Investigation of an interaction between the vesicle ... · Investigation of an interaction between...

Ulvi Ahmadov

Uppsats för avläggande av examen Tillämpningskurs Botanik

15 hp Institutionen för Biologi och miljövetenskap

Göteborgs universitet

Investigation of an interaction between the vesicle transport CPSAR1 gene and PORB, PORC, APX4, PSBO2, THF1, LHCB4.1, PAP2, and LHCB4.2 in chloroplasts using a yeast-two-hybrid system

Investigation of an interaction between the vesicle transport

CPSAR1 gene and PORB, PORC, APX4, PSBO2, THF1, LHCB4.1,

PAP4, and LHCB4.2 in chloroplasts using a Yeast-Two-Hybrid System.

Author: Ulvi Ahmadov

Supervisor: Assoc. Prof., Dr. Henrik Aronsson

Bachelor student in Department of Molecular Biology and Genetics in IzTech Erasmus Student at University of Gothenburg Summer Course Project

University of Gothenburg

8/20/2012

Investigation of an interaction between the vesicle transport CPSAR1 gene and PORB, PORC, APX4, PSBO2, THF1, LHCB4.1, PAP4, and LHCB4.2 in chloroplasts using a Yeast-Two-Hybrid System.

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Table of Contents

1. Introduction ..................................................................................................................... -4- 2. Materials and Methods ................................................................................................. -10- - 2.1 Amplification of genes of interest ................................................... -10- 2.1.1 Starting template ....................................................................................... -10- 2.1.2 Plasmid Isolation ...................................................................... -11- 2.1.3 Insert amplification – Polymerase Chain Reaction (PCR) .......... -11- 2.1.4 Introducing restriction sites....................................................... -12- 2.1.5 Gel electrophoresis ................................................................... -12- 2.1.6 Gel purification ........................................................................ -13- -2.2 Cloning ............................................................................................. -13- 2.2.1 Digestion .................................................................................. -13- 2.2.2 Ligation ..................................................................................... -14- 2.2.3 Transformation ......................................................................... -15- 2.2.4 Preparation of LB plate.. ........................................................... -15- 2.2.5 Colony PCR................................................................................. -15- -2.3 Checking possible mutations – Sequencing ..................................... -16- -2.4 Preparation of specific Y2H medias and plates................................. -17- 2.4.1 YPD (Yeast extract peoptone dextrose) .................................... -17- 2.4.2 SD (Selective dropout media) ................................................... -17- 2.4.3 Lithium acetate (1.0 M) ............................................................ -18- 2.4.4 PEG 3350 (50% m/v)................................................................. -18- 2.4.5 Single-stranded DNA Carrier (2mg/ml)...................................... -18- 3. Results ....................................................................................................... -19- -3.1 Co-IP analysis result ........................................................................... -19- -3.2 PCR amplification .............................................................................. -19- -3.3 Cloning................................................................................................ -20- -3.4 Sequencing.......................................................................................... -22- 4. Discussion ................................................................................................. -24- -4.1 PCR amplification .............................................................................. -24- -4.2 Cloning................................................................................................ -24- -4.3 Sequencing.......................................................................................... -26- 5. References ................................................................................................. -26-


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Abstract

Plant development goes through many important steps. One of them is thylakoid biogenesis. Recently Garcia et al. (2010) have shown the importance of a CPSAR1 chloroplast protein (homolog to the Obg protein family) in thylakoid biogenesis. Although the main function of CPSAR1 is still controversial, Aronsson and his colleagues succeeded to detect CPSAR1 proteins attached to the surface of the inner envelope membrane and in the stroma. It’s thought that CPSAR1 initiates vesicle formation and play a role in chloroplast vesicle transport. The next question comes after: Does CPSAR1 work alone or work/interact with some other proteins? Aronsson and his colleagues had continued further analyzes, isolated chloroplasts and by a co-immunoprecipitation method they discovered eight putative proteins (PORB, PORC, APX4, PSBO2, THF1, LHCB4.1, PAP4 and LHCB4.2) to possibly interact with CPSAR1. The next step was to verify the result; and that step was our project. We intended to use a Yeast-Two-Hybrid (Y2H) system to check any possible interaction between CPSAR1 with those eight proteins.

Before performing the Y2H tests there were some important steps that needed to be performed in advance; the genes of interest needed to be ordered, isolated, amplified by PCR and cloned. Except PORB and CPSAR1 (truncated version – 350 amino acids shorter), all genes of interest were successfully amplified by PCR with special genespecific designed primers, cloned and sequenced to verify no mutations being introduced. As a result, all the constructs (gene of interest + vector), chemicals, solutions and plates were prepared and made ready for the Y2H tests.

Because of time limit, we could not start the final Y2H test; however we could prepare everything for the Y2H tests. The only things remaining are the yeast vector transformation and screening, which will be performed by the permanent staff in the lab.


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1. Introduction

Plants are multicellular eukaryotic organisms of the kingdom Plantae. Although plant and animal kingdoms are eukaryotic, plant development differs from animal development in many ways. That is no surprise, because plants cannot move, they are immobile. Plant cells do not migrate as animal cells, development is continuous, they have a great plasticity ability, and can alterate between generations (e.g. haploid and diploid generations in plant life cycle). In addition of developmental differences, plants have chloroplast and they are phototrophic organisms and perform photosynthesis (except parasitic plants). Plants use sunlight to obtain energy via photosynthesis. Photosynthesis refers to a process of converting light energy to chemical bound energy. By photosynthesis plants use carbon dioxide and water; synthesize their food (sugar) and release oxygen as waste product (Figure 1, Bryant and Frigaard, 2006).

Figure 1. The plant photosynthesis overall reaction. Plant use sugar as fuel and release atmospheric oxygen.

Photosynthesis is not the only task of plant chloroplasts. Chloroplasts synthesize amino acids, lipid and phytohormones responsible for gravitropism. In addition to them, the chloroplast has its own DNA (Wataru et al., 2008). The chloroplast genome is mainly involved in protein synthesis and photosynthesis. Although the chloroplast has its own genome, still the genome cannot function alone and needs nucleus-encoded proteins and these proteins are transportedto chloroplasts by a chloroplast targeting sequences called the transit peptide (Vothknecht and Soll, 2005).

Chloroplasts resemble flat discs and covered by an envelope which consists of an outer and inner membrane. Between these membranes is an intermembrane space. Inside chloroplast there is an aqueous fluid, the stroma. Within the stroma there are stacks of thylakoids (grana). The interior spaces of thylakoids are called lumen. Photosynthesis occurs in the thylakoid membrane (Figure 2, Campbell, 2006).

