6.

05 pGLO Plenary BIOCHEMISTRY

Dr. Catherine Co-Reportoso | March 20, 2017 LE 6 TRANS 5

OUTLINE  pGLO: plasmid modified with 3 genes , and contains

genetic codes for the following:
I. Introduction o GFP: green fluorescent protein for bioluminescence
A. Definition of Terms  Originally AraA, AraB, and AraD (cluster of genes.
B. Experiment Proper Aka araBAD), responsible for catabolizing arabinose
C. Significance of Experiment sugar and encoding of digestive enzymes, were
II. Methodology transcribed
A. Materials  In the experiment, transcription of AraA, AraB, and
B. Procedure: Pre-lab AraD were replaced by transcription of GFP gene
C. Procedure: Experiment Proper
III. Results and Discussion o Bla: gene encoding for β-lactamase enzyme for
A. (-) pGLO / LB ampicillin resistance
B. (-) pGLO / LB / Amp  β-lactamase is an enzyme that degrades β-lactam
C. (+) pGLO / LB / Amp class of antibiotics
D. (+) pGLO / LB / Amp / ARA  Ampicillin contains β-lactam ring which is degraded
E. Questions #2-5 by β-lactamase rendering ampicillin ineffective
IV. Guide Questions
A. GQ #1-3 o araC: gene encoding for the AraC protein that is a
B. Transformation Efficiency constituent of the special gene regulation system by
V. References control of the arabinose promoter region (or PBAD);
requires arabinose to INDUCE GFP expression
LEARNING OBJECTIVES
 MECHANISM: “if pGLO transformation is successful, and [if]
1. To perform correctly the procedure known as genetic the bacteria are growing in arabinose, the colonies will appear
transformation using pGLO plasmid DNA neon green (Animo La Salle) under UV light”
2. Evaluate the effect of pGLO plasmid in E. coli
3. Justify the observed changes due to transformation
4. Explain how the genetically transformed bacteria turned green
5. Compute for the transformational efficacy
6. Discuss importance of gene transformation

LEGEND

Remember Lecturer Book Prev. Trans Notes

    

Figure 1. pGLO Map. (ori – Origin of Replication)

I. INTRODUCTION
C. Significance of Experiment
A. Definition of Terms
 Genetic transformation is used in a lot of areas in
biotechnology
 TRANSFORMATION – transferring genetic info from one
 In MEDICINE:
organism to another (should be of different species)
o Gene therapy is now being used to treat diseases
 PLASMID – small circular DNA found in bacteria that
caused by defective genes
transfers genetic info to:
o This is done by GENETICALLY TRANSFORMING “sick
o Allow adaptation to the new environment
cells” with the healthy copies of the defective genes
o Attain antibiotic resistance
II. METHODOLOGY
B. Experiment Proper
A. Materials
 Our experiment: GENETIC TRANSFORMATION using
pGLO
 LB Nutrient Agar (Luria-Bertani)
o nutrient agar that is a general medium for microbiologic
 E. coli HB101 K2: non-pathogenic bacteria used in study
experiment o used for routine cultivation of non-fastidious organisms
o Competent bacterial cell
o Capable of in-taking the set of new genes
 Arabinose
o Sensitive to ampicillin antibiotics
o carbohydrate isolated from plants; source of food of
bacteria
o hydrated and added into the LB nutrient agar
o required for the activation of GFP gene

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 Non-pathogenic E. coli K-2 strain III. RESULTS AND DISCUSSION
o host organism in the experiment/ vector containing
the recombinant GFP protein when transformed A. (-) pGLO / LB

 Transformation solution CaCl2

o neutralizes the repulsive negative charges of the
phosphate backbone of the DNA and the phospholipids
of the cell membrane
o allows the DNA to enter the cells

 Water and Ice Bath

o Used in introducing heat shock wherein test tubes
containing the bacteria are incubated in ice for 4 mins
then transferred immediately to a water bath at 42oC for
50 seconds then returned again to the ice.
o A sudden increase in temperature creates pores in the
plasma membrane of the bacteria hence increases
bacterial uptake of foreign
DNA

 Ampicillin
o antibiotic to which the bla gene has conferred
resistance
o belongs to the β-lactam group of antibacterial thus
susceptible to β-lactamase activity of BLA gene
Figure 2. (-) pGLO/LB result.
 UV light
o confirm presence of fluorescence brought about by GFP
 (+) Growth
B. Procedure: Pre-Lab o Growth of E. coli was observed due to the absence of
ampicillin
1. Prepare agar plates days 3 days prior to the experiment o There was no penicillin to stop bacterial growth
o Starter plate
o LB plate  (-) Fluorescence
o LB/amp plate o Absence of pGLO  no GFP gene that would code for
o LB/amp/arabinose plate the GFP protein  no fluorescence

