Grab-A-Cob:

An Integration of Photosynthetic Genes of Corn (Zea Mays) to Deinococcus Geothermalis for

Biofuel Production

Natural Science New Material Track

Biology Laboratory
(LBYMATB)

Damgo, Diomyka C.
Go, Troy Justin G.
Lim, Erwin Angelo C.
Oliva, Micah Rose T.
Paras, Jasmine Jeremy T.
Sayarot, Cecil Austin R.
Proponents, C31B

De La Salle University Manila

2401 Taft Avenue, Malate, Manila
 Chapter 1
The Problem and Its Setting
This chapter introduces the study conducted by the proponents. It comprises of the
following: background of the study, statement of the problem, general and specific objectives,
significance of the study and its scope and limitations.

Background of the Study

Here in the Philippines, corn (Zea mays) is one of the most abundant food crops. About
20% of the population use corn as staple food. The bulk of corn used in the Philippines is
processed into food (25%) and feeds (70%). Corn and other high-starch grains have been
converted into ethanol and in the past century, its use as fuel greatly expanded. Corn grain makes
a good biofuel feedstock due to its starch content and its comparatively easy conversion to
ethanol.
An extremely radiation resistant, moderately thermophilic bacterium known as
Deinococcus geothermalis, has been utilized already in the field of biotechnology for the process
of reducing solvents and heavy metals in radioactive sites.
After recombinant DNA was introduced in 1973, scientists have been able to move genes
from one place to another within a genome. Scientists refer to this technology as genetic
modification (GM). Different techniques are used to alter genes including DNA extraction, gene
transfer and microbial culture.
The Philippines goes the fossils way instead of taking advantage of its vast renewable
energy (RE) sources to power progress. However, the Philippines still lack source of energy.
Fossil fuel also became an issue because of high-carbon growth that has fueled increasing
greenhouse gas emissions (GHGs) which is the main cause of global warming.

General Objectives
This study was generally conducted in order to provide a sustainable resource for
automobiles with effort in sustaining the condition of the ozone layer via the integration of the
genetic components of maize with deinococcus geothermalis bacteria. Once it is proven to be
effective, this research study shall resolve the problem of prevalent carbon emission
and peoples dependence on fossil fuels.
Specific Objectives
This study was specifically intended to determine whether deinoccocus geothermalis is
an effective agent for further duplication and production of maize genes which have the ability to
generate biofuel. Specifically, the main objectives are the following:
1.4.1 To integrate the photosynthetic genes of zea mays to deinoccocus geothermalis.
1.4.2 To transform deinoccocus geothermalis into a photosynthetic bacteria which is capable of
self-production of biofuel.
1.4.3 To provide a cheaper alternative source of energy.
 Chapter 2
Review of Related Literature
This chapter presents the related literature, which is in-line to the study. It also contains
the different topics of issues addressed. Literatures were cited on the bases of their importance in
the conduct of the study.

Zea Mays
Global demand for oil and gasoline increases every year. Due to the abundance of corn in
countries such as the United States, corn was used as a raw material in making ethanol due to its
high starch content. Corn originated in Central America. However, due to the increased
production of corn, locals thought of a way on how to use these resources that will benefit others
which is to transform corn into biofuel.

Deinococcus geothermalis
Deinococcus geothermalis, which was obtained from the hot springs located in Naples,
Italy and in Sao Pedro do Sul, Portugal, is a gram-positive, extremely radiation resistant, and
moderately thermophilic bacterium. This is enveloped by a three-layered cytoplasmic membrane
enclosed in a cell wall which has a corrugated surface and an electron-dense inner layer. It is, in
fact, culturable on defined minimal medium and undefined rich medium. In biotechnology, the
ability of this bacterium has been utilized already in the process of reducing solvents and heavy
metals in radioactive sites.