Figure 2. Chloroplast structure. Outer and inner envelop membranes surround the chloroplast and separate it from the cytosol. Thylakoid formed stacks (granum) are located within the stroma.

Chloroplast biogenesis starts from proplastids in shoot meristems and is induced by light (Bauer, 2001). Proplastids lack internal membranes, so they are synthesized inside chloroplasts (Shimojima et al., 1997). Firstly, materials for new membranes need to be


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synthesized, and then a special transport system is needed for carrying new material. New materials could be from the nucleus (chloroplast targeted) or from the plastid envelop. Some proteins have been identified which might play an important role in vesicle transport inside chloroplast (Andersson and Sandelius, 2004).This report focus on the chloroplast protein CPSAR1 which is thought to be responsible for initiation of vesicles mediating thylakoid biogenesis. Ultimately it plays a role in embryo development since T-DNA insertion CPSAR1 mutant plants lack embryo development (Garcia et al., 2010).

Aronsson and his group has further investigated CPSAR1 and performed co-immunoprecipitation (Co-IP) in order to find possible interacting proteins since it is likely that CPSAR1 does not work alone. Immunoprecipitation is a technique which is used to isolate a known protein antigen out of solution by an antibody which binds specifically to the known antigen. Thus, Co-IP precipitate interacting protein complexes including the known protein antigen (Rosenberg and Ian, 2005). After Co-IP analysis using antibodies against CPSAR1, Aronsson and his colleagues discovered eight proteins already known to have a role in chloroplast biogenesis, development or photosynthetic processes. Those genes could be worthy to investigate to further confirm their interaction with CPSAR1: these proteins are PORB, PORC, APX4, PSBO2, THF1, LHCB4.1, PAP4 and LHCB4.2.

Here are shortly a descriptions of the genes of interest: PORB and PORC (NADPH: protochlorophyllide oxidoreductase B/C) proteins have a strictly light-dependent protochlorophyllide oxidoreductase activity and involved in chlorophyll biosynthesis. These genes are expressed from a very early stage of the embryo development and found in the , envelope and the thylakoid membrane. PSBO2 (oxygen evolving complex, photosystem 2 subunit O-2) encodes a protein which is a subunit of photosystem 2 and plays a central role in stabilization of the catalytic manganese cluster; in addition of that, it plays a role in photoinhibition. PSBO2 and PSBO1 are main protein encoding genes of the PSBO family. Those proteins are located in envelope, thylakoid, and stroma. LHCB4.1 and LHCB4.2 (light harvesting complex proteins of photosystem 2) encoding genes involved in photosynthesis, responding to blue, far red light and expressed especially in growth stages of plants. These proteins are located in thylakoid membranes. THF1 (chloroplast-localised thylakoid formation 1) protein encoding gene involved in vesicular mediated thylakoid membrane formation, being located in the stroma and the thylakoid membrane. It is expressed in very early stage of the embryo development and is vital for chloroplast biogenesis. APX4 (ascorbate peroxidise 4/thylakoid lumen 29 (TL29)) lumen protein coding gene is involved in oxidation-reduction processes, responds to oxidative stress and has peroxidase activity. It is localized in the nucleus, cytoplasm, and thylakoid lumen. PAP4 (plastid-lipid associated protein, a fibrillin family protein) protein encoding gene is involved in biological processes that are still unknown, located in thylakoid membranes.1

In order to confirm the Co-IP results, another method is valuable to use, Yeast-Two-Hybrid (Y2H) that could show protein interactions. Y2H is a technique used to screen and discover


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protein-protein interactions. The method is based on physical interaction (bindings) of two proteins; binding of a transcription factor onto an upstream activating sequence activates a downstream reporter genes (Figure 3). The transcription factor is split into two parts: a binding domain (BD) and an activating domain (AD) (Young, 1998).

Figure 3. Yeast-Two-Hybrid. For regular reporter gene transcription, GAL4 BD and GAL4 AD need to interact: A: main principle of Y2H. B, C: No interaction, results with no transcription D: bait and prey interact and transcription of the reporter gene

The two-hybrid vector and the yeast strain used must have same promoter system. Modern two-hybrid vectors utilize ADH1 (or truncated version of it – ADH1*) or GAL promoters. Depending on the experiment, ADH1 encoding vectors are used for higher expression; ADH1* used for lower expression; and GAL encoding vectors have the advantage of being tightly regulated by adding/removing galactose to/from the growth medium. It is also necessary that the yeast strain utilizes same promoter system as the DNA binding domain encoding vector does, so that the bait protein can bind to reporter gene promoter (Buckholz et al., 1999). For GAL4-based yeast strains, the galactose induction system must be knocked out – i.e. mutation of native genes encoding the GAL4 transcription factor and the GAL 80 transcription repressor. The GAL4 gene activates the transcription involved in galactose metabolism. The DNA binding domain binds to GAL4 region and the transcription activating domain interacts with DNA in the GAL1, GAL2 and GAL7 promoter regions to stimulate transcription (Bendixen et al., 1994).

Specific plasmid vectors are engineered in a way that one plasmid produce one protein of interest (bait) fused with the DNA binding domain (GAL4BD); another vector produce a protein of interest (prey) fused with a transcriptional activating domain (GALAD). Both vectors are introduced (mostly by transformation) to the yeast strain which carry the


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reporter gene coupled with GAL1, GAL2 and GAL7 promoter (Hurt et al., 2003). And if both protein products encoded by the bait and prey interact with each other, this interaction will lead to correct position for GAL4BD and GALAD, which will cause reporter gene activation (lacZ). After transformation, the transformants are plated on medium which allows detection of the reporter gene activation. For positive selections, there are some modifications which make Y2H a useful method (Keegan et al., 1996).

Some functional considerations need to be taken into account when performing Y2H, because the method is a long and complex which has many different steps and potential problems. Potential and technical problems start with the cloning work. Cloning is performed to create recombinant DNA from target species and allow their replication within other organisms (host organisms) and create identical copies of target recombinant DNA (Watson and James, 2007). Once the gene of interest is known, in order to start the cloning work, enough DNA needs to be prepared by Polymerase Chain Reaction (PCR). PCR is used to amplify a few copies of DNA up to millions copies. The PCR is based on thermal cycling – repeated cycles with cooling and heating of a polymerase reaction. Short DNA fragments (primers) are designed complimentary to target DNA and the polymerase replicates new strands starting just after the primers (reverse and forward). Newly synthesized strands then become template and the reaction continues exponentially. PCR has three main three; first one is denaturation, in that step the temperature is quite high and leads to denaturation of target DNA, but polymerases are heat-stable and not affected and primers are already single stranded. Second step is annealing; in that step the primers bind to both sense and antisense DNA strands in 5’3´ manner. Third step is elongation; in that step the polymerase starts to synthesize new strands from end of the primers by adding complementary nucleotides (Figure 4,Bartlett et al., 2003).

