FACULTY OF CHEMICAL ENGINEERING

GENETIC LAB ENGINEERING (CBE 561)

NAME

: RIAN AIDIL BIN MAT NASIR

MATRIC NO
GROUP
LAB NO./TITLE OF
EXPERIMENT
DATE PERFORMED
SEMESTER
PROGRAMME
LECTURER

: 2013208064
: EH2224D
: LAB 3 / BACTERIAL TRANSFORMATION

No.
1. Abstract

:
:
:
:

8-5-2015
4
EH2224D
MISS NORFAZILA BTE KHAIRUDIN

Content

Allocated Marks
5

2. Introduction

3. Objectives

4. Theory

5. Procedures

10

6. Apparatus

7. Results and Calculations (if any)

10

8. Discussions

30

9. Conclusions

10

10. Recommendations

11. References

12. Appendices

TOTAL

100

1 | Page

Marks Obtained

Remarks:
Checked by:

Date:

............
(MISS NORFAZILA BTE KHAIRUDIN)

1.0 ABSTRACT
In this activity,our group use a strain of E. coli that has been made competent to allow it to
incorporate and express a plasmid containing two genes. One gene codes for a green
fluorescent protein (GFP) and the other codes for ampicillin resistance. The source of the
GFP gene is the bioluminescent jellyfish Aequorea victoria. The ampicillin-resistance gene
allows us to select which of the E. coli cells have been transformed based on their ability to
grow in an environment that contains the antibiotic ampicillin.
This experiment was conducted to manipulate a variable present within the lab and
determine what affect this change would have on the results of the lab. This exercise exposed
on how to determine whether or not the calcium chloride transformation solution was a
critical ingredient in influencing the uptake and expression of the pGLO gene. In order to
accurately test this variable, we performed the complete pGLO lab as instructed in
the pGLO manual as a control, and then proceeded to manipulate the lab through duplicating
the lab setup, however using an altered transformation solution. The calcium chloride was
replaced with distilled water for the second set of plates, in order to compare the expression
of the pGLO gene in each case. In the end there was a total of 4 petri dishes. The first two
were prepared ahead of time, and filled with a solution containing nutrient agar solution, as
well as a combination of various solutions. These solutions included LB
broth, ampicillin and arabinose. Two of the plates, (LB/amp) and (LB/amp/ara) we covered in
a solution consisting of transformation solution, LB broth and pGLO plasmid DNA. The
other two, (LB/amp) and (LB) were covered in a solution consisting of only the
transformation solution and LB broth. These four plates acted as our control. We anticipated
that the LB/amp/ara plate containing the pGLO plasmid DNA would become fluorescent.
2 | Page

This result is expected because in theory, the transformation solution is an essential step for
giving the bacteria the ability to take up the pGlo plasmid, since the solution makes the
bacterial cell walls more permeable.
After the next two days,the result obtained should prove that LB/amp/ara plates will glow a
green colour because the presence of arabinose is not harmful to bacteria and pGLO plasmid
is the important component in produce the fluorescent protein, which causes them to glow a
brilliant green colour under IJV light. In this case, for the observation, IJV light must be used
to see the glow bacteria that cannot be seen with the naked eye.

Figure 1 above indicate scenario of bacterial transformation by using pGLO plasmid

2.0 INTRODUCTION

Generally,the process of bacterial transformation, and ones that very common for
eukaryotic cells, is used every day in biotechnology.By incorporating a gene of interest, the
scientist can express proteins that can be beneficial to the organism and or useful in other
areas of science. Due to the gene of interest is always inserted with a selectable marker,
determination which of the organisms have been successfully transformed will be likely
easier.
As mention earlier,transformation of cells is a widely used and versatile tool in genetic
engineering and is of critical importance in the development of molecular biology. The main

