Team:Saint Joseph/Description
How We Found Our Project?
This year, we started to work on IGEM by creating a club in October. Handan Kesim, our teacher, also our PI, explained the terms of the competition to the team members. We examined the projects of the teams who competed in 2018 and our previous project.
Since we are studying in different grades, not all of our team members possess the same level of knowledge in biology. That is why our teacher conducted lessons about genetics, DNA, protein synthesis, synthetic biology, gene transfer, competent cell and recombinant DNA.
We learned the rules of IGEM and how to get prepared for the competition with our studies after school.
The process of finding our project subject took time. Every team member came up with an idea, everyone did their research. We ran through each idea. We eliminated the ones we thought we wouldn’t be able to accomplish.
During our school’s half-term break, we took lessons from our instructor, professor Şule Arı (Istanbul University Biotechnology and Molecular Biology) about biotechnology and genetics. We did experiments which were similar to previous IGEM interlab experiments in a synthetic biology lab. We transferred a gene to a bacterium. This was an important lab practice for each team member.
One of our team members family is working in the textile industry, she told us that the dyes used in textile industry are polluting waters and they are trying to find a solution that is cheaper than the chemical cleaning. In response, in one of our meetings we decided to search if we could stop this pollution via biological ways. While searching for the pollution caused by textile dyes and how to clean it, we encountered laccase. We began our research on laccase. We learned that this enzyme is produced by many organisms, mushrooms, plants for example; rotting pumpkin, banana skin (Université des Frères Mentouri Constantine Faculté des Sciences de la Nature et de la vie) We talked to our PI and instructor about this idea and our instructor Professor Arı told us that this enzyme was multifunctional.
Our idea was to clean the waters polluted by textile dyes with laccase and we decided to carry this out in February. We liked the environmental part of this idea. Turkey will soon become one of the countries which has a risk of water pollution. Renewable water is going to become very important in the future. We read articles about laccase and we saw articles written by Turkish researchers and this made us happy. However they weren’t the same with our idea so we hoped that our project would be beneficial.
We are working with our school’s IT team to make our wiki better. We are learning coding language. We made groups like IT, lab and human practices to share the work between us. Each team member has to do their work and also follow other groups’ work. We share every task between us and we have a “Drive” file to follow what everyone has done.
Previous iGEM Studies on the Laccase Enzyme
We did some research about previous IGEM teams who worked on the laccase enzyme:
-
* Team:Bielefeld-Germany
Dangerous contaminants in the waterbody. Toxic compounds in natural water.
-
* Team:Hong_Kong_HKUST
The target of this module was to demonstrate the ability of Laccase, expressed by E.coli, to fragment PE into simple hydrocarbons.
-
* Team:Stockholm
Project Description: “We created Biotic Blue, a solution based on an enzyme produced by the common fungus Trametes versicolor. To be able to grow on wood, this fungus produces a class of multi-copper oxidases called laccases. Laccases degrade lignin, a building block in wood consisting of phenolic polymers.”
-
* Team:British_Columbia
Abstract - Lignin modifying enzymes onto the C.crescentus S-layed as one approach to pretreat raw biomass and make it more accessible to cellulose yield.
-
* Team:SHSBNU_China
Cleaning synthetic dyes in water using Biofilm made with CotA laccase.
-
* Team:Paris_Bettencourt
Paris Bettencourt Team produced synthetic enzymes to remove red wine stains from fabric. These enzymes are designed to replace perchloroethylene (PERC), a toxic solvent used in dry cleaning that will soon be banned in France. They used six enzymes including Bacillus pumilus’ BpuI laccase.
What We Have Done So Far?
-
Between 01/28 / 2019- 02 / 01/2019 we took theoretical lessons and lab training about synthetic biology and genetics. We learned about plasmid gene transfer, production of transformed bacteria and how to obtain a competent cell. These experiments were similar to Interlab experiments from last year.
-
Since paints are used in the automotive industry, we visited the Renault factory on 03/30/2019.
-
We went to Petit Prince Schools on 5/16/2019.
-
We organized a waffle day on DNA Day, 25 April 2019.
-
We started to ask the survey questions that we prepared in May in our school and around us and our survey will continue.
-
Our IT team is working on our wiki page.