Bio Fuel
Biofuel is an alternative source of energy to reduce pollution. Through a process of
biological carbon fixation, biofuel is produced. It is a renewable energy which can be obtained
from living organisms or from metabolic by-products (organic or food waste products).
Compared to fossil fuels, biofuels release lower levels of carbon.
DNA Extraction
DNA extraction is a technique used to separate DNA from cell. DNA is used for
diagnostic purposes to detect viruses or bacteria. It is basically the starting point for numerous
applications. In the case of obtaining the genetic component of corn, the method should yield
sufficient quality and quantity so that the sample may be suitable for experimentation.
Concentration of the DNA separated from corn is then used to combine with chemicals to
produce a new product, which may be beneficial in the future.

Gene Transfer
Gene transfer is the process of inserting copies of a gene in living cells in order to induce
synthesis of the gene. It is simply a technique in which foreign genes is introduced from the
genome of target cells. Gene transfer is developed for research to investigate gene functions.
Genes are the basic hereditary units of life. They provide information necessary to produce
proteins in the body. When a gene is introduced in a target cell, a new protein from the gene is
formed.

Microbiological Culture
Microbial culture is a method of growing and multiplying microbial organisms. This
allows the microbes to reproduce in an artificial culture media under controlled laboratory
conditions. In microbiology, this process is extensively used to isolate a pure culture of
microorganisms in order to support a research or a diagnosis.

Polymerase Chain Reaction

Polymerase Chain Reaction is a process, which enables the production of millions of
copies of specific DNA sequence in an approximate span of two hours. It is an in-vitro
amplification DNA protocol. Also, this method is one of the effective and efficient methods used
in diagnosis of diseases and virus.
Fractional Distillation
Fractional distillation is a technique used to separate a mixture into its component parts or
fractions, Through the use of fractionating column, chemical compounds are separated by their
boiling points. They are heated to a temperature at which two or more of the fractions will
vaporize. This method is commonly used in oil refineries using crude oil for the production of
gasoline fuel.

Ethanol Fermentation
Ethanol fermentation is also referred to as alcohol fermentation. It is a biochemical
anaerobic process of converting sugars such as glucose, fructose, and sucrose into cellular
energy. As a side-effect, this method produces ethanol and carbon dioxide. In the process of
brewing beer, this method is applied to allow the conversion of sugars into ethanol and carbon
dioxide by using yeasts.

Photosynthesis
Photosynthesis is the process used by plants, algae, and certain bacteria to convert light
energy, which normally comes from the sun, into chemical energy. Later on, the organism will
use this energy to fuel its activities. There are actually two types of photosynthetic processes
namely oxygenic photosynthesis and anoxygenic photosynthesis. In oxygenic photosynthesis,
light energy induces water to lose electrons and carbon dioxide to receive electrons. Ultimately,
this method produces oxygen along with carbohydrates. It is commonly used by plants, algae and
cyanobacteria. On the other hand, anoxygenic photosynthesis uses electron donors such as
hydrogen sulfide other than water. Absolutely, it does not produce oxygen. This process is most
common in purple bacteria and green sulfur bacteria.
 Chapter 3
METHODOLOGY

This chapter exhibits and displays the description of the research methods used for the
entire study. It shows information pertaining to the development of the research in a systematic
manner. This chapter also discusses the main subject of the research. It will contain the
following: Research Design, Procedure Flow Chart, and Experimentation and General
Procedure.