2. One day prior to the experiment: B. (-) pGLO / LB / Amp

o rehydrate the E. coli
o streak starter plates
o rehydrate pGLO plasmid DNA
o prepare LB broth aliquot solution

C. Procedure: Experiment Proper (Transformation Lab)

1. Label 2 test tubes: +pGlo and –pGlo

2. Transfer Calcium Chloride into the test tubes
o the positively charged calcium ions neutralize the
negatively charged lipid bilayer and DNA backbone,
allowing cell penetration 
3. Mix an entire bacterial colony in each test tube
4. Incubate in ice for 10 minutes
5. Heat Shock for 50 seconds in 42oC Celsius
o allows penetration of the cell membrane, allowing
bacterial uptake of the DNA plasmid 
o If heat shock is done for more than 50 seconds, the Figure 3. (-) pGLO/LB/Amp result.
transformants will decrease by 50% causing some
plates to not exhibit growth   (-) Growth
6. Incubation of both test tubes after adding 250µL of LB o Absence of pGLO  no ß-lactamase activity
broth for 10 mins. responsible for antibiotic resistance  absence of growth
o To allow the bacteria to express the bla gene and o Ampicillin was able to prevent the growth of colonies in
produce an initial amount of β-lactamase for antibiotic the plate, and there was no pGLO to resist it
resistance before being subjected to ampicillin.
o Failure to incubate after the  (-) Fluorescence
addition of LB broth will lead to zero growth of o Absence of pGLO  no GFP gene that would code for
bacteria  the GFP protein  no fluorescence
7. Streak solution onto agar plate
8. Incubate plates upside down in 37°C for 18-24 hours
o Incubation temperature is 37°C because it is the
optimum temperature for the growth of E. coli 

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C. (+) pGLO / LB / Amp
Figure 4. (+) pGLO/LB/Amp result.
 (+) Growth
o Growth is observed due to presence of pGLO plasmid,
which caused resistance to ampicillin due to the
presence of ß-lactamase activity
o Ampicillin contains β-lactam ring which is degraded by β-
lactamase rendering ampicillin ineffective
 (-) Fluorescence
o Absence of arabinose, which is needed for GFP gene
expression, caused the absence of fluorescence property
despite the presence of pGLO.
D. (+) pGLO / LB / Amp / ARA
Figure 5. (+) pGLO/LB/Amp/ARA result.
 (+) Growth
o Growth is observed due to presence of pGLO plasmid,
which caused resistance to ampicillin due to the
presence of ß-lactamase activity
 (+) Fluorescence
o Fluorescence is observed due to the presence of
arabinose, which enabled GFP expression
E. Questions #2-5
1. Draw, write your observations and rationale on each of
the 4 plates. (Refer to III. Results and Discussion  A-D)
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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?
o E. coli is a single-celled organism that reproduces
quickly, thus its transformation was evident throughout
the samples.
o Morphological characteristics of the colonies were not
altered. Before and after transformation, the colonies still
appeared as circular, raised, punctiform, and translucent
o Changed traits after performing the transformation
procedure:
 Fluorescent appearance in (+) pGLO/LB/Amp/Ara
plate
 Resistance to ampicillin in plates with (+) pGLO and
Amp
3. What evidence suggests that the changes were due to the
transformation procedures?
o Evidence is the growth itself, thus there was
transformation.
o The two plates compared would be the 2nd and the 3rd,
which were (-) pGLO/LB/Amp and (+) pGLO/LB/Amp
respectively 
 Both plates had ampicillin, but the difference
between them was that the 3rd plate had the
presence of pGLO, which caused resistance to
ampicillin because of the bla gene
o Note: The question is not asking about complete
transformation; it is asking for evidence that there was
simple transformation 
4. Compare your observation between the original pGLO
plasmid DNA and the plate with pGLO plasmid with the
use of the UV light. From this observation, what indicates
the source of fluorescence?
o The only difference is the addition of arabinose in the
plate with the (+) fluorescence, indicating that the source
of the fluorescence was the presence of arabinose
sugar
o When you shine the UV light on the original pGLO
plasmid DNA (vial), there will be no fluorescence
o Note: The original pGLO plasmid DNA refers to the one
in the vial with the original pGLO plasmid. During the
experiment, the vial should be observed with UV light 
5. Was your experiment on performing a genetic
transformation successful or not? Explain.
o Yes, because the expected results were acquired.
o (+) pGLO LB/Amp plates had growth (successful
induction of antibiotic resistance).
o Fluorescence was only present on (+) pGLO/LB/Amp/Ara
plate, indicating that the pGLO plasmid was incorporated
into the bacterial genome, as the traits of the pGLO
induced genes were expressed.
Table 1. Expected Results
Set-up Growth Fluorescence
1 (-)pGLO / LB + -
2 (-)pGLO / LB/ amp - -
3 (+)pGLO / LB/ amp + -
4 (+)pGLO / LB/ amp/ ara + +
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 IV. GUIDE QUESTIONS
B. Transformation Efficiency (TE)
A. GQ #1-3  To compute for transformation efficiency, divide the number of
successful transformants growing on the plate by the amount
1. What 2 external factors must be present to see the green of DNA used in transformation:
color/fluorescence
o Arabinose (in agar plate) Total # of cells growing on agar plate (transformants)
TE =
o UV light amount of pGLO DNA spread on agar plate (µg DNA)

