GENETIC ENGINEERING LABORATORY

Group 1

Laboratory 5. Bacterial transformation.

Abstract
On this practical bacterial transformation and electrophoresis was performed.
Transformation consists on the insertion of a gene in order to give an organism a particular
trait (change caused by genes). After the bacterial transformation is done, the single protein
"band" will be visualized as well as the phenotypic properties of a protein (fluorescent trait).
The procedure to be followed will be the pGLO transformation protocol, then, the GFP
fluorescence will be examined qualitatively on agar plates, and finally, the protein on
polyacrylamide gels will be identified. The objectives to be reached on this practical were to
become familiar with techniques required for bacterial transformation, to understand the
principle for bacterial transformation and the importance of plasmids for GE, to analyse the
effectiveness of bacterial transformation using the pGLO plasmid, to learn about protein
conformations and how different conformations can be identified using electrophoretic
techniques, to understand how proteins are separated during gel electrophoresis, and to
construct a standard curve and determine the molecular weight of an unknown protein. The
results that were expected were bacterial growth on -pGLO with LB and fluorescent bacterial
growth on +pGLO in LB/Amp/Ara.
Introduction
Genetic Information is used in many areas of biotechnology. Transformation literally
means "change caused by genes", and consists on the insertion of a gene into an organism
in order to change the organism's trait. On the other hand, a gene is a piece of DNA which
provides the instructions to codify a protein. This protein will give the organism a particular
trait. Biotechnologists are able to intentionally mutate a cell's DNA as a way to change it's
behavior using the ability to transfer DNA from one organism into another and make it
function there. This can make a cell produce novel protein products that the cells did not
make previously. (AP Biology Manual, 2001).
Transformation is the process by which foreign DNA is introduced into a bacteria
through plasmids, with the objective of sorting and replicating plasmids. Because of this,
nearly all plasmids carry both a bacterial origin of replication and an antibiotic resistance
gene for use as a selectable marker in bacteria. (AddGene, 2016).
Gene transfer in higher plants and animals are complex, however, the techniques of
gene transfer in Escherichia coli bacteria are simple, mainly because bacterial plasmidbased genetic transformation that makes genetic manipulation easier. (AP Biology Manual,
2001).

Image 1. Bacterial transformation http://media.collegeboard.com/digitalServices/pdf/ap/biomanual/Bio_Lab8-BiotechnologyBacterialTransformation.pdf

Flow diagram

DNARNAPROTEINTRAIT

Materials and equipment

1.E. coli strain K-12

9. Inoculation loops

2.Plasmid(pGLO),
lyophilised

10.
Petri
(coloured)

3.Ampicillin,
lyophilised

11.
Multicolor 19.Permanent
microcentrifuge
markers
tubes

27. Mini-PROTEAN
Tetra cell

4.L(+)Arabinose,
lyophilised

12. Foam micro test 20.Distilled water

tube holders

28. Power supply

5.Transformation
Solution

13. Hot plate

21.DTT

6.LB nutrient broth

14.UV pen light

22.Protein
Kaleidoscope
standards

7.LB nutrient agar

15.Water bath
Thermometer

& 23.BioSafe
Coomassie stain

8. Pipettes

16. 1 L flask

Results

17. 500 ml graduate 25. Laemmli buffer

cylinder

dishes 18.Micropipette 2-20 26. Mini-PROTEAN

l
TGX precast gel

24. TGS buffer

Image 2. This image shows the different growth of E. Coli with the plasmid and some other variants as ampicillin
and Arabinose. The fourth plate must shine but it does not because a bad temperature shock.

Image 3. In order to answers the questions the results of the team 3 of the laboratory are going to be observed.

1. Carefully observe and draw what you see on each of the four plates. Put your
drawings in the data table below. Record your data to allow you to compare
observations of the + pGLO cells with your observations for the non-transformed E.
coli. Write down the following observations for each plate.
2. How much bacterial growth do you see on each plate, relatively speaking?
LB Agar with pGLO- shows spread bacterial growth, while LB + Ampicillin with pGLOshows no growth. On the other side, the pGLO+ plates show colonial bacterial growth. The
LB+Ampicilline plate has a growth of X colonies, while the LB+Arabinose+Ampicillin plate
has a growth of X fluorescent colonies.
3. What colour are the bacteria?
LB Agar with pGLO- has a yellowish color on the growth, but with
consistency. On the other hand, pGLO+ plates have a clearer view of the grown
and with a more brilliant color. The growth in the LB+Ampicilline plate has white
while the growth in LB+Arabinose+Ampicillin plate has white colonies that
fluorescent under UV light.

a blurry
colonies,
colonies,
become

4. How many bacterial colonies are on each plate? (count the spots you see).

-pGLO LB has irregular bacterial growth, difficult to calculate because its spread, and
-pGLO
LB/AMP
has
no
growth.

