1 Construction of GFP Gene into Yeast Vector Farhana Muhammad Yusoff (30158) Resource Biotechnology Faculty of Science and Technology Universiti Malaysia Sarawak ABSTRACT The construction of Green Fluorescent Protein (GFP) gene into yeast vector is very important as it can ensure the successful of expression of GFP gene in a yeast, Pichia pastoris. The construction of this gene is integrated into pPICZ A vector which is yeast expression vector. The GFP gene that will be constructed into yeast vector is due to the easy to be manipulated genetically and culture than mammalian cells and can be grown to high cell densities compared to other expression system that available. The technique used in this study is mainly about amplifying the gene by using Polymerase Chain Reaction (PCR). The product was attempted to clone into pGEM-T and the transformation process was done by using heat shock method. Instead of using pGEM-T cloning method, the PCR product was subcloned directly into pPICZ A vector. The confirmation of positive transformant which contain of pPICZ A/ GFP was done to ensure the successful of construction of GFP gene into yeast vector by using colony PCR. This might be prior things in order to continue the next step in recombinant DNA technology. Key words: GFP, pPICZ A, pGEM-T ABSTRAK Pembentukan GFP gen ke dalam vektor yis adalah sangat penting kerana ia boleh memastikan kejayaan ekspresi GFP gen dalam yis, Pichia pastoris. Pembentukan gen ini disepadukan dalam pPICZ A vektor yang yis vektor telah diekspreskan. Gen GFP yang telah dibentuk ke dalam vektor yis adalah disebabkan oleh manipulasi secara genetik terutamanya daripada sel mamalia dan boleh berkembang kepada kepadatan sel tinggi berbanding dengan sistem ekspresi lain yang ada. Teknik yang digunakan dalam kajian ini adalah memperbanyakkan gen dengan menggunakan Polymerase Chain Reaction ( PCR ). Produk daripada PCR telah cuba untuk diklonkan ke dalam pGEM –T vektor dan proses transformasi itu dilakukan dengan menggunakan kaedah kejutan haba.Selain daripada itu, produk daripada PCR juga telah diklonkan terus ke dalam vektor pPICZ A . Pengesahan transformant positif pPICZ A / GFP telah dilakukan untuk memastikan kejayaan pembinaan GFP gen ke dalam vektor yis dengan menggunakan koloni PCR. Ini mungkin Antara perkara utama yang perlu dilaksanakan untuk meneruskan langkah seterusnya dalam teknologi DNA rekombinan. Kata kunci: GFP, pPICZ A, pGEM-T
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Construction of GFP Gene into Yeast Vector
Farhana Muhammad Yusoff (30158)
Resource Biotechnology
Faculty of Science and Technology
Universiti Malaysia Sarawak
ABSTRACT
The construction of Green Fluorescent Protein (GFP) gene into yeast vector is very important as it
can ensure the successful of expression of GFP gene in a yeast, Pichia pastoris. The construction of
this gene is integrated into pPICZ A vector which is yeast expression vector. The GFP gene that
will be constructed into yeast vector is due to the easy to be manipulated genetically and culture
than mammalian cells and can be grown to high cell densities compared to other expression system
that available. The technique used in this study is mainly about amplifying the gene by using
Polymerase Chain Reaction (PCR). The product was attempted to clone into pGEM-T and the
transformation process was done by using heat shock method. Instead of using pGEM-T cloning
method, the PCR product was subcloned directly into pPICZ A vector. The confirmation of
positive transformant which contain of pPICZ A/ GFP was done to ensure the successful of
construction of GFP gene into yeast vector by using colony PCR. This might be prior things in
order to continue the next step in recombinant DNA technology.
Key words: GFP, pPICZ A, pGEM-T
ABSTRAK
Pembentukan GFP gen ke dalam vektor yis adalah sangat penting kerana ia boleh memastikan
kejayaan ekspresi GFP gen dalam yis, Pichia pastoris. Pembentukan gen ini disepadukan dalam
pPICZ A vektor yang yis vektor telah diekspreskan. Gen GFP yang telah dibentuk ke dalam vektor
yis adalah disebabkan oleh manipulasi secara genetik terutamanya daripada sel mamalia dan
boleh berkembang kepada kepadatan sel tinggi berbanding dengan sistem ekspresi lain yang ada.