Figure 4. Polymerase Chain Reaction. The picture shows how PCR works; one cycle contains denaturation, annealing and extension (elongation) steps and cycles repeatedly many times and yield million copies of the target sequences in the end. The picture is revised from Lawley (2012).


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The gel electrophoresis is a method to separate mixed population of DNA/RNA/protein by charge or size (depending on gel type). DNA and RNA fragment mix are separated by length and proteins by charge by applying an electric field, and negatively charged nucleic acids move through the gel. Shorter fragments move faster, migrate further, because shorter fragments can easily migrate through the gel pores (sieving process). For proteins (or nanoparticles) the pores are too large, so separation occurs by charge (Sambrook and Russel, 2001). DNA Gel electrophoresis is mostly used for analytical purpose after PCR. Most common gel type is agarose because it is easy to cast and handle comparing to other gel types (Matt Lewis, 2011).

The following step, enzymatic digestion of amplified target DNA and vectors comes after PCR. A restriction enzyme recognizes and cuts target DNA within a specific sequence, called a restriction site. Such recognition sites are four to twelve nucleotides long. Many restriction enzymes have been identified and synthesized by companies specific to many distinct sequences; and as a result these enzymes can cut potential restriction sites common among almost any locus, gene or chromosome of interest. And also artificial plasmids (from companies) are designed to have a polylinker site (multiple cloning site) inside plasmids which enables insertion of any gene of interest and make cloning possible. The idea is to cut target DNA and vector with same enzymes in order to insert (ligate) the target DNA (insert) to a vector (Hartl et al., 2001). Sometimes it is not possible to find any restriction site (especially while digestion of inserts for ligation to vectors). In that case the restriction site could be introduced by using a restriction site introduced by specific primers using PCR. Such primers have complementary sequences to target and also a non complementary restriction (5’ end) site which would be introduced after a few cycles of PCR (Witchel, 1996). Restriction enzymes cut in two ways, either with cohesive ends (sticky) or non-cohesive end (blunt). In molecular biology, especially in cloning, sticky end digestion is desired since ligation is much more precise for sticky end cut DNA molecules (Sambrook et al., 2001). For ligation of digested insert with vector plasmid a ligase (enzyme) is needed. Ligases catalyze two large molecules by forming new chemical bonds (Subramanya et al., 1996).

The following step is introduction of a foreign DNA (vector plus insert in that case) into a host organism. Transformation is one of three methods of DNA introduction (others are conjugation and transduction) and refers to uptake of foreign DNA from the surrounding through cell membrane(s), and expression of exogenous DNA which results in genetic alteration of a cell. Transformation occurs naturally in some bacteria species. In molecular biology transformation occurs artificially. For bacteria, cells must be in a competence state which means ability to uptake foreign DNA. Competence ability could be naturally or it could be artificially induced (Chen and Dubnau D, 2004). Transformation of foreign DNA into eukaryotic cells is called transfection, because transformation refers to progression of cell to a cancerous state (Alberts and Bruce et al., 2002).

http://en.wikipedia.org/wiki/Bruce_Alberts


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In order to check the success of transformation/transfection selectable markers are used by biologists. Selectable marker is a gene which is a type of reporter gene. It could be an antibiotic resistance gene, nutritional or any other screenable genes. Antibiotic resistance gene is subjected to bacteria cells and by introducing plasmid vector with an antibody resistance gene would ensure only growth of positive transformant cells on media which has antibiotic. Nutritional selectable marker gene is used for yeast strains to screen positive transformants. In that case yeast media plates lack some amino acids and only cells with the plasmid would survive (Reyrat, 1998). Although cells are not supposed to grow on selective plates if they do not have foreign DNA (because exogenous DNA provides resistance or nutritional support), colony PCR is needed to ensure plasmid presence. The basic idea of PCR is every time same, in colony PCR the bacteria cells are checked and provide DNA. Because of high temperature in PCR, DNA release cells and primers bind, and polymerase synthesize new strands (Pavlov et al., 2006). However, positive result in colony PCR does not guarantee that there is not any mutation. One way to solve this problem is sequencing which means a process to read the nucleotide bases in the DNA sample (Olsvik et al., 1993). Most common method is chain-termination method, because it is very efficient and includes less toxic chemicals. For sequencing a DNA template and a suitable primer are needed. The idea is using addition to dNTPs (deoxynuclotides) using radioactively or fluorescently labeled ddNTP (dideoxynucleotides) to terminate elongation by polymerase, because ddNTPs lack 3’-OH group which is needed to make phosphodiester bond between two nucleotides and that results in DNA fragments that vary in size (one nucleotide). The newly synthesized DNA fragments are separated by size with one nucleotide resolution using polyacrylamide-urea by gel electrophoresis method. Then the gel is visualized by autoradiography or UV-light and the images are analyzed and the nucleotide order of DNA is discovered (Smith et al., 1986)

Functional considerations in Y2H do not end with cloning work, it continues with plasmid introduction into yeast cells. Because designed plasmids would be met with in vivo criteria inside yeast cells. The protein of interest should be expressed to be able to generate an interaction but not expressed too much which cause toxicity e.g. if both plasmid vectors have strong promoters In addition, expressed proteins definitely must be folded properly and they should be stable, localized into nucleus and capable of dimerization. It is not necessary expressed protein of interests are fully functional, if the protein of interest is known and putative interactive site is also know, even using smaller fragment is also possible, just a stability, localization and folding of protein of interest are important. If interaction is detected, that may not be end of project; there are some more technical obstructions. For validation, yeast cells need to be isolated and rescue plasmids, checking nucleotides by sequencing or carry out additional cloning and screen it again to ensure the result (Philips James, 2001).

