Microbial bioremediation and characterization of Arsenic resistant bacteria

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Microbial bioremediation and

characterization of Arsenic

resistant bacteria

Master thesis in Biomedicine (30 hp)

Report version 2

Author: Md. Aminur Rahman a09mdara@student.his.se Master in Biomedicine Supervisor: Prof. Abul Mandal abul.mandal@his.se

Examiner: Dr. Viktoria Lind viktoria.lind@his.se School of Life Sciences Skövde University BOX 408 SE-541 28 Skövde Sweden

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Abstract

Arsenic is a toxic metalloid existing everywhere in the nature. It is toxic to most organisms and considered as human carcinogen. Arsenic contamination leads to severe health problems with diseases like damage of skin, lung, bladder, liver and kidney as well as central nervous system. As arsenic can be found everywhere in nature it may come in contact with food chain very easily through either water or cultivated crops. My thesis works include studies of bioremediation of arsenic by microorganisms. In this experiment the test organisms were collected from the Hazaribagh tanning industrial area of Dhaka, Bangladesh. The whole laboratory works were performed with two types of bacterial strains. Genomic DNA isolation and restriction digestion of genomic DNA, plasmid DNA isolation, Growth response to different concentrations of Arsenic, minimum inhibitory concentration (MIC), plasmid degradation procedures were carried out during this experiment. The MIC value for amoxicillin of these test organisms was 300 µg/ml and they are able to degrade 5 mM arsenite (AsIII) and 40 mM arsenate (AsV). Though the experiment was carried out with two bacterial strains but by observing all experimental data such as restriction digestion, growth response to the arsenic before and after treated with ethidium bromide and minimum inhibitory concentration it can be concluded that these two strains were not different. These bacteria are able to survive in high concentration of antibiotics and arsenic (AsV_{and As}III_{). Loss of plasmid resulted no growth on media} containing arsenic. These results support that plasmid contains important genes that are responsible for surviving bacteria in stress conditions.

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Abbreviations

As Arsenic

AsIII Arsenite AsV _Arsenate

CFU Colony forming unit DMAA Dimethyl arsenic acid DNA Deoxyribonucleic acid

LB Luria-Bertani

MIC Minimum inhibitory concentration MMAA Monomethyl arsenic acid

ND Nano drop

SDS Sodium dodecyl sulphate EtBr Ethidium bromide

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1. Introduction

The existence of the heavy metals disturbs the ecology of sediments (Montuelle et al., 1994) and the biodegradation of organic chemicals by microorganisms (Said and Lewis, 1991). Arsenic (As) is a toxic metalloid particle, available ubiquitously in earth’s crust. In addition, As is also present in the surface, ground water and atmosphere (Moore et al., 1977). Natural processes such as weathering of rocks and volcanic emissions as well as human activities like mining, combustion of fossil fuels, smelting of ores or the application of arsenical herbicides, pesticides and wood preservatives are the sole sources leading to As contamination in the environment (Smedley and Kinniburgh, 2002).

The most common inorganic forms of arsenic in the environment are Arsenate (AsV) and arsenite (AsIII). On the other hand, the environmental organic arsenicals may derive from herbicides, pesticides and preservatives. In the bio-remediation process, change of the oxidation state of arsenic alters the solubility of the oxyanion (Ahman et al., 1997). It is demonstrated that AsIII is 1000 times more toxic comparing with AsV and more mobile (Dowdle et al., 1996). AsIII _{readily forms precipitates when reacts with metal sulphides} (Newman et al., 1997) or hydrous oxides of iron (Rittle et al., 1995) and as a result it can be removed easily from the solution.

In industrialized areas, there are high concentrations of arsenic and heavy metals have been found in wastes and soils that establishing a serious ecological risk (Singh and Steinnes, 1994). It is broadly circulated all over the earth’s crust, ranging from trace levels to hundreds of milligrams per kilogram (Smedley and Kinniburgh, 2002). Background soils concentrations of arsenic are typically bellow 15mg/kg but it can exceed 2000mg/kg in some severe arsenic contaminated areas (Smith et al., 1998).