3 | Page

aims of this technique is to introduce a foreign plasmid into bacteria, the bacteria then
amplifies the plasmid thus making large quantities of it.
Basically,the starting materials is bacterium Escherichia coli (E.coli).It is widely used in the
laboratory for recombinant DNA research. It is a common inhabitant of the human colon and
can be found easily in many areas of our environment. It has a single circular chromosome
that contains about five million DNA base pairs, only 1/600th the haploid amount of DNA in
a human cell. E.coli may also contain small circular DNA molecules called plasmids.A
plasmid is described as a small circular piece of DNA ( approximately about 2,000 to 10,000
base pairs) that consists crucial genetic information for the bacteria to grow.These plasmids
carry genetic information and are extrachromosomal, which mean they are not part of the
bacterial genome. Bacteria,which usually grew in the same environment like molds and
fungi, evolved to make proteins that inactivate the toxins produced by these other organisms.
The bacteria share this important information by passing it among themselves in the form of
genes in plasmids. Therefore, the natural function of a plasmid is to transfer genetic
information vital to the survival of the bacteria. It is this characteristic of plasmids that is
exploited for use in transformation.. By using plasmids the scientist can change or transform
the genetic information available to the bacterial cell. When plasmids contain multiple genes,
these genes will always be transferred together. In the laboratory,the plasmid used is called
pFluoroGreen (plasmids are always denoted by a lowercase "p" before the name), which
has a gene for Ampicillin resistance (AMPR) and a gene for GFP (green fluorescent protein).
The gained ampicillin resistance will allow the E.coli to grow on media that contains
ampicillin and the GFP will allow it to bioluminesce (to glow, light produced by a living
organism).
The purpose of green fluorescent protein (GFP) gene is being used because of the
bioluminescence (ability of an organism to produce color and light) that is produced when the
gene is expressed and energy is transferred to it within its host. This energy transfer results in
a change in conformation (shape) of the protein, producing the light and color. The GFP gene
naturally occurs in specialized photogenic cells in the umbrella of the jellyfish Aquorea
Victoria. This fluorescent protein can be expressed in E.coli bacteria and will produce a green
light and can especially be seen when exposed to a long wave U.V. light source.
The transformation process itself is done by the inserting a foreign plasmid into a bacteria
cell which results in a new genetic trait.Bacterial transformation involves the transfer of
4 | Page

genetic information on a plasmid by the direct uptake of this exogenous, or foreign, DNA into
the bacteria cell of interest that results in the acquisition of a new genetic trait that is stable
and heritable. Because bacterial cells contain enzymes called endonucleases, better known as
restriction enzymes, which degrade linear pieces of foreign DNA starting at the ends and
working inwards, the DNA is contained on a plasmid. Plasmids are circular and immune to
endonuclease attack. Plasmids are also used because they will be replicated and transmitted to
daughter cells. Plasmids can be engineered through a process that starts with cutting DNA
with restriction enzymes to include a piece of foreign DNA that contains a specific gene of
interest. Plasmids are very convenient vectors for introducing new genetic material, or
transferring genes, into bacterial cells. By putting a gene of interest into a plasmid the
transformed bacteria will express that gene and scientists can use that expression for the
production of a needed protein or for selection purposes. Bacterial transformation is essential
to the field of molecular biology in that it allows for the propagation, genetic expression, and
isolation of recombinant DNA molecules
For bacteria to take in a plasmid, they must first be made "competent" to take up DNA,
which won't normally pass through a bacterial cell's membrane. There are a few species of
bacteria that go through the transformation process naturally. Most bacteria need to be
manipulated so they become competent, or able to take up the plasmid. Competency is a
physiologic state, which changes the structure and permeability of the cell membrane so the
plasmid DNA can enter the cell. Competence can be induced in E.coli by treating with
chloride salts of the metal cations calcium, magnesium, and rubidium and with the assistance
of sudden cycles of heat and cold. This process is called chemical competency.This is done
by creating small holes in the bacterial cells by suspending them in a solution with a high
concentration of calcium. DNA can then be forced into the cells by the procedures followed
during this experiment.In the molecular view,the cations in the solution disrupt the bacterial
cell wall and membrane to create small holes through which DNA can enter. The cold
conditions also serve to create gaps in the lipid structures of the cell membrane that allow
DNA to enter. The heat shock produces a thermal gradient that sweeps into the cell and brings
along the DNA; after this step the DNA has entered the competent cell.On other
ways,competency can also be achieved through the use of electrical pulses called
(electroporation), which transiently changes the cell membrane structure and allows foreign
DNA into the cells without killing most of the cells. Electroporation can also be used to
introduce foreign DNA into other types of cells such as plants and animal cells. It tends to be
5 | Page