In our experiment we decided to use pAS1 plasmid vector, a popular TRP-1 marked GAL4-based BD vector for bait protein; pGAD24 vector, another useful LEU-2 marked GAL4-based AD vector for prey proteins. Because both vectors have nutritional markers, the positive


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selection of reporter gene activation is possible and plates with lacking both (Selective Dropout (SD)) would ensure growth of yeast cells with both plasmid vectors. In Y2H there are also some yeast strains which are modified and engineered for the high efficient transformation. We have chosen the Y187 yeast strain which engineered and commercially sold by ClonTech Company (Fromont-Racine et al., 2008). The genotype of Y187 is MATα, ura3-52, his3-200, ade2-101, trp1-901, leu2-3, 112, gal4Δ, met–, gal80Δ, URA3::GAL1UAS-GAL1TATA-lacZ.2

2. Materials and methods

2.1 Amplification of genes of interest

2.1.1 Start template

The genes of interests (PORB, PORC, APX4, PSBO2, THF1, LHCB4.1, PAP4 and LHCB4.2) are ordered from the Arabidopsis Biological Resource Centre at the Ohio State University in USA. The genes of interests were already inserted in pUNI51 vectors (has Kanamycin resistance) which were introduced to cells of the PIR1 E. coli strain. Because the main research of Aronsson and his colleagues were CPSAR1, they provided us with CPSAR1 and also a truncated CPSAR1 (lack 350 amino acids from the N-tail which shall not play a role in interaction and furthermore it might cause troubles in yeast introduction and proper folding) inside p123 vector which was introduced to DH5α E. coli strain cells.

2.1.2 Plasmid isolation

In order to start pre-step to cloning, plasmids needed to be isolated. One day before isolation process bacteria cells were cultured overnight in Lysogeny Broth (LB) nutritional rich media (for growth of bacteria cells) with Kanamycin (50 µg/L) :

1. 20 g LB media (20g/L) 2. Fill up to 1 litre deionised H2O 3. Autoclave for 20 min 4. 1 ml 50 mg/L Kanamycin (when it is about 50 ̊C)

-and two colonies added to 5 ml liquid LB broth media from each gene from ordered E. coli plates, E.coli was cultured at 37 ̊C overnight.

Next day all the plasmids with our genes of interest were isolated by Qiagen Plasmid Isolation Kit. The main isolation steps were:

1. 3 min centrifuge at 14 000 rpm (revolution per minute) 2. Resuspension of pellet by Resuspension buffer 3. Cell lyses by lysis buffer 4. Neutralization of lysis buffer by Neutralization buffer 5. Washing all debris away


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6. Elute DNA by Elution buffer.

After isolation, the plasmids were run and checked in 1% agarose gel. Then the concentration of each plasmid was detected by NanoDrop. All the samples were stored at -20 ̊C.

2.1.3 Insert amplification – Polymerase Chain Reaction (PCR)

After plasmid isolation, all genes were amplified up to enough amounts (1.5-2 µg) for Cloning experiment i.e. for being inserted into vectors. The amplification step was based on PCR and we analysed the products by running them on 1% agarose gel to verify their correct size. For this insert amplification we used a Phusion High Fidelity polymerase to increase yield and efficiency. Reactions were either based on 20 µL aor50 µL for all genes. A typical PCR of 20uL reaction is shown below (for 1 reaction):

1. DNA 100 ng 2. Phusion HF 5X Buffer 4 µL 3. dNTP 0.4 µL (10 mM) 4. Forward primer 1 µL (10 mM) 5. Reverse primer 1 µL (10 mM) 6. Phusion HF polymerase 0.2 µL 7. dH2O up to 20 µL

-and the PCR program was used and it was followed for all insert amplification steps:

1. Initial denaturation 98 ̊C for 1 min; 2. Denaturation 98 ̊C for 10 sec; 3. Annealing 60 ̊C for 20 sec; 4. Extension 72 ̊C for 1 min; 5. Go to step 2; Repeat 34 cycles; 6. Final Extension 72 ̊C for 10 min; 7. Wait 4 ̊C continuosly; 8. End.

2.1.4 Introducing restriction sites

Because we aimed to digest the inserts later for downstream cloning steps, we used specific primers (forward and reverse) to introduce our restriction sites. The primers were designed to introduce the desired restriction site by adding sequences from 5’ end. Below there are the restriction sites introduced to inserts by PCR:

I. CPSAR1: Nco1 and BamH1 II. CPSAR1: Nco1 and Xma1

III. Truncated CPSAR: Nco1 and Xma1 IV. PORB: EcoR1 and Pst1 V. PORC: EcoR1 and Sal1

VI. APX4: EcoR1 and Pst1


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VII. PSBO2: EcoR1 and Pst1 VIII. THF1: Xma1 and Pst1

IX. LHCB4.1: EcoR1 and Pst1 X. PAP4: EcoR1 and Pst1

XI. LHCB4.2: EcoR1 and Pst1

2.1.5 Gel electrophoresis

We used agarose (1%) for Gel electrophoresis. Agarose gels were used for checking and separating the templates, PCR success, checking sizes of PCR products, rescue right size PCR products by gel purification, checking positive colony PCR. The 1% agarose gel contains 1 g agarose (1 g/ml) dissolved in 100 ml 1X TAE buffer (40 mM Tris base, 1% Acetic acid, 2 mM EDTA, pH 8.1-8.3). In order to run the samples in the gel, they were prepared before running. The standard 12 µL gel sample per each well used is shown below:

1. DNA 200 ng (sample) 2. 6X Fermentas Loading Dye 2 µL (color marker) 3. GelStar® 1.8 µL (fluorescent DNA detecting stain) 4. dH2O up to 12 µL

The samples were run at 60-120 V (depending on gel length) for around 1 hour. The optimal voltage was five times the gel length. The band sizes were detected by a New England BioLabs Inc. DNA 1 kb Ladder:

1. DNA 1 kb Ladder 2 µL 2. 6X Fermentas Loading Dye 2 µL 3. GelStar® 1.8 µL 4. dH2O 6.2 µL

The gel was screened by exposing it to UV-light and analysed by a GeneSnap computer software program from Syngene.

2.1.6 Gel purification

In order to save and rescue PCR products run on gel, which corresponded to our gene of interest, we cut the gel containing the band of interest and cleaned the gel piece by a Qiagen Gel Extraction Kit. The main steps were:

1. Resolubilize the gel by Resolubilization buffer 2. Rescue DNA by Binding buffer 3. Washing away gel and other artefacts 4. Elute DNA by Elution buffer.

The columns were spun by centrifugation at 5000 g force in each step. The elution buffer was heated up to 30 ̊C before use in order to increase yield.


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2.2 Cloning

Cloning of genes of interest was performed using four stages: Digestion, Ligation, Transformation and Colony PCR:

2.2.1 Digestion

As mentioned before, all genes of interest were amplified by PCR (2 µg) before the digestion step and we called them ‘inserts’ for vectors. Each gene was digested by the specific restriction enzymes already introduced by PCR by the help of specific primers. In the Yeast-two-hybrid system CPSAR1 is referred as the ‘bait’ protein and the other proteins are called ‘prey’ protein, thus they need different vectors. Because of that we decided to use pAS1 for CPSAR1 and pGAD424 for the preys.