There are many ways to cope high levels of arsenic by microorganisms ranging from reduced uptake, adsorption, and methylation (Say et al., 2003) to dissimilatory arsenate respiration. The most common and well-described method is reduction of arsenate to arsenite and extraction of the resulting arsenite by soluble reductases and membrane associated pumps (Rosen et al., 1991; Ji et al., 1994). The metals have an effect on microorganisms by reducing their number, diversity, biochemical activity and changing the community structure (Kandeler et al., 2000; Ellis et al., 2001), though metal exposure can lead to the establishment of resistant microbial populations (Jackson et al., 2005b). In industrialized areas, these populations may be involved in the changing of mobility of metals by their accumulation, reduction, and in situ immobilization by extracellular precipitation (Collard et

al., 1994; Roane, 1999).

It has been reported that the concentration of soil arsenics in As contaminated areas ranges from 57 to 83 mg/kg dried soil whereas the concentration of As in

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soil from non-contaminated area ranges from 4 to 8 mg/kg dry soil (Abedin, Feldman et al., 2002).

In some mammals, including human, the arsenites (AsIII) are methylated and converted to some relatively less harmful substances like monomethyl arsenic acid (MMAA) and dimethyl arsenic acid (DMAA). In human and other animals, these methylated arsenic acids can be excreted through urine (Hughes, Devesa et al., 2008).

A number of microorganisms containing the ars genetic system and they are capable of using the reduced inorganic form (arsenite) and the oxidized form (arsenate) in their metabolism. They are capable of resisting arsenic toxicity by the ars genetic system (Jackson et al., 2003; Oremland and Stolz 2003). The study analysis shows that many bacterial divisions appear to have representatives that indicate arsenate resistance. This proposes that arsenic resistance may not be confined to organisms occupying arsenic-laden environments, and even arsenic-free environments might harbor arsenic resistant bacteria. These bacteria could be important in transforming arsenic which are available in industrial areas and environments by pollution or unexpected redox changes.

There are some studies in which arsenic resistant microorganisms have been found from arsenic-contaminated areas or soils (Macur et al., 2004; Anderson and Cook 2004). Microbial interaction may play a crucial role in the environment by the process of biogeochemical cycling of toxic metals, as well as by cleaning up or by remediating metal-contaminated environments. The success of these bioremediation processes depend upon the better understanding of how microbial populations respond to different concentrations of metals. The responses to the metals analyzed should be involved to some vital soil biological processes such as, those involved in C and N cycling (Martensson and Torstensson 1996).

Industries are the main sources for the pollution of environments all over the world basically in south Asian countries. In Bangladesh there are hundreds of leather tanning industries are identified as the red sources of pollution in Dhaka city of Bangladesh (Mohanta et al., 2010). The amount of discharged wastes

from these tanneries is approximately 18000 liters in liquid form where as 115 tons of solid wastes every day. Because of high decomposition rate in summer, serious air pollution occurred in the whole industrial areas due to production of obnoxious, intolerable odor. A recent study showed that approximate 60,000 tons of raw hides and skin are processed in each year and around 9500 liters of untreated effluents are released to the environment every day in Bangladesh (Rusel et al., 2006). These effluents cause severe contamination of ground water resources and create negative impacts on crops and health of human being. Some heavy metals like cadmium, chromium and lead contaminated with these effluents have been found to be carcinogenic as well as arsenic contaminated are considered as carcinogenic (Tamburlini et al.,2002) depending upon the dose and exposure duration. These are not only poisonous for human but also toxic for the aquatic life (WHO, 2002).

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In this experiment, we used two bacterial samples from an area with known pollution problems, namely arsenic, where pollutants have been released by the tanning industry. The main aim of our investigation was to compare the similarities between these two bacteria and determination of the minimum inhibitory concentration. In addition, to determine the level of arsenic sensitivity and resistance as well as to establish if resistance is due to the reduction of arsenate to arsenite.

2. Methods

Various types of microorganisms were collected by the research group Mohanta et al. (2010) from the tanning industry area Hazaribagh, Dhaka the capital city of Bangladesh as a source of inocula for the isolation of microorganisms which are capable of degrading effluents. In these current experiment bacteria samples were collected from the research team Mohanta et al. (2010).