Teknik yang digunakan dalam kajian ini adalah memperbanyakkan gen dengan menggunakan
Polymerase Chain Reaction ( PCR ). Produk daripada PCR telah cuba untuk diklonkan ke dalam
pGEM –T vektor dan proses transformasi itu dilakukan dengan menggunakan kaedah kejutan
haba.Selain daripada itu, produk daripada PCR juga telah diklonkan terus ke dalam vektor pPICZ
A . Pengesahan transformant positif pPICZ A / GFP telah dilakukan untuk memastikan kejayaan
pembinaan GFP gen ke dalam vektor yis dengan menggunakan koloni PCR. Ini mungkin Antara
perkara utama yang perlu dilaksanakan untuk meneruskan langkah seterusnya dalam teknologi
DNA rekombinan.
Kata kunci: GFP, pPICZ A, pGEM-T
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1.0 INTRODUCTION
Green Fluorescent Protein (GFP) gene that is mainly from jellyfish, Aequorea victoria has
becoming one of the most widely used reporter proteins. Recent application have been
found regarding the use of GFP gene which is the GFP genetically modified cats will aid
human and feline medical research. Based on several researches, scientists use genetically
modified animals that have been inserted with GFP gene for the study of HIV/ Aids (Jha,
2011). Therefore, there are many important reasons for choosing GFP gene in this project.
GFP is the protein of choice compared to other fluorescent proteins is due to its fluorescent
is more stable and species-independent. It also allows a simple detection under UV light
and can be monitored non-invasively in living cells (Kain et al., 1995).
In this project, the yeast expression system being used to produced recombinant
protein. Nowadays, the developments of Pichia expression, which is the yeast system that
was used in this project, had an impact on not only the expression levels, but also the
bioactivity of various heterologous proteins (Macauley-Patrick et al., 2005). There are
some advantages of yeast compare to mammalian and bacteria which is usually E. coli
expression system. Pichia pastoris that will be used is easier to be manipulated genetically
and culture than mammalian cells and can be grown to high cell densities. Due to the
eukaryotic type of cells, P. pastoris, can provide correctly folded recombinant proteins that
have undergone the post-translational modifications required for functionality. Based on
the research that has been made by the researchers, the P. pastoris system has strong
promoters to drive the expression of a foreign gene of interest. Hence, this will produce
large amount of the target protein with technical ease and low cost rather than most
eukaryotic systems (Daly and Hearn, 2005).
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Obviously, the construction of GFP gene into yeast expression vector will provide a
platform to established P. pastoris expression system for production of other recombinant
protein. The construction will involve the amplification of GFP gene by PCR there are also
other methods which are cloning, ligation and transformation. The PCR will be used in this
research to amplify the desired gene. The PCR process is the process of amplification of
primer-mediated enzyme for specifically cloned or genomic DNA sequence (Innis,
Gelfand, Sninsky, and White, 1990). The construction of GFP gene into yeast vector
requires some important steps. The objective of this project is to construct GFP gene into
yeast vector which is P. pastoris, before the successful of the next step of recombinant
technology which is expression step. Most of the research made for GFP gene are basically
was constructed successfully into large and ever-growing number of species, for example
bacteria, fungi, plants, insects, and nematodes (Chalfie et al., 1994). However, in this
project, the construction will be made into P. pastoris, which is the yeast species.
Therefore, the objectives of this study are:
1. to amplify the GFP gene by using PCR method
2. to clone the GFP gene into pPICZ vector
3. to extract plasmid of positive transformant
4. to confirm the insertion by using colony PCR
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2.0 LITERATURE REVIEW
2.1 Green Fluorescent Protein (GFP)
Green Fluorescent Protein (GFP) is amazingly becoming useful for studying living cells,
and recently, scientists are making it even more useful. Scientist using GFP to create
biosensors for example is for molecular machines that can detect the levels of ions or Ph,
then the report is done by fluorescing. There is one example of fluorescent protein which is
β-glucuronidase (GUS) gene that also has been used widely. The transformed tissue can be
identified histochemically, but it is a destructive test and not suitable for assaying the
primary transformats. However, GFP gene shares none of these problems (Haseloff, 1999).