We cut pAS1 and CPSAR1 with Nco1 and Xma1; PORB, PSBO2, LHCB4.2, PAP4 and pGAD424 with EcoR1 and Pst1; PORC, LHCB4.1, APX4 and pGAD424 with EcoR1 and Sal1; THF1 with Xma1 and Pst1.

-The digestion set for all inserts for a 40 µL reaction:

1. DNA 34 µL 2. Enzyme 1 µL 3. Enzyme 2 1 µL 4. Buffer 4 µL 5. BSA 1 µL (only for Xma1 usage)

- The digestion set for vectors for 20 µL reaction:

1. Vector 15 µL 2. Enzyme 1 1 µL 3. Enzyme 2 1 µL 4. Buffer 2 µL 5. BSA 1 µL (only for Xma1 usage)

-After overnight digestion of inserts and vectors, we checked on gel for any star activity, overdigestion or other potential problems had occurred. For comparison of vectors, we run both cut and uncut intact plasmids, because intact plasmids have more than one conformational structure and mostly they supercoil (their size were smaller than cut plasmid)

After digestion, the digestion mix for all genes were cleaned from enzymes by adding binding buffer, then washed by washing buffer and eluted with elution buffer. The samples’ concentration was checked by NanoDrop, and then stored at -20 ̊C.


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2.2.2 Ligation

This step combines the inserts with specific vectors. After digestion, we ligated the insert with the vectors also digested by same restriction enzymes. We set the ligation reaction to a 3:1 ratio (insert vs. vector). The calculation for the reaction was based on the equation:

ng of Insert = ((ng of Vector*length of Insert in kb)/length of Vector in kb)*3/1

The ligation reaction was set at 20 µL for about 6 hours at room temperature:

1. Insert 100-300 ng 2. Vector 250-300 ng 3. Buffer 4 µL 4. T4 Ligase µL 5. dH2O up to 20 µL

CPSAR was ligated with pAS1 and the other constructs were all ligated with the pGAD424 vector.

2.2.3 Transformation

Once the ligation was completed after 6 hours, we immediately switched to the transformation step. This step refers to transformation of the constructs to a competent E. coli strains. We used DH5α, TOP10 or JM109 E. coli strains as competent cells. The protocol used:

1. Adding 5 µL ligation mix to 25-100 µL competent cells 2. 20-30 min incubation on ice 3. 25-50 sec heat-shock at 42 ̊C (depending on strain) 4. 5 min incubation on ice 5. Adding 250-950 µL SOB (depending on strain) 6. 1-1.5 hour shaking at 37 ̊C 7. Plating new plates by adding 50-400 µL the transformed E. coli strains 8. 16-24 hours growth at 37 ̊C

2.2.4 Preparation of LB plates

The LB plates were prepared for plating the transformed E. coli strains. In order to select the deserved colonies, we prepared LB plates with antibiotic (Carbenicillin). Both vectors (pAS1 and pGAD424) had ampicillin resistance, so we used another homolog of ampicillin, called as carbenicillin, because it is much more stable than ampicillin. The plates made like that:

1. 20 g LB broth media (20 g/L) 2. 8 g of Agar (for thickening, 8 g/L) 3. Deionised H2O up to 1 litre 4. Autoclave for 20 min


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5. Add 1 ml of 50 mg/L carbenicillin (when at approximately 45C ̊) 6. Pouring to 50 ml Petri dishes

The agar was solidified and plates stored at +4 ̊C prior to use.

2.2.5 Colony PCR

Successful transformation step was observed as colonies growth in the plates (because of antibiotic resistance due to pAS1 and pGAD424). Thus, a colony PCR was performed to check colonies whether they had retained the construct. The colony PCR is based on adding colonies to a PCR reaction mix by touching the colonies by pipette tip and then the reaction is carried out with Tag Polymerase. For a 20 µL reaction:

1. Tag Polymerase Buffer 2 µL 2. dNTP 0.4 µL (10 mM) 3. Forward primer 1 µL (10 mM) 4. Reverse primer 1 µL (10 mM) 5. Tag Polymerase 0.1 µL 6. dH2O 15.5 µL

Selected colonies were moved into new plated with LB Carbenicillin (50 µg/L) in order to find positive selected colonies later on for usage. The PCR program used:

1. Initial denaturation 96 ̊C for 1 min; 2. Denaturation 96 ̊C for 30 sec; 3. Annealing 60 ̊C for 30 sec; 4. Extension 72 ̊C for 1 min (2 min for CPSAR); 5. Go to step 2; Repeat 30 cycles; 6. Final Extension 72 ̊C for 10 min; 7. Wait 4 ̊C for continuously 8. End

About 20-30 colonies for each gene were tested and positive colonies were one observed based on a PCR product of correct size , These positive colonies were grown in liquid LB media (+Carbenicillin) overnight and the constructs were isolated by a Qiagen Plasmid Isolation KIT. The plasmid concentrations were measured by NanoDrop. The isolated plasmids (with genes of interest) were stored at -20 ̊C.

2.3 Checking possible mutations – Sequencing

Two construct from each positive colony of genes of interest were sent for sequencing to GATC BioTech Company to confirm correct gene. For CPSAR we sent three constructs. The sequencing is based on PCR and only one primer could be added to each reaction:

1. DNA 500-600 ng 2. Primer 5 µL (final molarity was 5 µM) 3. dH2O up to 10 µL


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For the constructs PORB, PORC, APX4, PSBO2, THF1, LHCB4.1, PAP4 and LHCB4.2, a reverse primer of gene of interests and a pGAD424 sequencing primer 1 were used. For the CPSAR, CPSAR forward/reverse sequencing primer and pAS1 sequencing primer were used. The sequencing analysis was performed using the free software ChromasLite and the BLAST algorithmic program from NCBI. The sequences were read by Chromas and blasted against the Arabidopsis thaliana genome. The main focus was to verify that our sequences (from sequencing) were similar to the original one in genome.

2.4 Preparation of specific Y2H medias and plates

For Y2H media and solutions are needed for growth and transformation. The samples prepared were in enough amounts and saved for the Y2H tests.

2.4.1 YPD (Yeast extract Peptone Dextrose)

The YPD media was used to grow yeast strains. There was not any selection marker included in the media. The media prepared:

1. Difco peptone 20 g/L 2. Yeast extract 10 g/L 3. Autoclave for 20 min 4. 20% 100 ml distilled Dextrose (glucose solution)

-for YPD plates agar is added (thus, called YPDA):

1. Difco peptone 20 g/L 2. Yeast extract 10 g/L 3. Agar 8 g/L 4. Autoclave for 20 min 5. 20% 100 ml distilled Dextrose 6. Pouring to 50 ml Petri Dishes

The liquid YPD and plates were sealed and stored at 4 ̊C.