2.1. Determination of minimum inhibitory concentration (MIC)

MIC is the lowest concentration of an antimicrobial which inhibits the visible growth of microorganism after overnight treatment. In this experiment, the MIC of antibiotics, amoxicillin was determined by agar method. The different concentrations (150, 200, 250, 300, 400 µg/ml) of amoxicillin were taken to the LB-agar medium and bacteria were incubated at 37°C for overnight and again for 48 hours at 37°C.

2.2. Plasmid DNA extraction

The plasmid DNA from two types of bacteria (Strain A and B) were isolated following the standard protocol QIAprep® Miniprep handbook (Qiagen 2006). To get good concentrations of plasmid DNA bacterial strains were freshly cultured in Luria-Bertani (LB) broth at 37° C for 14 hours. Again the plasmid DNA was isolated after treatment with ethidium bromide following the same protocol.

2.3. Chromosomal DNA isolation

Fresh cultured bacteria were used as the source of chromosomal DNA. The chromosomal DNA from two types of bacteria (Strain A and B) were isolated following the standard protocol DNeasy® Blood & Tissue handbook (Qiagen 2006). After isolation and purification of chromosomal DNA the concentration of chromosomal DNA was measured by the Nanodrop® ND-1000 Spectrophotometer (Saveen Werner, USA).

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2.4. Restriction digestion of chromosomal DNA

Restriction digestion of chromosomal DNA was performed to find the similarity and difference between two strains of bacteria. DNA was digested by two restriction enzymes EcoRI and AscI. For cleavage analysis, the digested chromosomal DNA fragments were run in 1.0% agarose gel electrophoresis at 70 volt power for 2 hours. The reaction mixtures of restriction digestion test with EcoRI/AscI restriction enzymes have showed in Table 1. DNA and restriction enzymes were added separately in the master mixture.

Table 1. Reaction mixture of restriction digestion test with EcoRI/AscI restriction enzymes

Reagents Final conc. Volume (μl)* Master Mix (μl) **

DNA 500 ng 3.0 12.0

Buffer 4 1X 2.5 10.0

EcoRI/AscI 1.0 04.0

dH2O 18.5 74.0

Total 25 100

* = volume was taken for one reaction **= volume was taken for four reactions

2.5. Plasmid degradation

Generally plasmid of bacteria can be degraded by ethidium bromide (EtBr), sodium dodecyl sulphate (SDS), acriflavin (Trevors 1986). In this experiment LB agar medium were prepared containing 100 mg/ml ethidium bromide. Then the fresh bacteria were cultured in LB agar containing 100 mg/ml ethidium bromide at 37°C for overnight.

2.6. Survivability test of bacteria in As.

LB-agar plates were prepared with different concentrations of arsenic (both arsenite and arsenate). Freshly cultured bacteria were grown in arsenic containing LB plate at 37°C for overnight and it was observed the growth response of bacteria in different concentrations.

3. Results and discussion

Many bacterial heavy metal resistances have been studied for several years especially in contaminated areas. Resistance to poisonous heavy metals has been establishing in bacteria from clinical and environmental origins (Coral et

al., 2005). Arsenic is a naturally occurring toxic element existing all around in

nature which is also used in a number of industrial processes. It is very much toxic element to almost all organisms and considered as human carcinogen, categorized by the international agency for research on cancer (National Research Council, 2001; Naidu et al., 2006). The contamination of arsenic leads to severe health problems with diseases like damage of lung, skin, bladder, kidney and liver as well as central nervous system (WHO 2000;

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Mukherjee and Bhattacharya, 2001, Chakraborti et al., 2003). Though arsenic is highly toxic, a number of microorganisms are able to use either the reduced form arsenic (arsenite) or oxidized form of inorganic arsenic (arsenate) in their metabolism and even more microorganisms are capable of resisting arsenic toxicity throughout the ars genetic system. The objectives of this study were determining the resistance of bacteria to heavy metal like arsenic and to determine the presence of plasmid mediated heavy metal resistance.