-
Our laboratory team started the experiments which is similar to lab work in February, on the 10th of June
-
We have continued reading articles concerning our project
Our Subject : Cloning of Trametes versicolor Laccase Gene in E. coli
Laccase is an enzyme that reduces lignin from the class of “multiple copper” oxidases found in higher plants, fungi and some microorganisms. The laccase enzyme, first isolated by Gabriel Bertrand in 1894 from the sap of Japanese varnish tree, reduces an electron in phenolic and phenolic like substrates. Yoshida (1883) made the first research on the laccase enzyme. The laccase enzyme is used as a biocatalyst in industries like:
However, laccase production is not sufficient for industrial use today and the production is expensive. Especially in industrial areas, production of large amounts of laccase enzyme is required for biological cleaning of contaminated areas (e.g. textile wastewater mixed with dyes). (Hildén et al., 2009; Guo H., et al., 2017).
In order to meet the industrial requirements, it is necessary to develop the reproducible and cheap methods of isolation or to optimize the conditions of laccase production. It is also necessary to do research about the microorganism sources which produce laccase actively and to choose the most relevant kind.
The studies of laccase are mostly about the decolorization of wastewater in the textile industry. It is determined that the most active form of laccase enzyme is produced by Trametes versicolor.
The aim of this project is to design the signal peptide for T. versicolor laccase enzyme and to clone the gene carrying this signal peptide sequence in Escherichia coli. PelB, the signal peptide to be used, will direct the laccase enzyme to the periplasmic domain in the E. coli bacteria, the host organism to be used in the study. Thus, it is aimed to increase the amount and the activity of total enzyme purified from the host organism.
The Scope
Trametes versicolor laccase enzyme is 520 amino acids in length and 55.644 kDa in weight. The enzyme has a high stability at pH 5-8 and 25° and moderate stability at pH 6 and 10-30 °C. (Kurniawati and Nicell, 2008). In a study, it was determined that laccase can convert 80% of 500µM phenol sample under optimum conditions in 6 hours.The pelB signal peptide to be used in this project will benefit from the ability to secrete the protein into the periplasmic domain in E. coli. With the design of signal peptide by bioinformatics tools, it is aimed to shorten this period and produce more efficient production. An increase in laccase production of about 1.3-1.7 times is expected due to the increase in the yield of the enzyme to be released into the periplasm and the increase in the amount of product and substrate formed. The aim of the project is to reorganize the laccase gene to make a new approach to yield enhancement studies in this field and to use enzymes for the recycling of dye mixed water.
Although ¾ of the world is covered with water, there are very few clean water sources. In addition, considering the amount of water used in all areas of the industry, water shortages are foreseen within a few years (Witze A., 2019). Cleaning of waters contaminated by textile waste paints is a high cost job. Textile companies, which had to make extra expenses due to the necessity of recycling the dyed water, went in search of cost effective recycling methods. The high cost of electrochemical methods for the recovery of water from the paint revealed the necessity of preferring alternative biological methods. In this context, laccase enzyme has a very important place. In this project, it is aimed to obtain a biotechnological product that promises biological contribution to the elimination of environmental pollution through the recovery of water mixed with paint and to a wider scope of economy.
Work-Flow
The procedures followed in the project are listed below:
1. Bioinformatics
design
Once the laccase gene sequence is obtained from the GenBank database, E. coli will also be optimised for the correct expression. Following optimisation, the vector from which the laccase gene will be cloned will be selected and designed in the Benchling program. This design process:
Adding the restriction enzymes to the expression vector and both ends of the gene to ensure proper insertion of the gene into the expression vector, Designing the pelB signal sequence to direct the gene to the periplasm,Simulation of cutting and ligation process with restriction enzyme in computer environment and revealing the structure of the recombinant DNA molecule targeted to be obtained.
2.
Complementation (sufficiency for transformation) of
Bacterial Cells to be Cloned
For the transformation of the cloning vector carrying the expression vector and the synthetic gene to be used in the experiments, the cells should be made competent. Competence will be carried out by chemical method based on CaCl2. This method will be used before each transformation to bacterial cells. Competence of bacteria is necessary to obtain free DNA in the environment.
* That’s general description of calcium chloride treatment
3. Production of
Cells and Replication of Genetic Material for Cloning
Before and after the creation of the recombinant DNA, it needs to be replicated and stocked. This process is necessary to ensure that the subprocess operations to be applied to the vector, gene to be transferred and recombinant DNA can be performed and not interrupted. The E.coli DH5α strain will be used for cloning. E.coli DH5α cells will be grown on LB (Luria Bertani) medium and ready for transformation. Both the expression vector and the laccase gene (in the cloning vector) will first be amplified by transforming into E. coli DH5α cells. The same procedure will also be applied to the recombinant DNA molecule resulting from the cleavage of the cloning vector with the expression vector and the laccase gene by restriction enzymes and ligation.