2.4.2 SD (Selective Dropout media)

The media used for selection of positive transformed yeast colonies. The selective marker of the pAS1 plasmid vector was Tryptophan (TRP1), and for pGAD424 it was Leucine (LEU2). Thus, the SD media prepared lacked either tryptophan (for checking auto activation of bait) or tryptophan and leucine (for checking colonies which have both constructs). The media prepared:

1. Yeast nitrogen base without amino acids 6.7 g 2. Dropout amino acid mix lacking TRP/LEU+TRP 1.92 g/1.54 g 3. Adjustment of pH to 5.8-6 4. Agar 9 g 5. Distilled water 900 ml 6. Autoclave for 20 min 7. 20% 100 ml distilled Dextrose 8. Pouring to 50 ml Petri Dishes


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The plates were sealed and stored at 4 ̊C

2.4.3 Lithium acetate (1.0 M)

A lithium acetate solution was made for yeast transformation:

1. Adding LiAc 5.1 g 2. dH2O 50 ml 3. Autoclave for 20 min

It was kept at room temperature and different concentrations of LiAc solutions were made depending on the yeast transformation protocol to be used.

2.4.4 PEG MW 3350 (50% m/v)

Polyethylene glycol solution was made for yeast transformation:

1. PEG 50 g 2. Dissolving PEG in 30 ml dH2O by heat 3. dH2O up to 100 ml 4. Autoclave for 20 min

The PEG 3350 solution was stored at room temperature.

2.4.5 Single-stranded carrier DNA (2mg/ml)

SS-DNA is used in yeast transformation for increasing the yield, it binds to yeast cell walls and prevent the plasmid to leave the cell. SS-DNA solution:

1. Salmon sperm 200 mg 2. TE buffer 100 ml 3. Stirring for 2 hours at 4 ̊C

SS-DNA solution was stored at -20 ̊C and before use DNA was denaturated in a boiling water bath for 5 min and chilled on ice for some time prior to use.

Note: Yeast two-hybrid tests will be included after some time.

3. Results

Note: During the project time there were many steps with many genes and trials, thus I got many analyse results in gel pictures and the DNA concentration. It would be too long and unnecessary to show all pictures and NanoDrop results, thus I had to show some of them as examples for all genes and repeated steps.


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3.1 Co-IP analyze result

The genes of interest were selected based on previous Co-IP results performed by Aronsson et al. The Co-IP gel analysis showed mass of precipitated proteins with different weights. Preserum were used as control in order to eliminate background noise and artefacts. Gel screening resulted in five protein bands with different size, which were not in preserum, and after proteomics analyses putative proteins were identified which had precipitated with CPSAR1 by using the antibody specific for CPSAR1. Mass spectrometer analyses resulted with lots of ribosomal proteins (which were already ignored) and some essential genes, which are important for chloroplast and chloroplast biogenesis. From 5 bands, proteins from second, third and fourth band were in same size like in SDS gel analyse; proteins from first and fifth band did not match with SDS result, so they were omitted. From those three bands eight putative proteins have been identified (Figure 5). Here is the Co-IP result from Aronsson et al.:

antibody Preserum

Preliminary Data after MS AnalysisCo-Immunoprecipitation

1) 103

2) 46

3) 34

4) 24

5) 12.5

KDa

Figure 5. Co-IP result. The figure shows the Co-IP result of Aronsson et al. while investigating any interacting proteins of CPSAR1 for vesicular transport. The result shows some proteins precipitated with CPSAR1. First well shows Co-IP result of isolated chloroplasts and second well is control - preserum to detect background noise. The proteins have been detected after removingthe background. Eight intersting proteins were detected all essential for chloroplast biogenesis. (The picture was taken from Nadir Z. Khan’s project and used by permission from him ).

3.2 PCR amplification.

The starting templates were checked as afirst step. They all worked well and we could detect all constructs (pUNI51 vector + gene of interest) in a 1% agarose gel. The genes of interest (except truncated CPSAR1 (1.52kb)- our collegues had it before) were inside a pUNI51 plasmid. Firstly the constructs were isolated and then run on 1% agarose gel to test whether they existed or not (Figure .6). The pUNI51 plasmid vector is 2.55 kb and the genes of interest: CPSAR1 is 1.97 kb, PORB and PORC are 1.23 kb, APX4 is 1.10 kb, PSBO2 is 1.02 kb, THF1 is 0.93 kb, LHCB 4.1 is 0.90 kb, PAP4 is 0.75 kb and LHCB 4.2 is 0.71 kb. After gel electrophoresis we had all templates needed to start the PCR amplification pre-step to cloning:


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Figure 6. Start template control. . All the start templates, plasmids, were run on 1% agarose gel. The picture shows that the isolation was successful and we had templates to start amplification of genes of interests. The letters corresponds to: L: 10 kb DNA ladder; 1: pUNI51 + CPSAR1; 2: pUNI51 + PSBO2; 3: pUNI51 + PORC; 4: pUNI51 + THF1; 5: pUNI51 + APX4; 6: pUNI51 + PAP4; 7: pUNI51 + PORB; 8: pUNI51 + LHCB4.1; 9: pUNI51 + LHCB4.2

The genes of interests were amplified by PCR successfully and for checking the reaction success, PCR products were separated on 1% agarose gel for each gene. Once we observed band(s) in right size(s), then the PCR product were cleaned from enzymes and the concentration were measured. Here is one representative example of all the PCR amplification result, this one showing the CPSAR1 gene (Figure 7):

Figure 7. CPSAR1 amplification. The picture shows the amplification step for CPSAR1. In order to clone the gene, we made several PCR reactions to gain enough final yield: L: ladder; 1, 2 and 3: CPSAR1 with introduced Nco1 and Xma1 restriction sites; 4, 5 and 6: CPSAR1 with introduced Nco1 and BamH1 restriction sites

3.3 Cloning

All the genes of interest were amplified up to 1.5–2 µg DNA by PCR. Once there were enough DNA, the digestion was set and after digestion it was checked on 1% agarose gel and below is some representative results of them (Fig.8):


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Figure 8. Checking the inserts after restriction enzyme digestion.

Although it is hard to detect a few base pairs size difference after digestion, still gel electrophoresis can show whether any overdigestion or star activity occurred: L: ladder; 1: CPSAR1 (with BamH1 restriction site); 2: PORB; 3: PORC; 4: APX; 5: PSBO2; 6: THF1; 7: LHCB 4.1; 8: PAP4; 9: LHCB 4.2.