3.1. Determination of minimum inhibitory concentration (MIC)

Minimum inhibitory concentrations (MICs) are the lowest concentration of an antimicrobial that will prevent the noticeable growth of a microorganism after overnight incubation. The MIC value of amoxicillin for the isolated bacterial strains was 300 µg/ml which indicating a high concentration of antibiotic was needed for inhibiting the growth of bacterial species. The experimental data for the MIC values are shown in Table 2.

Table 2. Observation of minimum inhibitory concentration (MIC) of amoxicillin

Test organism Conc. Of amoxicillin (µg/ml) Growth response

Bacteria strain A 150 ++ Bacteria strain B 150 ++ Bacteria strain A 200 ++ Bacteria strain B 200 ++ Bacteria strain A 250 ++ Bacteria strain B 250 ++ Bacteria strain A 300 -- Bacteria strain B 300 -- Bacteria strain A 400 -- Bacteria strain B 400 --

++ indicates the growth of bacteria -- indicates no growth of bacteria.

In this experiment we have observed that in lower concentrations of amoxicillin both types of bacteria can grow numerously but with the increasing of amoxicillin concentration the rate of bacterial growth was decreased. The lowest concentration of amoxicillin preventing the growth of both types’ of bacteria is considered to be the minimal/minimum inhibitory concentration (MIC). Therefore, 300 µg/ml amoxicillin is the bacteriostatic agent for both bacterial strains. The same results have been published in the previous study of microbial bioremediation of effluents from tanning industry in Bangladesh (Mohanta et al., 2010).

3.2. Restriction digestion

Restriction digestion was carried out with genomic DNA of both types of bacteria. Firstly genomic DNA was isolated and purified. The concentration of genomic DNA has been shown in Table 3. The concentrations of two bacterial strains were almost same (Table 3).

Table 3. Concentrations of genomic DNA of two bacteria and their purity ratio after Nanodrop test

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Bacterial Strain Conc. (ng/µl) A260/280 A260/230

Bacterial Strain A 44.3 2.08 0.52

Bacterial Strain B 43.4 2.04 0.71

The genomic DNA was treated with two restriction enzymes EcoRI and AscI. The ratio of A260/280 and A260/230 indicate the purity level of the experimental sample. From Table 3 it can be observed that the A260/280 ratio seems to be correct but the A260/230 ratio should be more than 2.0 but here too less so these samples were contaminated. This contamination might be with some cell debris, cell wall, plasmid DNA or the contamination with other equipments. The result of restriction digestion has been shown in gel Figure 1. From this gel Figure 1 it can be observed all lanes contain smears that are due to contamination or DNA degradation. There were not any visible differences between lane 1 and 2 as well as lane 4 and 5. Therefore by observing the DNA bands, it can be concluded that these two bacterial strains were may be same bacterium. Lane 3 and lane 6 were for control bacteria Erwinia cartovora.

Figure 1. Restriction digestion test of bacteria by two restriction enzymes EcoRI and

AscI. Lane L indicates the 2 log DNA ladder. Lane 1 and 4 showed band for bacterial

strain A, lane 2 and 5 showed band for bacterial strain B whereas lane 3 and 6 showed band for a control bacteria Erwinia carotovora.

3.3. Effect of Arsenic on bacterial growth

Resistances of poisonous metals in bacteria perhaps reproduce the degree of environmental contamination with these substances and may be straightly connected to exposure of bacteria to them (Aiking et al., 1984). Arsenic is both an essential micronutrient and a toxic heavy metal for most living cells. Numerous examples of plasmid and chromosomal heavy metal resistance systems in bacteria have been reported (Cervantes et al., 1994). Baath (1989) established that though exposure to metals enhances resistances, the fact that resistant bacteria are found in environments never exposed to high

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concentrations of heavy metal tolerant species already exist in nonpolluted habitats. Both types of bacteria were grown on LB-agar media in different concentrations of arsenite (AsIII) and arsenate (AsV) along with control. The growths of bacterial colonies were observed after 1 day and two days after culture. The results of the effects of AsIII_{in bacterial growth have been} publicized in Table 4 whereas the results of the effects of AsV_{in bacterial} growth have been publicized in Table 5.