The GFP was found by Osamu Shimomura, one of the professors at the Priceton
University, USA during a visit to US Pacific coast for a vacation in the late 1960’s. He
collected some specimen of Aequorea victoria and was known to glow with bluish colour
that can turn green. The size of the GFP is a relatively small which about the half of the
size of serum albumin and also the half of the size of haemoglobin in the blood stream
(Ward, 2009). The gene contains 238 amino acids and the residues 65 to 67 (Ser-Tyr-Gly)
in the sequence of GFP will spontaneously form the fluorescent chromophore p-
hydroxybenzylideneimidazolinone. The chromophore is resulting from the process of
spontaneous cyclization and oxidation of the residues. It also needs the native protein fold
for both formation and fluorescence emission (Ormo et al., 1996). Besides, the GFP is in
barrel structure to keep the chromophore away from solvents. Thus, the GFP are able to
fluorescing under almost any conditions (King and May, n. d.). Figure 1 shows the
structure of GFP gene.
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Figure 2.1: The chemical structure of the GFP gene (Prashera, Eckenrodeb, Wardc, Prendergastd, &
Cormierb, 1992).
The first function of GFP was discovered in the 1970’s by Morin and Hastings
which was to turn the bioluminescence flash of jellyfish, hydroids, and sea pansies from
blue to green. After that time, there are a lot of functions has been found. In science
research, the GFP gene is used widely in scientific research because GFP is the only
fluorescent protein which is the only coloured protein that can be transfer genetically into
other cells, tissues, organs, and organisms after the gene transplant. In fact, many other
proteins have colours and fluorescence, but none of them can be cloned. They only can just
being inserted, through single gene into another organism. Hence, the unique GFP gene is
used widely rather than any other gene. By using GFP gene, scientist and researchers can
watch what is happening in cells in real time. This is done by monitoring a non-invasive,
non-toxicfluorescent marker for GFP. Before the findings of GFP, scientist usually kills the
organisms, preserve them, and stain them in toxic chemicals to see what is happening
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inside the cells and tissues. Now, the ways of research have been upgraded by the use of
GFP gene as it can report the development and details of cellular metabolism in cells that
are still living. GFP makes the whole organism which is every cell in the body becoming
green-fluorescent. The GFP gene also can be linked to the cellular control factor which is
called the promoters. There is an example of research in Columbia University which is on
the nerve growth of round worm. In the research, the GFP have been linked to the
promoter for nerve growth. Thus, the Columbia scientists can study on what triggers others
cells to grow into nerves. Based on the study, the nerves begin to grow when the cells
begin to glow green (Ward, 2009). According to Zupan, Trobec, Gaberc-Porekar and
Menart (2004), the fused GFP to the N- or C- terminus of proteins, GFP can be used to
express their intracellular location and arrangement. It also is to determine the level of
gene expression due to GFP fluorescence and to study the transportation and secretory
processes that happen in the cell.
2.2 The Yeast Expression Vector
The vector used for this project is yeast vector which is P. pastoris. The yeast is a
methylotrophic yeast species and it also is widely used for the production of recombinant
protein (Cregg et al., 2009). P. pastoris is chosen because of its capability of
accomplishing post-translational modifications that will be result to the proper folding of
numerous foreign proteins. In recent research, there are only a few reported cases of
protein expression in the form of insoluble particles (Sreekrishna et al., 1988). Compare to
other yeast vector, P. pastoris is known as an excellent expression host. There is also a
promoter which is derived from alcohol oxidase I (AOX1) gene from P. pastoris that is
very unique to suite the control of expression for foreign gene. The type of yeast vector
used in this project is pPICZ expression vector. Basically, a yeast, P. pastoris, is a single-
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celled microorganism which can manipulate and culture in easy way. However, the nature
of yeast is, they are also a eukaryote and able to do many post-translational modifications
that usually performed by higher eukaryotic cells. Moreover, P. pastoris system can
generally being easier, faster, and less expensive to use rather than expression systems that
comes from higher eukaryotes like mammalian tissue culture or insects cell systems. This
is because the higher eukaryotes systems usually give higher expression levels.
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3.0 MATERIALS AND METHODS
3.1 Materials
Low Salt LB medium with Zeocin and pPICZ vector was obtained from Invitrogen, and
pGEM-T Easy vector was obtained from Promega. GFP gene in the plasmid of pAGS/GFP
and E. coli XL1Blue competent cell was obtained from Department of Molecular Biology
UNIMAS. The extraction of plasmid materials; Solution I (50 mM Glucose, 1.8 M Formic
Acid, 25 mM Tris-HCl pH8), Solution II (0.2 N NaOH, 1% SDS), Solution III (3 M KAc,
10 mM EDTA pH8) was used for plasmid extraction. Agarose gel,
phenol/chloroform/isoamyl alcohol, T4 DNA ligase, T4 DNA Ligase buffer, water,
nuclease-free, phosphorylated linkers was used during ligation process.