more efficient than chemical compentency, but it requires a special instrument called an
electroporator. Rapidly growing (log-phase) cells take up foreign DNA most efficiently after
either of these treatments that make the cells competent and able to be transformed.
The selection must be made.Bacteria that have taken up the plasmid and transformed must
be selected, or isolated. The plasmid is too small to be seen, so it is useful for it to contain a
gene that expresses a characteristic that can be seen or interpreted. One such characteristic is
antibiotic resistance. By growing the bacteria on solid media which contains an antibiotic,
only the cells that were transformed with a plasmid containing the gene for antibiotic
resistance will grow and the others will die. Plasmids can have multiple genes inserted into
them. The genes on a plasmid cannot be separated during transformation. If a plasmid had a
gene for antibiotic resistance and a gene for the expression of some protein, then the cells that
survive on an antibiotic plate would be known to also contain the other gene of interest. In the
case of this particular lab, the cells that survive on the media plates that contain ampicillin
will have obtained the gene for ampicillin resistance from the plasmid used in the
transformation and will also have the gene for GFP and will glow when exposed to long wave
UV light.
Mostly plasmids that used in bacterial transformations consist of genes that provide
resistance to various antibiotics. Ampicillin resistance (AMPR) is a well known used gene
when transforming bacteria. Ampicillin is a derivative of penicillin and inhibits bacterial
growth by interfering with the synthesis of cell walls. The gene for ampicillin resistance
produces an enzyme called beta-lactamase that is secreted by the cell and breaks down the
ampicillin in the surrounding media. Because of this extracellular secretion from transformed
cells, some untransformed cells can grow in the zones around colonies of transformed cells
and are called "satellite colonies".When compare,they are much smaller and found around the
larger transformed colonies.

3.0 OBJECTIVES
By the end of the experiment,students should be able to -:

6 | Page

1. Understand recombinant DNA techniques, in particular the transformation

procedure using the heat shock method.
2. Understand the uses of marker or reporter genes in molecular biology experiments
and how to screen for a gene of interest.
3. Understand that DNA can be transferred to another organism and therefore change
the observable characteristics of that organism.
4. Become familiar with sterile technique and decontamination procedures that are
used to handle bacteria.
5. Learn how to calculate transformation efficiency.

4.0 THEORY
Transformation Efficiencies
Transformation efficiencies are a way to determine how many cells were transformed per
microgram (ug) of plasmid DNA used. The colonies (on plate #4 for this lab) we count after
the overnight incubation originally grew from one transformed cell, called a transformant.
The calculation for transformation efficiency is:
Number of transformants (colonies) X final volume at recovery (ml) = number of
transformats/ ug of plasmid DNA

volume plated (ml)

ug of plasmid DNA

In research laboratories the transformation efficiencies will usually be between 1x104 to

1x107 transformants per ug of DNA. Transformations are never 100% efficient. The
efficiencies can be lowered due to shortened incubation times or temperature variations while
making the cells competent, shorten incubation times when uptake of the plasmid DNA is
taking place, or possibly by a shorten "recovery" phase before plating transformed bacteria

7 | Page

for overnight growth. There are many possible factors that can decrease the transformation
efficiencies, which have to be optimized and controlled in the laboratory.

5.0 PROCEDURES
Basically,the experiment was conducted for about three days until the result is obtained-:
Day one
1. Five empty plates had been prepared by the students.
2. The agarose powder is put in the beaker containing about 700 ml
3. The mixture then is stirred
4. After that, the mixture is put under the microwave it reached boiling point of water.
5. Then, the mixture which is the agarose solution is pour into the first two plates and these
plates are marked with the LB on top of it by using the marker pen.
6. The leftover of agarose solution in the beaker is mixed with the ampicillin solution before
stirring.
7. The mixture is poured into the second two plates.
8. The first of this plate is marked with LB/Amp (+), while the other one is marked with
LB/Amp (-).
9. The leftover of the mixture of agarose solution and Ampicillin in the beaker is mixed
with arabinose solution before stirring.
10. The new mixture obtained is poured into the last plate.
11. All of the agarose plates are kept cool until the solutions change into solid.