Table 4. Observation of bacterial growth responses in different concentrations of arsenite (AsIII₎

Bacterial strain Conc. Of AsIII _(mM) _{Growth responses}

Bacteria strain A 0 ++ Bacteria strain A 1 ++ Bacteria strain A 5 + Bacteria strain A 10 -- Bacteria strain A 15 -- Bacteria strain B 0 ++ Bacteria strain B 1 ++ Bacteria strain B 5 + Bacteria strain B 10 -- Bacteria strain B 15 --

++ indicates the growth of many bacteria + indicates the growth of less bacteria -- indicates no growth of bacteria.

Table 5. Observation of bacterial growth responses in different concentrations of arsenite (AsV)

Bacteria strain Conc. Of Asv _(mM) _{Growth responses}

Bacteria strain A 0 ++ Bacteria strain A 20 ++ Bacteria strain A 40 + Bacteria strain A 50 -- Bacteria strain A 75 -- Bacteria strain B 0 ++ Bacteria strain B 20 ++ Bacteria strain B 40 + Bacteria strain B 50 -- Bacteria strain B 75 --

++ indicates the growth of many bacteria + indicates the growth of less bacteria -- indicates no growth of bacteria.

From the Table 4 and Table 5 it can be observed that bacterial strain A and bacterial strain B both was showing the same growth responses to arsenite and arsenate. Hence it can be assumed from the growth responses result that these two strains might be same bacterial strain because there is no difference between the growth responses on different concentrations of arsenite (AsIII_{) and} arsenate (AsV) but to be confirmed some more experiments should be done like DNA-DNA hybridization, restriction digestion, plasmid degradation, PCR amplification. Here it is clear that 10mM AsIII and 50 mM AsV is the bacteriostatic agent for these bacteria. The sensitive and resistant levels of these two strains have been shown on the basis of arsenic. However the

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sensitive and resistant ranges can be varied when used other bacterial strains or other metal ions (Coral et al., 2005). Therefore, the bacterial ars genetic systems are not capable to work at the concentrations of 10mM AsIII and 50 mM AsV. Bacterial strain A and bacterial strain B was able to grow at high concentrations of Arsenic (Arsenite and arsenate) in solid medium, which might be important for the capacity of this bacterium to survive in different source of pollution with elevated heavy metal levels, this result is supported by the study of El-Deeb B., 2009. Malik and Jaiswal (2000) recommended that the occurrence of a high metal-resistant population caused from increasing environmental pollution. Bogdanova et al. (1998) also found that transposable elements transport metal resistance genes have been connected to the circulation of these resistances in environment.

3.4. Plasmid isolation

Plasmids DNA were isolated from the bacterial strains after 14 hours incubation at 37°C. The concentrations and purity ratio of plasmid DNA were shown in Table 6.

Table 6. Concentrations of plasmid DNA of two bacteria and their purity ratio after Nanodrop test

Bacterial Strain Conc. (ng/µl) A260/280 A260/230

Bacterial Strain A 55.5 1.98 2.13

Bacterial Strain B 43.3 1.96 1.68

The plasmid DNA was run in the 1% agarose gel electrophoresis along with 2 kb DNA ladder to predict the DNA length. The gel image of plasmid DNA has been shown in Figure 2. From Figure 2 it can be observed that both bacteria contain plasmids and the plasmid length is more than 10.0 kilo base (Kb).

Figure 2. Agarose gel electrophoresis of plasmid DNA. Lane L indicates the 2 log DNA ladder whereas lane 1 and lane 2 indicates band of bacterial strain A and strain B respectively.

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The plasmids were isolated from the both strains carrying plasmid-encoded resistance. According to electrophoresis findings, strain A and strain B have plasmid. The molecular size of the plasmids isolated from the both strains was found to be more than 10kb (Figure 2).

3.5. Plasmid degradation

After degradation of plasmid DNA from the bacterial strains by EtBr the DNA were run in the 1.0% agarose gel electrophoresis to predict the confirmation whether the plasmid degraded or not. Gel image has been shown in Figure 3.

Figure 3. Agarose gel electrophoresis of plasmid DNA after plasmid degradation. Lane L indicates the 2 log DNA ladder. Lane C indicates the control plasmid whereas lane 1,2,3 and lane 4,5,6 indicates band of bacterial strain A and strain B respectively.