For the vectors, comparison of cut (digested) and uncut vectors would have differences in gel electrophoresis, because uncut intact plasmid vector has more than one conformational structure. Thus in gel electrophoresis it is highly expected to see more than one band unlike cut plasmid (Figure 9). Below is one example which is used for pGAD424:

Figure 9. Comparison of cut and uncut plasmid vector. The figure shows how we ensured the digestion of the vectors as an example of pGAD424 vector and it helped to compare intact and cut vectors. Note that intact plasmids have more than one band and differ from cut plasmid: L: ladder; 1: Intact pGAD424; 2: Cut pGAD424 (with Ecor1 and Sal1); 3: Intact pGAD424; 4: Cut pGAD424 (with Xma1 and Pst1).

Once the restriction enzyme digestion was successful, a ligation reaction was performed to check the reaction success. The ligation mix was loaded on a gel. Below is one representative result (Figure 10):

Figure 10. Checking ligation reaction success.

In order to introduce constructs to bacteria cells, one has to be sure to have constructs with correct size. The picture shows how we checked as in the example of the CPSAR1 + pAS1 construct. Note that CPSAR1 is 2 kb, and the pAS1 vector plasmid is 7.1 kb. To see one clear band was optional: L: ladder; 1:CPSAR1 + pAS1 (ligation mix).


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Transformation has been performed with different competent cells, thus the efficiency varied, still all genes of interest could have been introduced and the positive colonies have been detected by running colony PCR products on 1% agarose gel from transformed bacteria cells. Except truncated CPSAR1 and PORB, transformation step worked very well and we had enough positive colonies for the other genes of interests. Here is one example from LHCB4.1 and LHCB4.1 colony PCR results after transformation (Figure 11):

Figure 11. Checking positive transformants after colony PCR. Figure shows how positive colonies (with the introduced plasmid vector) were detected by running colony PCR products on 1% agarose gel in LHCB 4.1 (upper part of the gel) and LHCB 4.2 (lower part of the gel) examples. LHCB4.1/4.2 constructs were introduced to the TOP10 E. coli strain and by colony PCR the labelled colonies were investigated. Note that LHCB4.1/4.2 are around 0.9 kb and we got 14 positive colonies from LHCB4.1 and 16 positive colonies from LHCB4.2.

3.4 Sequencing

From each gene we got five to sixteen positive colonies. Those positive colonies were labelled and then the constructs were isolated from them. The concentrations of plasmid vectors were around 600 ng and they were prepared for sequencing. For sequencing, two good colonies (the one who had highest concentration and good gel picture) were selected and run on 1% agarose gel before sending to sequencing (Figure12):

Figure 12. Checking constructs before sequencing. Figure shows the isolated plasmid vectors carrying the genes of interest (constructs). The constructs were successfully isolated and run on 1% agarose gel. As a result they were in correct size and good for sequencing: L: ladder; 1,2 and 3: CPSAR1+pAS1 construct (totally should be around 9kb); 4 and 5: PORC+pGAD424 (~8kb); 6 and 7: APX4+pGAD424 (~7.8kb); 8 and 9: PSBO2+pGAD424 (~7.7kb); 10 and 11: THF1+pGAD424 (~7.6kb); 12 and 13: LHCB4.1+ pGAD424 (~7.6kb); 14 and 15: PAP4+pGAD424 (~7.4kb); 16 and 17: LHCB4.2 9~7.4kb).


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The sequencing analyses showed that cloned templates were good enough to use for Yeast-two-hybrid. Here are the results shown in Table 1 after sequencing gene by gene:

Serial number

Sample name-number of colony

Primer name Max. Identity

Result

1 CPSAR1-1 CPSAR1 forward primer 100% No mutation 2 CPSAR1-1 pAS1 sequencing primer 100% No mutation 3 CPSAR1-2 CPSAR1 forward primer 100% No mutation 4 CPSAR1-2 pAS1 sequencing primer 99% No mutation 5 CPSAR1-3 CPSAR1 forward primer - Failure 6 CPSAR1-3 pAS1 sequencing primer - Failure 7 PORC-1 PORC reverse primer 100% No mutation 8 PORC-1 pGAD424 sequencing primer 100% No mutation 9 PORC-2 PORC reverse primer 100% No mutation

10 PORC-2 pGAD424 sequencing primer 100% No mutation 11 APX4-1 APX4 reverse primer 100% No mutation 12 APX4-1 pGAD424 sequencing primer 100% No mutation 13 APX4-2 APX4 reverse primer - Failure 14 APX4-2 pGAD424 sequencing primer 100% No mutation 15 PSBO2-1 PSBO2 reverse primer 100% No mutation 16 PSBO2-1 pGAD424 sequencing primer 100% No mutation 17 PSBO2-2 PSBO2 reverse primer 100% No mutation 18 PSBO2-2 pGAD424 sequencing primer 100% No mutation 19 THF1-1 THF1 reverse primer 100% No mutation 20 THF1-1 pGAD424 sequencing primer 100% No mutation 21 THF1-2 THF1 reverse primer 100% No mutation 22 THF1-2 pGAD424 sequencing primer 100% No mutation 23 LHCB4.1-1 LHCB4.1 reverse primer 99% No mutation 24 LHCB4.1-1 pGAD424 sequencing primer 100% No mutation 25 LHCB4.1-2 LHCB4.1 reverse primer 100% No mutation 26 LHCB4.1-2 pGAD424 sequencing primer - Failure 27 PAP4-1 PAP4 reverse primer 100% No mutation 28 PAP4-1 pGAD424 sequencing primer 100% No mutation 29 PAP4-2 PAP4 reverse primer 100% No mutation 30 PAP4-2 pGAD424 sequencing primer 100% No mutation 31 LHCB4.2-1 LHCB4.2 reverse primer 100% No mutation 32 LHCB4.2-1 pGAD424 sequencing primer 100% No mutation 33 LHCB4.2-2 LHCB4.2 reverse primer 100% No mutation 34 LHCB4.2-2 pGAD424 sequencing primer 100% No mutation 35 CPSAR1-3 CPSAR1 reverse primer 99% No mutation 36 CPSAR1-3 CPSAR1 reverse primer 99% No mutation 37 CPSAR1-3 CPSAR1 reverse primer - Failure

Table 1. Sequencing result. The table shows sequencing analysis of constructs for checking any possible mutation which can be result of many steps in cloning. Two-three colonies from each gene of interest with two-three different primers were sequenced and totally 37 positive constructs have been checked. As a result, except CPSAR1 (3rd colony), there was not any significant mutation on genes of interest sequences and all of they were good enough to use for Y2H.