2. How do these 2 factors cause genetically transformed  Numerator: Count the number of colonies
bacteria turn green? o Each colony can be assumed to have grown from a
o Arabinose single successfully transformed viable cell (colony-
 Allows expression GFP (Green Fluorescent forming units or CFU)
Protein) gene
 Arabinose will be taken up by the bacteria, then it  Denominator: You need to multiply the following
will bind to AraC leading to conformational change  1. Total amount of DNA used
 Conformational change promotes the binding of
RNA polymerase to the promoter (pBAD), which = (conc. DNA plasmid) x (initial volume)
causes the transcription of the GFP gene (originally µg
= 0.08 µL x (10µL) = 0.8 µg DNA
it will transcribe AraA, AraB, and AraD) into
messenger RNA (mRNA), followed by the 2. Fraction of DNA that was actually transferred to the agar
translation of this mRNA into GFP; this process is plate
called GENE EXPRESSION   Recall that after transformation, we did not use all of
 As they produce more and more protein, the cells the solution to grow onto LB/Amp/Ara plate
would be able to expressing GFP fluorescence: a  Divide the volume of pGLO cell solution transferred
brilliant green (100 μL) with the total volume pGLO cell solution in
 GFP gene will be turned off if there’s (-) Arabinose transformation (510 μL)
 Therefore if you transfer the bacteria in plate 3 to
100 µL 100 µL
arabinose containing media, GFP will be turned Fraction of DNA = =
250 µL CaCl2 + 250 µL Broth + 10 µL plasmid DNA 510 µL
on
 The final equation for our experiment can be computed as
follows: 
𝐂𝐅𝐔
𝐓𝐄 =
𝟏𝟎𝟎
𝟎. 𝟖 µ𝐠 𝐱
𝟓𝟏𝟎

 If a different method was used, TE can be computed using the

general equation:
colony − forming units
TE =
vol. pGLO cell sol′n transferred to agar
(conc. plasmid x vol. plasmid) x
total vol. of pGLO cell sol′n

Sample Computation: (Refer to Figure 7)

Figure 6. (+) Regulation of GFP expression by araC and Arabinose (BioRad)

o UV light
 When exposed to long wavelength of UV light, the
electrons in GFP’s chromophore are excited to a Figure 7. (+) pGLO/LB/Amp/ARA counting. 2021C group 8; CFU = 34
higher energy state. (Absorption of UV light)
 When they drop down to a lower energy state, they 34 CFU
emit a longer wavelength of visible fluorescent green TE =
µg 100 µL
(0.08 x 10 µL) ( )
light µL 510 µL

transformants transformants
3. What is the advantage for an organism to be able to turn = 216 Or = 0.216 x 103
µg DNA plasmid µg DNA plasmid
on or off particular genes in response to certain
conditions? *Note that usually TE is a very large number so it is often expressed in
o Gene regulation allows adaptation of bacterial cells scientific notation x 103.
towards different conditions.
o Prevents wasteful overproduction of unnecessary V. REFERENCES
proteins. In this way, the organism conserves energy by 1. Biochemistry Experiment 7: Genetic Transformation using pGLO
regulating the production of proteins especially those not (Manual)
needed in their current state. 2. 2021C Reporting PPT
3. 2020C Trans
4. Dr Reportoso’s Briefing PPT
5. BioRad pGLO™ Bacterial Transformation Kit lit1660033edu
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