8 | Page

12. The four agarose plates except one of the LB agarose plate, had been closed by using
parafilm tape.
13. These four agarose plates are kept in the incubator at 37 degree Celcius.
14. The agarose plate that did not closed yet is streked with the bacteria E.coli by using
sterile loop.
15. The LB agarose plate is kept closed by using parafilm tape.
16. This plate then is kept in the refrigerator.
Day two
1. The results obtained from the transformation lab was observed under normal room
lighting. Then, the lights was turn out and UV lamp was hold over the plates.
2. In the data table, the results was recorded and observed the changes of the bacteria on the
four plates.. Include a description of the bacterial growth, colony color (room light and
UV), and number of bacterial colonies (1 spot = 1 colony)

6.0 APPARATUS &MATERIAL

micropipette
Water bath (with floating tube racks)
Streak plates of E.coli
pGREEN plasmid
Crushed ice
Distilled water
37C incubator
Parafilm
Waterproof marker
2 plates with LB medium#
2 plates with LB medium + ampicillin#
Disposable pipettes
Disposable inoculating loops

7.0 RESULTS & CALCULATIONS

9 | Page

Plates

Observation
-

Cells grown since there is many

colonies on the plate

-

Does not have green color after

been under the UV lamp. White color.

Bacterial

Does not have green color after been

under
the UV lamp.

-No bacterial growth

Cells with pGLO and plasmid are

grown and glow.

10 | P a g e

Have green color under UV lamp.

Two colonies have grown.

CALCULATIONS

We used 0.3 mg of pGLO at a concentration of 0.03 g/ L

Therefore, we used 0.3 mg of DNA
Total number of cells/colonies = 2
DNA (g) = (concentration of DNA (g/l) x (volume of DNA in l)
Volume spreadon LB/amp
Totalvolume test tube

Fraction of DNA used =

100 L
500 L

= 0.2
pGLO DNA spread (g) = Total amount of DNA used (micro g) x Fraction of DNA
= 300 g x 0.2
= 60 g

Transformation efficiency =

Total number of cells growing on the agar plate

Amount of DNA spread onthe agar plate

2
= 60 g
= 0.033 transformant/g

11 | P a g e

8.0 DISCUSSION

In the control lab a different outcomes were observed in each of the four plates. In the
LB/amp/arabinose agarose plate containing the +pGLO sample, fluorescent green colonies
dformed. This is due to the gene which codes for the fluorescent protein, GFP, is located near
the beta lactamase gene on the pGLO plasmid, which give protectsion towards bacteria from
the antibiotic ampicillin.As the cell produced beta lactamase to deactivate ampicillin, the GFP
gene was also transcribed, producing the fluorescent protein observed. In the LB/amp plate
containing the +pGLO sample white, non- florescent cells were observed. While these genes
contained the pGLO plasmid and the GFP gene they could not express the GFP gene because
they were not grown in the presence of arabinose. While the presence of ampicillin causes the
cell to transcribe the beta lactamase and GFP genes.In order to activate the GFP
operon,arabinose is needed. Thus, with absence of arabinose in the agarose gel, GFP cannot
be transcribed and of course the cells will not fluoresce. In the LB/amp agarose plate treated
with the -pGLO sample, no cells grew detected. This is because without the pGLO plasmid
and the beta lactamase gene the cells cannot deactivate the ampicillin in the gel. Therefore all
the cells were wiped out. In the LB plate containing the -pGLO sample small colonies were
seen spread over the entire plate. Because there was no ampicillin to kill the cells, all the cells
survived and the entire plate was covered, in contrast to the other plates where individual
colonies represented the cells which had taken up the plasmid.
The transformation efficiency of our control test was 0.033 transformant/g.This lower
number could be a result of sources of error that may be present within the methodology of
this lab or potential human error. Our variable test had an even lower transformation
efficiency of 0 transformants per microgram. This could be attributed to the lack of
transformation solution, but the tests should be repeated to reach a definitive result.
Due to the multiple solutions and bacterial plates used in this lab there it is likely that some
cross contamination occurred. Though many precautions were taken, such as using disposable
pipettes and sterile loops, there is always a chance that these tools could be contaminated
before use, or that a new substance, suchas bacteria, was introduced from the environment.
12 | P a g e