3.2 Method
3.2.1 Competent Cell Preparation
The culture containing 5 ml Luria broth with 5 µl of 50 mg/ml ampicillin and a single
colony or thawed frozen glycerol stock of Escherichia coli strain XL1Blue were grown
overnight at 37°C with shaking at 250 rpm. The culture was transferred to Erlenmeyer
flask that contains 50 ml of pre-warmed Luria broth media without the presence of any
antibiotics. The culture was allowed to grow again at 37°C with shaking at 250 rpm until
the OD of 600 reached the reading between, 0.45 to 0.5. The flask was put on ice for 20
minutes and centrifugation was performed at 3500 rpm for 5 minutes at 4°C in a cooled
McCartney bottles. The supernatant was removed and the cells were re-suspended in a 12.5
ml iced-cold 100 mM CaCl₂. Centrifugation was performed for 5 minutes at 3500 rpm.
Incubation on ice for 1 hour also was performed (Sambrook, Fritsch, and Maniatis, 1989).
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3.2.2 Plasmid Extraction
The overnight bacterial culture was transferred into a 2 ml microcentrifuge tube and was
centrifuged for 2 min at 8000 rpm. The supernatant was removed and re-centrifuged the
pellet for 1 min. Any traces of liquid media were removed from the tube. The cell pellet
was re-suspended by using 100 μl of Solution I by vortexing about 10 seconds and was
kept on ice. Solution II was added about 100 μl into the cell suspension and was mixed
gently by inverting 10X. The tube was left at room temperature for exact 5 min. Solution
III then was added into the tube and mix by inverting 10X. The solution was centrifuged at
10000 rpm for 5 min. The supernatant was transferred carefully by pipetting into a 1.5 ml
microcentrifuge tube. The DNA was precipitated by adding 2 volumes of cold absolute
ethanol and the tube was inverted at least 10X. Centrifugate at 13000 rpm for 5 minutes.
The pellet was washed with 500 μl of 70% ethanol and was re-centrifuged again at 13000
rpm for 2 min. The pellet was air dried and re-suspended the pellet in 50 μl of ultrapure
water. The 5 μl plasmid DNA was checked or determined by Agarose Gel Electrophoresis
(AGE). A 1% agarose gel was used and AGE was performed at 105 V for 30 min.
3.2.3 Purification of Extraction Product
An equal volume of phenol/chloroform/isoamyl alcohol was added to the DNA solution to
be purified in a 1.5-ml microcentrifuge tube and was vortex vigorously 10 seconds as well
as centrifuged for 15 seconds at room temperature. The top part which was the aqueous
phase containing the DNA was removed carefully by using a 200 µl pipette and was
transferred to a new tube. About 1/10 volume of 3M sodium acetate, pH 5.2, was added to
the solution of DNA and was mixed by vortexing briefly or by flicking the tube several
times with a finger. About 2 to 2.5 volume (calculated after salt addition) of ice-cold 100%
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ethanol was added and then was mixed by vortexing and place in crushed dry ice for 5 min
or longer. It was spin for 5 min in a fixed-angle microcentrifuge at high speed and the
supernatant was removed. About 1 ml of room temperature 70% ethanol was added. The
tube was inverted several times and was centrifuged back. The supernatant was removed.
Air was allowed to dry for 15 minutes. DNA pellet was resuspended in 100 µl of ultrapure
water.
3.2.4 Polymerase Chain Reaction
Before applying PCR process, the primer for the GFP gene was designed. After the
forward and reverse primer was designed, the process was preceded to PCR. The PCR
amplification, the specific PCR primers were chose to prime the nucleic acid template to
make the polymerase to be attached to it. This is the first step for the duplications of the
template (Mackay, 2013). The protocol of PCR is based on PCR Master Mix by Promega.
The PCR was performed after all reactions were prepared, 1 X reaction mix was allowed,
when the DNA template was added. The amplification process was performed by
following the cycling profile which is: 94ºC for 5 minutes, 35 cycles at 94ºC for 30
minutes, and lastly, 72ºC for 10 minutes in 1 cycle. The reaction product was visualised by
using agarose gel electrophoresis and the product was stained with ethidium bromide.