4. Creation and
Replication of Recombinant DNA
The cloning vector containing the laccase gene and the expression vector in which the gene is intended to be cloned will be cut with restriction enzymes forming the sticky end. From the cut products analyzed by agarose gel electrophoresis, the expression vector and laccase gene will be isolated and subjected to sticky end ligation. The resulting molecule after ligation will be the recombinant DNA molecule to be used for laccase enzyme expression. The ligation reaction will be transferred to competent E. coli DH5α cells and cloned. Then, isolation and control of the recombinant DNA molecule that grows in the DH5α strain will be performed. For this purpose, isolated DNA samples will be sent for sequencing. Recombinant DNAs confirmed after sequencing will be available for enzyme production.
5. Production of
Cells for Expression
In order for the recombinant vector carrying the laccase gene to produce the laccase enzyme by gene expression, the appropriate E.coli strain will be used as previously mentioned. For this purpose, the cells stored at -80 ° C will be amplified and rendered competent for transformation.
6. Transformation
into Cells Produced for Expression
The recombinant plasmid will be transformed into competent cells. The transformation will be done by heat shock method. Transformant cells contain plasmid which have kanamycin résistant region. After transformation cells will be inoculated into LB Agar medium which contains kanamycin antibiotic. By this way transformant cells will be selectable. Plasmid isolation will then be performed from these cells. Isolated plasmids will first be scanned on agarose gel, then sequencing will be done to see if they are suitable for the design. After verification of the isolated plasmids, the necessary processes for protein production will be initiated.
7. Induction of
Transformed Cells for Expression
One colony will be selected from cells successfully transformed in the previous step and will be resuspended with antibiotic in liquid medium. Cells will be incubated at 30 °C and after incubation cells will be induced by the IPTG method for gene expression overnight. After induction expression control will be done and the potentiel secreted by the cells will be analyzed.
8. Protein
Isolation and Analysis
After expression, cells will be lyses to release proteins. Since there are histidine tags at the end of the laccase gene, proteins will have histidine tags at the end. Using the ability of nickel particules can interact with histidine tags, protein will be separable by Magne-His purification method. Separated proteins will be analyzed by SDS-PAGE method. After this analysis, the proteins will be subjected to further chromatographic analysis.
9. Enzyme
Substrate Treatment and Activity Determination
The laccase enzyme, isolated and analyzed as indicated, will be treated with the textile dye substrate at the required temperature and pH in the required solution. The laccase enzyme is expected to degrade the dye at this stage. The activity of the enzyme will be analyzed with spectrophotometer and reported.
Researches
The scientific articles we have read during our research
Synthetic dye
decolorization by three sources of fungal laccase
It was observed that decolorization efficiency of all dyes was enhanced by increasing of HBT(hydroxybenzotria- zole, HBT is a synthetic laccase mediator assisting in lac- case oxidation of different substrates by facilitating of electron transfer from O2 to laccase substrate) concentration from 0.1 mM to 5 mM.
Decolorization of a wide range of synthetic and textile dyes using laccases from the genus of Trametes (from basidomycete family) has been investigated in recent years. For example, Maalej-Kammoun et al. studied on malachite green decolorization ability of a newly iso- lated strain of Trametes sp. Furthermore, the laccase from genetically modified Aspergillus oryzae (DeniLite IIS) was applied for elimination of a large number of reactive tex- tile dyes and other xenobiotics.
The laccase of P. variabile was the most efficient enzyme with 72.2% removal of bromophe- nol blue (a triphenylmethane dye) after 30 min treat- ment in absence of HBT. However, decolorization percent by using two other sources of fungal laccases did not increase higher than 25.3% even after 3 hours in- cubation.
In the present study, the pure laccase of T. versicolor (Syn. Coriolus versicolor) could not efficiently decolorize the tested synthetic dyes except for RBBR and methylene blue during 3 h of incubation. Three sources of fungal laccase were ap- plied for decolorization of six synthetic dyes among which the laccase with the origin of P. variable was able to remove all tested dyes. The laccase from A. oryzae was not able to decolorize examined dyes except for methylene blue and RBBR. In absence of HBT, RBBR was the sole synthetic dye efficiently removed by laccase from T. versicolor.
Production de la
laccase par des mycètes filamenteux sur milieu à base de
déchets de citrouille (cucurbita sp.)
In a research that Université des Frères Mentouri Constantine Faculté des Sciences de la Nature et de la Vie had done which focuses on laccase on microbiology area reveals about the cases for laccase production and usage. Laccase is an oxidation enzyme which includes copper. Laccase enzyme has been discovered by Yoshida on Rhus vernicifera plant on 1883. Also this enzyme is being used on paper, textile, food, pharmaceuticals, organic syntheses industries with similar purposes. In the research that was conducted by Algerian university, production of laccase from pumpkin was stressed. It was proved that by rotting pumpkin, two fungi named Trametes sp. and Chaetomium sp. can produce laccase. Both of these fungi proved to be producing laccase.