While this could be improved by using a culture hood or wearing gloves, cross
contamination, especially from the environment, can never fully be prevented.
The transfer pipettes used during much of the procedure werent very accurate, since any
variation of the pressure applied to the pipette would change the volume. This inaccuracy
could be eliminated by using micropipettes, which are accurate to the microliter.
This lab has many future prospects. Genetic modification does not have to be limited to
florescence. Organisms can be modified to have all sorts of interesting and unique traits. For
instances, plants can be given plasmids so that they gain certain traits, such as resistance to
disease or extreme weather. This can lead to better crop yield and shelf life. A lab extension
of the pGLO lab may involve modifying more complex organisms than bacteria. For
example, one could attempt to influence the expression of the GFP gene in fish, or another
multi cellular organism. Another potential lab extension might include the isolation and
purification of GFP. This could be done using column chromatography. It may also be
worthwhile to repeat the experiment without the transformation solution to ensure the results
are the same. This would further explain the effect of the calcium chloride solution.

9.0 CONCLUSION
This experiment was successful in determining the transformation efficiency and results of
genetic transformation.The plasmid should have resistance to the antibiotic on the plates that
contained ampicillin within it.The pGLO bacteria that doesnt have plasmid couldnt survive
on the ampicillin plates,which resulted in no growth of the control.Another plate of pGLO
thrived in a plate that didnt contain the antibiotoic.The two plates that contained the plasmids
both had bacterial resistance towards the antibiotic.Furthermore the plate that had
LB/ampicillin/arabinose gave the colonies a fluorescent green glow under ultraviolet
light.Transformation efficiency is the quantitative value that describes how effective the
transfer of plasmids into bacteria.The number represents the number of transformed colonies
produced per microgram of DNA added.Most of group achieve the ideal transformation
efficiencies.

13 | P a g e

10.0

RECOMMENDATION

In order to ensure the result obtained accurately,some recommendation is suggested to get

rid several error that may exist during conducting the experiment and .These includes
disinfect your work area before and after working with bacterial cultures.This is because
many microorganisms thrive under the same conditions as the harmless E. coli strain being
used in this experiment, therefore it is important to maintain sterile conditions to minimize
the possibility of contamination with foreign bacteria or fungi.Next,the heat shock must be
done about 50 seconds in order to prevent the DNA from denatured.In addition,sterile
technique will be used throughout this experiment. As kept in our mind that everything that
comes in contact with E. coli bacteria has been, or must be, pre-sterilized including:
calciumchloride solution, Luria broth, transfer pipettes, inoculating loops, spreaders, culture
tubes, and petri plates.Next,always open wrappers of sterile transfer pipettes and loops so that
you do not touch the tipend of the pipet or loop. Open at the opposite end.Do not reuse
pipettes or transfer loops.Thats the reason why we use the disposal ones.Last but not
least,wash hands before leaving the laboratory. It also been suggested that student should be
take note on factors influencing transformation efficiency include technique errors, the
temperature and length of the incubation period, the growth stage of the cells, and using the
correct mass of plasmid DNA.

11.0

REFERENCES

i.

Bacteria modified to combat HIV. (2005, November 13). BBC News. Retrieved

ii.

January 15, 2012, from http://news.bbc.co.uk/2/hi/health/4692905.stm

http:// www.apsnet.org/EDCENTER/K-

iii.

12/TEACHERSGUIDE/PLANTBIOTECHNOLOGY/Pages/Activity4.aspx
BIO-RAD. (n.d.). Retrieved 18 May, 2015, from Bacterial Transformation:
http://www.bio-rad.com/en-us/product/pglo-bacterial-transformation-kit

14 | P a g e

iv.

Blaber, D. M. (n.d.). Genetic Transformation. Retrieved 18 May, 2015, from

mikeblaber.org:http://www.mikeblaber.org/oldwine/BCH4053l/Lecture07/Lecture07.h
tm

12.0

APPENDICES

The figures below are listed of apparatus and material being used during experiment

LB broth

Inoculating loop

15 | P a g e

Microtube

Incubator

Petri dish with agar

16 | P a g e