Research Design

DNA Extraction

Polymerase Chain Reaction

DNA Recombination

Bacterial Gene Transformation

Microbiological Culture

Ethanol Fermentation

Fractional Distillation

The study follows a basic research design. It shows the possibility of a mass production
of biofuel by utilizing D. Geothermalis as the instrument. Also, it provides an alternative option
to consider in the production of biofuel.
The first step is the DNA extraction of corn to obtain the DNA of the plant, which
contains the necessary genetic codes in producing biofuel. The next process is polymerase chain
reaction wherein we replicate the extracted DNA to be in possession of copies of the DNA.
Afterwards, the copies of DNA undergoes DNA recombination to cut them into fragments and
insert them into the vectors extracted from D. Geothermalis, which will serve as carriers of the
specific genetic codes that produces biofuel. Then, bacterial gene transformation takes place
wherein D. Geothermalis engulfs the recombinant DNA. It adapts to the foreign body it
enveloped and transforms to a recombinant bacterium, which possesses the new genetic material.
Next, bacterial culture is done to replicate copies of the recombinant bacterium. The next process
is fermentation wherein the proponents feed the bacteria with feedstock as an input. As a result,
biofuel is produced. Lastly, the mixture with the biofuel is put under the process of distillation to
separate the biofuel from the rest of the liquid.
Procedure Flowchart
This section shows in a systematic manner the course of experimentation through figures
and charts.

A. Procedure for DNA Extraction

Figure 1
Schematic Diagram of DNA Extraction Method

Corn (Zea mays) Kernels

DNA
Extraction

Blending of Kernels

Addition of liquid detergent

Addition of enzymes

Addition of ethyl alcohol

Extraction of DNA
B. Procedure for Polymerase Chain Reaction
Figure 1
Schematic Design of Polymerase Chain Reaction Method

Corn (Zea mays)

DNA Extract

Polymerase
Chain Reaction

Denaturation

Primer Annealing

Extension/Elongation

Elongation

Amplified Corn (Zea mays)

DNA
C. Procedure for DNA Recombination
Figure 1
Schematic Design of Recombination

Bacterial Strain
Corn (Zea mays)
DNA Extract Deinococcus Geothermalis

DNA
Recombination

Fragmentation of DNA

Isolation of bacterial plasmids

Fragmentation of bacterial
plasmids

Ligation of DNA and bacterial

plasmids fragments

Recombinant DNA
D. Procedure for Bacterial Gene Transformation
Figure 1
Schematic Design of Bacterial Gene Transformation

Recombinant DNA
(Ligated DNA and bacterial plasmid)

Bacterial Gene
Transformation

Introduction of recombinant
DNA to bacterial strain

Exposure of bacterial strain to

recombinant DNA

Closure of bacterial strain to

foreign materials

Recombinant D. Geothermalis
E. Procedure for Microbiological Culture
Figure 1
Schematic Design of Microbiological Culture Method

Recombinant D. Geothermalis

Microbiological
Culture

Preparation of Inoculum

Preparation of Inoculum Plate

Transfer of recombinant
bacteria to the plates

Culture of recombinant D.
Geothermalis
F. Procedure for Ethanol Fermentation
Figure 1
Schematic Design of Ethanol Fermentation Method

Culture of recombinant D.
Geothermalis

Ethanol
Fermentation

Introduction of recombinant
bacteria to water

Photosynthesis in recombinant
D. Geothermalis

Conversion of carbohydrates to
bioethanol

Bioethanol mixture with other

liquid substances
G. Procedure for Fractional Distillation
Figure 1
Schematic Design of Fractional Distillation Method

Bioethanol Mixture with other

liquid substances

Fractional
Distillation

Exposure of the bioethanol

mixture to heat

Vaporization of the bioethanol

mixture

Condensation of all substances

in the bioethanol mixture

Bioethanol
Experimentation and General Procedure

A. Procedure for DNA Extraction

The corn (Zea mays) kernels are crushed using a blender to open its cell wall with
blender and salt. The proponents then, add liquid detergent and enzymes to isolate the
DNA from other chemical compounds. Lastly, they pour ethyl alcohol in preparation for
the final step of the process. The DNA condenses and floats on the topmost of the
mixture, and it is extracted and put in an Eppendorf tube.

B. Procedure for Polymerase Chain Reaction

The DNA extract is heated usually to 95 to render it single-stranded. The two
primers bind the appropriate complementary strand. There are temperature variations due
to the different sizes of the DNA. DNA polymerase extends the primer by its polymerase
activity. The temperature is optimized for the particular polymerase utilized. The
proponents repeat the process of denaturation, annealing, and primer extension numerous
times to replicate numerous copies of the DNA. It is placed under automated thermal
cyclers, which conducts the PCR reaction for the researchers.