Dye removal by
dead biomass of newly isolated Pleurotus ostreatus strain
The removal of dyes from wastewaters has admitted attention within environmental research. Different methods are functional for the remediation of dye wastewaters. These include physicochemical methods, like adsorption, chemical oxidation, precipitation, coagulation, filtration, electrolysis, photodegradation, biological, and microbiological methods. Adsorption on activated carbon is an effective method for the removal of color, but it is too expensive.
Living and dead fungi have been shown to be capable of removing dyes due to the presence of varied functional groups on the biomass. However, dead cells offer several advantages over living cells. Dead fungal biomass can be stored easily and kept for prolonged periods. Fungal biomass holds distinct advantages over other microorganisms. In this study, dead biomass of newly isolated Pleurotus ostreatus strain was used as a biosorbent to remove the indigo carmine dye. The aim of this research was to develop effective adsorbents for dye- removal technology. So, effect of varied operating parameters on dye removal such as initial pH, dye concentration, time, agitation rate and adsorbent amount were studied. Freundlich and Langmuir models are the most commonly used isotherms.
The most effective biomass amount for dead biomass was 0.2 g and 0.5 g. Dye removing capacity increased from 12 to 66% as the dead biomass amount was increased from 0.1 to 0.5 g/20 mL at 60 min. Similar results were reported et al12 for decolorization of astrazone blue by dead biomass of Phanerochaete chrysosporium. It was possible to increase the decolorization of malachite green by increasing the adsorbent dose of the agro-industry waste17. Also, other similar results were reported et al18 and Gupta et al19 for decolorization of quinoline yellow and three anionic dyes, respectively. The maximum dye removal value of 89% was observed at an optimum pH of 2, after 120 minute of treatment with dead biomass. Dye adsorption by dead biomass was maximum (89%) at the pH of 2, which decreased to 82% at pH 8. The effect of the agitation rate on decolorization capacity of dead bbiomass was carried out at 50 mg/L initial dye concentration with 0.2 g/20 mL adsorbent mass (100 mesh) at 30°C for 60 minute time and pH 2.0. There was also an observed decrease in pore sizes in the dead biomass, and this can be affection to the fact that the porous structure plays a role in indigo carmine biosorption.
Laccase
production by newly isolated white rot fungus Funalia
trogii: Effect of immobilization matrix on laccase
production
In this study, Cu-impregnated apricot stone-based activated carbon was used effectively for immobilisation of F. trogii cells. This matrix highly induced the laccase production of this fungus in low- cost molasses media in a short time. The obtained laccase enzyme had also high textile dye decolorization activity as shown by a single step detection method on native PAGE gels and also in aqueous textile dye solutions. Therefore, this newly prepared immobilisation matrix impregnated with copper could be used to immobilise white rot fungi for high amount of laccase enzyme production.
Overexpression of
a Laccase with Dye Decolorization Activity from Bacillus
sp. Induced in Escherichia coli
Escherichia coli is one of the most effective expression systems for the production of recombinant proteins [Makrides, 1996]. However, ligninolytic enzymes like laccase are generally regarded as the most difficult proteins to express in bacterial systems [Bulter et al., 2003].
Yano et al. [2009] tried to express recombinant laccases in sev- eral conventional expression systems, including E. coli, Saccharomyces cerevisiae, and Pichia pastoris, but the laccase gene cannot be successfully expressed in the overexpression system. The laccase from Haloferax volcanii was successfully overexpressed in E. coli, but no laccase activity was detected [Uthandi et al., 2010]. The undetectable laccase activity may be partially due to the fact that the laccase from a halophilic archaeon H. volcanii is halophilic and after expression it needs to be dialyzed against buffer containing 2 M of NaCl to activate the enzyme [Uthandi et al., 2012]. It has also been reported that the haloarchaeal proteins, glycoproteins, and enzymes with elaborate metal clusters are all extremely difficult to over- express in conventional expression systems, such as E. coli and S. cerevisiae [Madzak et al., 2005; Uthandi et al., 2010]. Furthermore, most of the recombinant laccases were kept in cytoplasm or formed insoluble intracellular fractions,even though the laccase activity was detected in the E. coli expression system [Santhanam et al., 2011]. In this study, we report the cloning and successful heterologous production of the thermo-alkali stable laccase of Bacillus sp. in E. coli. The overexpression system was highly induced to produce soluble and intracellular laccase in the presence of copper ion (Cu2+). This is a promising approach to producing laccase for industrial applications.