+pGLO LB/amp has colonial growth, but the number of colonies is lower than the +pGLO
LB/amp/ara.
5. Which of the traits that you originally observed for E. coli did not seem to become
altered? In the space below list these untransformed traits and how you arrived at this
analysis for each trait listed. (Original trait & Analysis of observations)
The bacteria remained white since it was untransformed and after transformation.
Also, the size of the colonies remained the same before and after the transformation.
6. Of the E. coli traits you originally noted, which seem now to be significantly
different after performing the transformation procedure? List those traits below and
describe the changes that you observed. (New trait & Observed change)
Fluorescence in the fourth growth and the way how the bacteria grow only by
separate uniform colonies instead all together.

7. If the genetically transformed cells have acquired the ability to live in the presence
of the antibiotic ampicillin, then what might be inferred about the other genes on the
plasmid that you used in your transformation procedure?
It can be inferred that none of the other genes are the ones that makes the host
resistance to the ampicillin. Also, as the ampicillin resistance gene was expressed, the other
genes in the plasmin are going to be expressed too, but they might be not detectable if the
proteins do not change the function of the cell.
8. From the results that you obtained, how could you prove that the changes that
occurred were due to the procedure that you performed?
It can be proved because the different plates simulate steps. Naturally, the bacteria
wouldnt live in a media with ampicillin and arabinose. After the transformation, the bacteria
becomes resistant to the media and is able to survive and grow at this conditions. If the
transformation hadnt been done, the bacteria wouldnt survive.
9. What are the two possible sources of fluorescence within the colonies when
exposed to UV light? The LB agar or the Arabinose
Explain:
Recall what you observed when you shined the UV light onto a sample of original
pGLO plasmid DNA and describe your observations.
Which of the two possible sources of the fluorescence can now be eliminated?
LB agar
What does this observation indicate about the source of the fluorescence?

We rule out the LB agar because all the plates have the LB agar but not all are
glowing. And just in one plate where the LB agar+amp+arabinose+pGLO were present it
glows. So we already know that the amp is an antibiotic who allow the grow of a specific
colony of bacteria and the last option which can make it glow is the arabinose.
Describe the evidence that indicates whether your attempt at performing a genetic
transformation was successful or not successful.
The transformation was successful because in the plate with only amp+LB agar there
was not growth because of the lack of the plasmid, however in the plates with
LB+amp+pGLO and LB+amp+pGLO+Arabinose there was a growth of E. Coli because the
plasmid allows the bacteria be resistant against the antibiotic.
11. Look again at your four plates. Do you observe some E. coli growing on the LB
plate that does not contain ampicillin or arabinose?
Yes, the LB plate shows spread yellowish bacterial growth. The bacterias are not
organized in colonies, but the streak of the medium is clearly seen in the bacterial growth.
12. From your results, can you tell if these bacteria are ampicillin resistant by looking
at them on the LB plate? Explain your answer.
No, because to know if a bacteria is resistant to the ampicillin, this antibiotic must be
added to the plate in order to observe if the bacteria grow. If this happens, it is demonstrated
that the ampicillin resistance gene is being expressed because of the presence of ampicillin.
13. How would you change the bacterias environmentthe plate they are growing on
to best tell if they are ampicillin resistant?
To assure that the bacteria is ampicillin resistant, this antibiotic must be added to the
plate media. If the antibiotic is not present on the plate, it cant be assured that the bacteria
is resistant. If the bacteria survives and grows in a media with antibiotic presence, it means
that the bacteria effectively is resistant. Ampicillin must be added to the media to prove that
the bacteria is ampicillin resistant.
14. Very often an organisms traits are caused by a combination of its genes and its
environment. Think about the green colour you saw in the genetically transformed
bacteria:
What two factors must be present in the bacterias environment for you to see the
green colour? (Hint:one factor is in the plate and the other factor is in how you look at
the bacteria).
The arabinose in the plate and a UV light to be seen.
What do you think each of the two environmental factors you listed above are doing
to cause the genetically transformed bacteria to turn green?