C. Procedure for DNA Recombination

The proponents add restriction enzymes to the DNA extract to cut the DNA into
fragments. Plasmids are extracted from deinococcus geothermalis to serve as carriers of
the new genetic codes. These plasmids are also combined with restriction enzymes to cut
them into fragments. Afterwards, the researchers recombine the DNA and bacterial
plasmid fragments. Thus, it forms a recombinant DNA, which contains the specific
genetic code for photosynthesis.

D. Procedure for Bacterial Gene Transformation

The recombinant DNA is introduced to deinococcus geothermalis. They are then
put under a cold bath but exposed to a heat shock to open the membranes of the bacteria.
The recombinant DNA enters the bacteria and it is adapted to its system. Lastly, the
proponents place the recombinant bacteria into a cold bath to close its membrane. As a
 result, recombinant bacteria are transformed with a new genetic code from the corn (Zea
mays).

E. Procedure for Microbiological Culture

The recombinant deinococcus geothermalis is replicated through bacterial
culturing. The proponents prepare the inoculum and the inoculum plate wherein the
bacteria will grow. An appropriate agar is utilized to provide the bacteria proper nutrients
and environment to grow. The recombinant deinococcus geothermalis is mixed with the
agar to have a uniform distribution. The agar is poured on the petri dishes to have a sterile
environment to grow without any competition from other bacteria.

F. Procedure for Ethanol Fermentation

The recombinant deinococcus geothermalis is able to undergo photosynthesis.
Thus, they are capable of providing their own food, particularly starch. This carbohydrate
is converted into bioethanol through the process of fermentation. Bioethanol is produced
with other chemical compounds and are integrated with other liquid substances.

E. Procedure for Fractional Distillation

The proponents separate bioethanol from other substances through a continuous
and successive vaporization and condensation, which is the process of fractional
distillation. In this manner, bioethanol vaporizes earlier than water and condenses in a
container. Afterwards, water vaporizes and condenses in a separate container. Bioethanol,
which can be utilized in automobiles to lessen their carbon emission, is produced through
this process.
 Chapter 4
Summary and Conclusion
This chapter presents the summary of the proposal and the researchers findings and the
conclusions that were based from the investigation and study conducted.

Summary
The proposal aims to address an alternative source of energy, which only requires
sunlight as an input for the conversion to bioethanol. If it is proven so, the proposal can be
utilized to prevent a worldwide crisis in biofuel production and climate change.
The proponents extracted DNA from zea mays, which is replicated numerous times
through polymerase chain reaction. The authors integrated the photosynthetic genetic code of zea
mays to deinococcus geothermalis by recombining their DNA, ligating the DNA extract and
bacterial plasmid. It is then introduced to the bacteria so that it will engulf the recombinant DNA
and adapt the foreign material to its system thus, becoming a recombinant bacterium. The
bacteria is reproduced and grown in a culture media through microbiological culture. The
exponential growth of the bacteria occurs in this procedure. The proponents mix the replicated
copies of deinococcus geothermalis into water and expose it to sunlight. The bacteria undergo
photosynthesis, which produces its own food supply in the form of carbohydrates. The bacteria
convert these carbohydrates into bioethanol. In this step, bioethanol is not yet separated with
other liquid substances and chemical compounds. Therefore, it is subjected to fractional
distillation wherein the mixture is continuously vaporized and condensed to separate bioethanol
from the other liquid substances. Thus, this is creating bioethanol which can be used as a fuel for
automobiles and lessen its carbon emissions.

Conclusion
Deinococcus geothermalis is an effective agent for production or duplication of maize
genes. It is capable of undergoing photosynthesis to produce carbohydrates, which are necessary
in producing bioethanol. It is a cheaper and alternative source of energy since it does not need
any input in the production of biofuel.
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That In All Things, God May Be Glorified!