3.2.5 Gel Extraction of PCR Product
The bigger AGE well was performed and the 50 µl was loaded into each well. The gel slice
containing the DNA fragment was excised by using a clean scalpel or razor blade. The gel
was cut as close as possible to minimize the gel volume. The gel slice was placed into pre-
weighed 1.5 ml tube and weighed back. The weight of the gel slice was recorded. This step
of gel extraction was done by using GeneJET Gel Purification Kit by Thermo Scientific.
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Then, 1:1 volume of Binding Buffer was added to the gel slice (volume: weight). The gel
mixture was incubated at 50-60˚C for 10 minutes or until the gel slice was completely
dissolved. The tube was mixed by inversion every few minutes to facilitate the melting
process. The gel was make sure completely dissolved. The gel mixture was vortex briefly
before loading on the column. Up to 800 µl of the solubilised gel solution was transferred
to the GeneJET purification column, then it was centrifuged for 1 minute. The flow-
through was discarded and the column was placed back into the same collection tube. The
Wash Buffer of 700 µl was added to the GeneJET purification column. It was centrifuged
for 1 minute. The flow-through was discarded and placed back into the same collection
tube. The empty GeneJET purification column was centrifuged for an additional 1 minute
to completely remove residual wash buffer. The GeneJET purification column was
transferred into a clean 1.5 ml microcentrifuge tube. Elution Buffer of 50 µl was added to
the center of the purification column membrane for 1 minute. The GeneJET purification
column was discarded and the purified DNA product was stored at -20˚C.
3.2.6 Ligation of pGEM-T and pAGS/GFP gene
Table 3.2.6: The ligation mixture of pGEM-T and pAGS/GFP gene
Ligation for pGEM-T vector Standard
Reaction
Positive
Control
Negative
Control
2X Rapid Ligation Buffer, T4 DNA
Ligase
5 µl 5 µl 5 µl
PGEM-T Easy Vector 1 µl 1 µl 1 µl
PCR Product 3 µl - -
Control Insert DNA - 2 µl -
T4 DNA Ligase 1 µl 1 µl 1 µl
Deionised water to a final volume of 10 µl 10 µl 10 µl
The reactions were mixed by pipetting. The reactions were incubated 1 hour at room
temperature. Alternatively, the reactions were incubated overnight at 4˚C.
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3.2.7 Cloning of PCR product into pGEM-T Vector
The cloning of the PCR product into pGEM-T vector was lead to the formation of
fragment that consists of pGEM-T/ pAGS/GFP gene. The ligation reaction was set up with
the sample contained of 5 µl of 2X rapid ligation buffer, 1 µl of pGME-T vector, 3 µl of
insert DNA restricted with EcoRI and 1 µl of T4 DNA ligase. The total volume of the
mixtures was about 10 µl per sample. All the reactions were mixed by pipetting gently.
The 1.5 ml of autoclaved Eppendorf tubes was used for ligations. The mixtures were kept
in refrigerator (4ºC) and incubated overnight.
3.2.8 Transformation of pGEM-T/pAGS/GFP Gene in E. coli competent cells
The transformation process was done by using heat shock method based on the
Sambrook’s method of transformation. Before the process is done, the shaker was
preheated 37ºC as well as the water bath to exactly 42ºC. The ligation reaction mixture was
removed from the refrigerator and equilibrates to room temperature for 1 minute. Each
ligation of 2 ml was added to the bottom of a sterile 5 ml Falcon round-bottomed tube that
has been pre-cooled on ice. XLIBlue competent cell was removed from freezer and was
placed in a 50% ice/deionised water bath for 5 minutes. The mixture was mixed by flicking
gently. Competent cells for about 50 ml was added to the Falcon round-bottomed tubes on
ice using wide-bore pipette tips and was pipetted gently. Then, it was left on ice for 20
minutes. The cells were heat shocked for exactly 45 seconds at 42ºC in a water bath. The
cells was returned to ice for 2 minutes. SOC media was added to each transformation and
was mixed by flicking gently. The tubes were closed completely to make it airtight. The
tubes were put in an incubator-shaker at 37ºC for 90 minutes. After 60 minutes, LB with
ampicillin plates were put in 37ºC incubator oven inverted with lids off to dry for 30
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minutes. After the plates are dry and the transformations have been incubated for 90
minutes in the shaker, it was taken to a laminar flow hood. A sterile pipette tip was used;
the transformation cultures were added and spread onto the LB with ampicillin plates. This
was performed for about 4 plates per sample. The plates was sealed with parafilm strip and
placed in a 37ºC dry oven for overnight (Sambrook, Fritsch, and Maniatis, 1987).