The pGLO have a gene that produce a fluorescent protein(GFP) in presence of the
sugar arabinose and the UV light is necessary because the bacteria glow with this beams.
What advantage would there be for an organism to be able to turn on or off particular
genes in response to certain conditions?
It will become easier for the organism to adapt in new environments. Also, it will
increase the organisms survival rate. With the ability to turn on or off particular genes, an
organism can manipulate its own vital needs

Finally, for the calculation of the efficiency the appendix of Lesson 4 procedure was
developed. The number of fluorescent ufc calculated was 192, and the total amount of pGLO
plasmid DNA was .8 microliters. To determine the fraction of pGLO plasmid DNA that got
spread onto the LB/amp/ara plate, the following formula was used:
Fraction of DNA used = Volume spread on LB/amp plate (l)
Total sample volume in test tube (l)
Fraction of DNA used= 100 microliters/510 microliters= 0.196
pGLO DNA spread in g = Total amount of DNA used in g x fraction of DNA used
pGLO DNA spread in g=99.96 microliters
The transformation efficiency was calculated with the following formula:
Transformation efficiency = Total number of colonies growing on the agar plate
Amount of DNA spread on the agar plate (in g)
Transformation efficiency= 192/99.96 = 1.92 ufc/microliters

Discussion
During this practical, the main educative objectives were successfully met, the team
understood the main steps and theory about the bacterial transformation; thats why the
techniques of gene transfer in Escherichia coli (E. coli) bacteria are simple and appropriate
for the teaching and learning laboratory (Rapoza and Kreuzer 2004).
As was mentioned before, genetic transformation occurs when a cell takes inside and
expresses a new piece of genetic material, DNA in this case, this new genetic information
provides the organism with a new trait which is identifiable after transformation (Bio-Rad,
n.d.). The kit of work for this practical included the pGLO plasmid that contains the gene for
GFP and a gene for resistance to the antibiotic ampicillin; pGLO also incorporates a special
gene regulation system that can be used to control expression of the fluorescent protein in
transformed cells.

Once all the steps was successfully realized, the final step was see the results of the
transformation. It was a surprise to realize that none of the transformed bacteria grew;
analyzing the process, it was clear that the only possible cause of this results was a mistake
during the process of making the cells competent. To effect transformation of E.coli, the cells
need to be made competent, this is achieved by shocking the cells in ice-cold solution of
calcium chloride , then the transformation of competent cells is carried out by mixing the
plasmid DNA with the cells (Nicholl, 2008), during the process heat shock is used to
increase the bacterial uptake of foreign DNA by increasing the permeability of the cell
membrane, also important is the rapid temperature change and the duration of the heat
shock; here is where the mistake was made, because the team didnt realized the heat
shocks in exactly the indicated time. This mistake produced that the plasmid do not get into
the bacteria, so it did not give the necessary resistance to grow in the medium with
antibiotic.
One clear example the production of insulin from bacteria; a healthy human gene for the
hormone insulin can be put into bacteria, under the right conditions, these bacteria can make
authentic human insulin; this insulin can then be used to treat patients with the genetic
disease, diabetes, because their insulin genes do not function normally (Bio-rad), saving
millions of lives every year. This is just one of many examples that represent the huge
importance of this techniques.

Conclusions
The results of the pratical shows a bad performance of the bacterial transformation
because a carelessness in the process, specifically during the heat shock, in which the
proper times of heat and cold wasn't exactly done. This results on a inhibition of the growth
of the bacteria in the antibiotic plates because the plasmid wasn't incorporated into the
bacteria, which produces a lack of resistance to the antibiotic and therefore no bacteria shine
in the Ultraviolet (UV) Lamp.

References
AddGene. (2016). Bacterial Transformation. [online] Available at:
https://www.addgene.org/plasmid-protocols/bacterial-transformation/
[Accessed Oct. 17, 2016]
AP Biology Manual (2001). Biotechnology: Bacterial Transformation. [online] Available at:
http://media.collegeboard.com/digitalServices/pdf/ap/bio-manual/Bio_Lab8BiotechnologyBacterialTransformation.pdf
[Accessed 15 Oct. 2016].
Bio-Rad. (n.d.). pGLO Bacterial Transformation Kit. [online] Available at: http://www.biorad.com/webroot/web/pdf/lse/literature/1660033.pdf [Accessed 15 Oct. 2016].
Nicholl, D. (2008). An introduction to genetic engineering. Cambridge: Cambridge University
Press.