3.6. Growth response of bacteria after plasmid degradation

Bacteria can grow in LB agar plate containing 100 mg/ml ethidium bromide. When again bacteria were cultured from this plate to arsenic containing LB-agar then the bacterial growth response is negative. The bacterial growth response has been shown in Table 7.

Table 7. Observation of bacterial growth responses after treated them with ethidium bromide

Bacteria species Conc. Of Arsenic (AsIII_{and As}V₎ _{Growth response}

Bacteria strain A 00 mM AsIII ₊₊

Bacteria strain A 05 mM AsIII _--

Bacteria strain B 00 mM AsIII ₊₊

Bacteria strain B 05 mM AsIII _--

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Bacteria strain A 10 mM AsV _--

Bacteria strain B 00 mM AsV ₊₊

Bacteria strain B 10 mM AsV _--

++ indicates the growth of bacteria -- indicates no growth of bacteria.

From Table 4, Table 5 and Table 7 it can be concluded that both types of bacteria can grow at the concentration of 5mM AsIII and 20 mM AsV before treatment with ethidium bromide but after degradation of plasmid by ethidium bromide bacteria are not able to grow in stress condition. Therefore plasmid is an important factor for bacteria to survive in any stress condition. So plasmid contains crucial genes or enzymes such that bacteria can survive in the presence of Arsenic. Parallel transfer of resistance genes between bacteria of different species and genera occurs easily and frequently in nature (Slayers and Cuevas, 1997). This result is the similar with the result of Mohanta et al., (2010) though their study was concerned some heavy metal like chromium, cadmium and lead-nitrate. Degradation of the plasmid in the isolated strain was done by using ethidium bromide in order to determine the presence of metal ion resistance genes on plasmid DNA. This study also revealed the high efficiency of ethidium bromide in degradation the plasmid. Cured and uncured cultures were compared for their resistance against arsenic ions. The comparison clearly showed the tolerance to AsIII and AsV appeared to be associated with plasmid, as confirmed by the conjugation data. Similar study was carried out by El-Deeb B. (2009) with toxic metals (Zn2+ and Cd2+). An evaluation of parental strains and its degraded derivative exhibited that with the loss of plasmid, of both bacterial strains cannot grow in AsIII and AsV containing medium. Metal biotransformation or accumulation is another mechanism for metal decontamination in bacteria (Gadd, 1992). A number of fungi and bacteria that collect an ample range of metal have been designated (Fourest et al., 1994; Hernandez et al., 1998).

4. Conclusion

Microorganisms have been measured not only for use in bioremediation of metals but also as accumulators of metals from dilute solutions. A great number of bacteria, algae and yeast are accomplished of accumulating metal ions in their cells to concentrations some orders of degree higher than the background concentrations of these metals. These methods have a probable use in extracting rare metals from dilute solution or removing toxic metals from industrial effluents. Though the experiment was carried out with two bacterial strains but by observing all data such as restriction digestion, growth responses to the arsenic before and after treated with ethidium bromide it can be decided that these two strains might be same but some more experiments should be done like DNA-DNA hybridization, PCR amplification, gene sequencing of two strains and their alignments. In view of the results of arsenic resistance experiments, it was concluded that these bacteria were able to survive in high concentration of antibiotics and arsenic (AsV and AsIII). Loss of plasmid, results no growth of bacterial colony on media containing arsenic.

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5. Future perspectives

A comparative analysis was performed for the heavy metal arsenic but the analysis for some other heavy metals like lead, chromium, cadmium should be further conducted. Moreover, characteristics of arsenic uptake by the test organisms should be performed for the bioremediations.

6. Acknowledgements

I would like to thank Prof. Dr. Abul Mandal (Department of Molecular Biology, University of Skövde, Sweden) for supervising me and for giving me an opportunity to complete my master thesis in his laboratory. I also would like to thank Noor Nahar (PhD fellow) for guiding me in the laboratory. I thank Viktoria Lind for evaluation my thesis. I would like to thank all faculty members at the School of Life Sciences for creating a friendly environment during my whole studies and master thesis at the University of Skövde.

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