3.2.9 Blue/White Screening
The white colony was picked about 5 colonies and subcultured. The subculture was
incubated overnight and the extraction of plasmid was done as in 3.2.2.
3.2.10 Restriction Enzyme Digestion
The specific Restriction Enzyme was selected to digest the plasmid. The double digestion
of restriction enzyme was used to cut both GFP gene and PPICZ A vector at the same
reaction tube. The appropriate reaction buffer for enzymes was used. Plasmid, Restriction
Enzyme, buffer and dH2O was combined in a microcentrifuge tube. The combination was
mixed gently by pipetting. The tube was incubated at appropriate temperature which is
37ºC for 1 hour.
3.2.11 Gel purification
Gel purification was done as in 3.2.5 method.
3.2.12 Ligation of pPICZ A and GFP gene
The enzyme that was used for the ligation of pPICZ and GFP gene is T4 DNA ligase. The
protocol that was used is based on the Fermentas protocol for T4 DNA ligase. The
following reaction mixtures were as followed:
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Table 3.2.12: List of reaction mixture for 1:4 ration of 30 µl.
pPICZ A vector 5 µl
GFP gene (insert) 20 µl
10X T4 DNA Ligase buffer 3 µl
T4 DNA Ligase 2 µl
Total Volume 30 µl
The mixture was mixed thoroughly and spins. Then, it was incubated for 1 hour at 22ºC.
The heat was inactivated at 65ºC for 10 minutes or at 70ºC for 5 minutes (Linker ligation,
2013).
3.2.13 Transformation of pPICZ A and GFP gene into E. coli competent cells
The transformation was done as in 3.2.6 method.
3.2.14 Confirmation of positive transformants by using colony PCR
The confirmation of successful transformants was done by picking 8-10 small colonies in
the plates that contain transformants of pPICZ A/GFP gene. The colonies was streak in the
plates before put into PCR tubes. The PCR protocols was done as in 3.2.4.
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4.0 RESULTS AND DISCUSSION
4.1 The primer design for PCR
The construction strategy was designated to ensure the successful of construction of GFP
gene into pPICZ. The GFP gene was design to be inserted into multiple cloning sites in the
pPICZ vector. In the multiple cloning sites, there are C-terminal peptide which contains c-
myc epitope and a polyhistidine (6xHis) tag. The C-terminal peptide is where the
expression of recombinant protein occurs. There is some cloning consideration of that has
been done before construct the GFP gene into the pPICZ vector. For better initiation of
translation, the insert contain an initiation ATG codon as part of a yeast consensus
sequence (Romanos et al., 1992). In this project, the yeast consensus sequence that has
been chosen is provided below. The ATG initiation codon is shown underlined.
(G/A)NNATGG
For the expression of the gene as a recombinant fusion protein, the clone was designed and
must be frame with the C-terminal peptide containing the c-myc epitope and the
polyhistidine tag. On the other option, if the expression of the gene without the C-terminal
peptide, the stop codon was included.
After all the consideration was done, the design of the specific primer for GFP gene
was conducted. The first step of primer design was conducted after getting the GFP
sequence. The primer was designed 20 nucleotides before the start codon which is ATG
codon. Before designing the primer for PCR, the site of restriction enzyme from pPICZ
and GFP gene sequence were identified. The restriction enzyme site in GFP gene was
identified by using NEBcutter, (n. d.) in the internet, while the restriction enzyme site for
pPICZ was identified by using the map in Invitrogen manual. The selection of restriction
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enzyme in the GFP gene must be the same to the restriction enzyme site in the sequence of
the pPICZ vector. The restriction enzymes that were chosen are Not1 and Xho1. The Xho1
was designed at the upstream of the gene inside the forward primer while Not1 was
designed at the downstream of the gene inside the reverse primer.
Figure 4.1.1: The restriction enzyme site available in GFP gene sequence by using NEBcutter