sugar
arabinose to the transformed cells will activate the gene for GFP, thereby turning the said gene on.
Arabinose does this by binding to the regulatory protein, araC, located on the P promoter. Binding causes a
conformational change of the araC which facilitates transcription of the gene by RNA polymerase. When
exposed to UV light, the electrons in GFPs chromophere are excited to a higher energy state. When they
drop down to a lower energy state they emit a longer wavelength of visible fluorescent green light at 509
nm.
Selection for cells that have been transformed with pGLO DNA is accomplished by growth on antibiotic
plates. Transformed cells will appear white (wild-type phenotype) on plates not containing arabinose, and
fluorescent green when arabinose is included in the nutrient agar (Bio-Rad).
3. What advantage would there be for an organism to be able to turn particular genes on or
off in response to certain conditions?
BAD

The ability to turn particular genes on and off is called gene regulation. This is an important mechanism
that allows adaptation to differing conditions and prevents wasteful production of unnecessary proteins.
This is exemplified well by genes that code for enzymes that participate in carbohydrate catabolism.
Therefore, as in the experiment, if the sugar arabinose is present in the growth medium, the bacteria are
able to beneficially produce the enzymes needed for sugar break down. On the other hand, if arabinose is
absent in the nutrient media, enzyme production for such processes would be very energetically wasteful
(Bio-Rad).