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4. Discussion

4.1 PCR Amplification

Garcia et al. (2010) proposed the presence of putative CPSAR1 partners and this lead to another project attributed to find putative CPSAR1 partners. Y2H have been performed on that reason and fortunately some proteins have been detected and identified. Genes of interest were actually quite important genes for chloroplast and its development, Vesicular transport system inside chloroplast is very essential for chloroplast development, thus assays on this subject is important to perform.

Up to Y2H tests, the genes of interest were able to be cloned and prepared successfully. The start templates were ordered and fortunately we had not any problem with start template. The plasmids were isolated with two different isolation kits and the best one selected (for later use) and the elution buffer heated in advance in order to increase yield. At later steps we could detect all the desired genes of interests after a few PCR trials.

All designed primers worked very well for each individual gene of interest. Primers were designed to introduce restriction sites to genes of interest. The restriction sites added for our desire gene and also compatible with plasmid vectors which have been used in cloning (pAS1 and pGAD424). The PCR program for amplification was finally 60 ̊C in all reactions although we initially tested lower and higher temperatures.

High Fidelity Phusion polymerase (Finnzymes) was a perfect choice to eliminate nonspecific PCR product. In the amplification part, the step which needed luck was gel cleaning. Sometimes we observed more than one band and we had to cut the gel including DNA with right size, clean and rescue the DNA from gel. That part is hard, because sometimes one loses a lot of DNA after cleaning and thus not enough to use as template. Still none of genes of interest have failed to be amplified in PCR amplification step and we were able to amplify all genes of interest up to enough amounts for the following cloning steps.

4.2 Cloning

All the genes of interest were successfully digested and ligated to vectors. For the transformation, all genes except PORB and the truncated CPSAR1 were successfully introduced.

Digestion step was quite successful; we already introduced the restriction sites into our genes of interest and checked several times the cDNA of genes of interest to avoid any possible false digestion. Our vectors already had the restriction sites which we introduced to our genes of interest. All the enzymes were suitable for double digestion, but we had some doubts about Xma1, fortunately it also worked well and we also found out that even though BSA was it also worked without BSA. To conclude, digestion reactions of all genes of interest and vectors were successful.

Ligation step worked well as we just set the reaction and fortunately nothing wrong or unexpected event happened. Sometimes we had to check the ligation mix because of failure of transformation. The T4 ligase was quite successful and stable enough. The reaction time was not so strict, overnight or 5-6 hours both worked fine which made our work much easier.


Page 24

The competent cells were definitely the key of success of the transformation; competent cells from companies (even differs from one company to another) and from handmade stock differs dramatically. Once we made our own competent cells but they were not good. Company made competent cells made the transformation step much easier and much more efficient. All constructs were introduced to E. coli competent cells; the most efficient competent cells were JM109 cells from Promega , then comes TOP10 and DH5α from Invitrogen and the less efficient was self-made Top10 competent cells. Another advantage of using company made competent cells were higher number of positive transformants, so from each plate we got more than enough positive colonies. The heat shock time was a tricky part; because bigger constructs (like CPSAR1+pAS1) needed more time and bigger pores. Depending on the competent cells, we almost exposed ten more seconds for CPSAR1-pAS1 construct and could increase yield, and efficiency of transformation. To conclude, all genes were introduced, cultured into plates and bacteria cells survived.

The hardest and trickiest part of cloning was colony PCR and fortunately we could succeed to find out the positive colonies without much experience. It was hard to put some DNA (inside colony) and avoid bubbles. We tapped, behaved extra gentle and have done analyses gene by gene, sometimes plate by plate and avoid harm from polymerase activity. Except PORB and the truncated CPSAR1, we could get lots of positive colonies from all constructs. Once we used company made competent cells, we got much efficient transformation, and also lots of positive transformants in the same way. The key of success were firstly numbering every colonies which were analysed with colony PCR, thus it was not complicated to find positive colonies after PCR. Once we got positive result from PCR, we detected the number of colony and isolated it for sequencing.

4.3 Sequencing

Sequencing ensured the positive constructs from the colony PCR. Both forward and reverse primers for each construct have showed there were no mutation (except one CPSAR1 construct) and we got two constructs per each gene for future Y2H tests.

Primers could sequence maximum 1.4 kb, sometimes much shorter, so we had to combine results from both forward and reverse primer results in order to conclude something. My personal thought is it would be better to have a third primer (for middle part) for CPSAR1. Still forward (CPSAR F.P and pAS1) and reverse primers could sequence around 1.2 kb that is actually enough in that case. We now have starting templates for Y2H ready for tests.

4.4 Yeast-two-hybrid

Because of the time limit, we could not start the two-hybrid tests which would take quite some more weeks. I wish my colleagues would have time for that and continue our job from where we stopped. When we look the whole summer course, I think we were quite successful and lucky to do so much work. To be able to prepare everything for Y2H is a great work and actually a tough part.


Page 25

Acknowledgments

I am very deeply thankful to my supervisor Assoc. Prof. Dr. Henrik Aronsson for giving me the opportunity to improve myself, learn new techniques and to gain self-confidence. His constant support and guidance helped me to adopt science life easily.

I am very deeply honoured to know and work with my colleagues: Christopher Ayres, Sazzad Karim, Nadir Z. Khan, Mohammed Alezzawi, Selvakumar Sukumaran and Emelie Lindquist. I want to thank for their helps, support and advices. They were for me as brothers and a sister, never begrudged their limitless helps from me and never gave up teaching us. Without their help and deep knowledge it would be really hard to succeed something.

I am very grateful to Filipe de Souza and Noriaki Tanabe for their support and help as good friends and colleagues.

After all, I thank University of Gothenburg, Sweden for providing such good experience, and I should not forget the Erasmus Exchange Program, it was the reason how I met with such good people and had chances to improve myself.

5. References

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Campbell, Neil A.; Brad Williamson; Robin J. Heyden (2006). Biology: Exploring Life. Boston, Massachusetts: Pearson Prentice Hall. ISBN 978-0-13-250882-7.

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Fromont-Racine, Micheline; Rain, Jean-Christophe, Legrain, Pierre (1997). Toward a functional analysis of the yeast genome through exhaustive two-hybrid screens. Nature Genetics 16 (3): 277–282. Harry Witchel (1996). Inclusion of restriction sites in PCR primers, Bristol.Ac.Uk Hartl, D.L., Jones, E. W. (2001) Genetics: Analysis of Genes and Genomes, 5th Edition. ISBN 0-7637-0913-1

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Sambrook, Joseph; David Russell (2001). Molecular Cloning: A Laboratory Manual. New York: Cold Spring Harbor Laboratory Press.

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