Laccase
production by Pleurotus ostreatus and its application in
synthesis of gold nanoparticles
In this work, the production of fungal laccase was optimized from local isolate of Pleurotus ostreatus using solid state fermentation. Factorial design was used to study the effect of several nutrients on enzyme production. Purification and characterization of the enzyme and the effect of temperature, pH and gamma radiation on fungal growth and enzyme production was investigated.
Optimization of production conditions yielded an enzyme with activity over 32,450 IU/g of fermented substrate. Factorial design was capable of establishing the conditions that multiplied the activity of the enzyme several folds, consequently, reducing the cost of production. The enzyme was capable of decolorizing several dyes with over 80% reduction in color confirming the aromatic degrading capability of laccase. The enzyme was also used in the synthesis of gold nanoparticles, proving that laccase from Pleurotus ostreatus has a strong potential in several industrial applications.
Decolorization of
Textile Dyeing Wastewater by Phanerochaete chrysosporium
One of the possible alternatives for the degradation of wastewaters is the use of white-rot fungi. They can degrade a wide variety of structurally diverse pollutants. The indigo dye is extensively used by textile industries and is considered a recalcitrant substance which causes environmental concern. This dye was shown to be decolorized by ligninolytic enzymes but no study has been published on wastewater-containing indigo-dye decolorization activity of pellets from white-rot fungi.
In this paper, the effect of various culture conditions on textile dyeing wastewater decolorization activity of live and dead pellets under optimum conditions was investigated. The indigo dye and textile dyeing wastewater were obtained from GAP Textile Co. , the white-rot fungus Phanerochaete chrysosporium was used as fungus, the pellet preparation has been made (the fungus was cultured), after 1 week spore suspension was prepared and used for cultivation of inoculum, fungal pellets were harvested by filtration from culture media and wet pellets were used in decolorization for further experiments. Dead pellets were also used for decolorization. The pellets were used at 3 different concentrations. The effect of temperature agitation and the amount of live pellets on decolorization was studied. The capacity of live pellets was examined using only indigo dye in the water given that this dye is the main component of wastewater used in our study. Live and dead pellets were used in a repeated-batch mode under optimum conditions. The degree of decolorization was quantified by measuring changes of absorbance at its max. wavelength.
The live pellets showed a rapid wastewater decolorization activity. Intact pellets absorbing dyes were deeply colored whereas those subject to degradation remained pale blue. This observation shows that decolorization by live pellets involves microbial metabolism. The longevity of decolorization activity of live and dead pellets was further investigated in repeated-batch decolorization mode. The live pellets showed high and stable decolorization activity. Immobilized Phanerochaete chrysosporium could maintain high decolorization activity in a long-term operation. Recently they reported that live pellets of the white-rot fungus ‘Funalia Trogii’ significantly decolorized Atrazine dye during a long-term cultivation.
The results suggest that Phanerochaete chrysosporium pellets can contribute to the decolorization and degradation of dyes in textile-industry effluents.
Highly Stable
Laccase from Repeated-Batch Culture of Funalia trogii ATTC
200800
The effect of temperature, pH, different inhibitors and additives on activity and stability of crude laccase obtained from repeated-batch culture of white rot fungus Funalia trogii ATCC 200800 was studied. The crude enzyme showed high activity at 55-90 degrees C, which was maximal at 80-95 degrees C. It was highly stable within the temperature intervals 20-50 degrees C. The half life of the enzyme was about 2 h and 5 min at 60 degrees C and 70 degrees C, respectively. pH optimum of fungal laccase activity was revealed at pH 2.5. The enzyme from F. trogii ATCC 200800 was very stable between pH values of 3.0-9.0. NaN3 and KCN were detected as the most effective potent enzyme inhibitors among different compounds tested.
The fungal enzyme was highly resistant to the various metal ions, inorganic salts, and organic solvents except propanol, at least for 5 min. Because of its high stability and efficient decolorization activity, the use of the crude F. trogii ATCC 200800 laccase instead of pure enzyme form may be a considerably cheaper solution for biotechnological applications.
References
- [1] Levin L, Papinutti L, Forchiassin F: "Evaluation of Argentinean white rot fungi for their ability to produce lignin-modifying enzymes and de-colorize industrial dyes", Biores Technol 2004; 94:169–176.
- [2] Kurniawati S. ,and Nicell J. (2008), Characterization of Trametes versicolor laccase for the transformation of aqueous phenol, Bioresource Technology, 99(16), pp.7825-7834.
- [3] Nyanhongo G., Gomes J., Gübitz G., Zvauya R., Read J. and Steiner W. (2002), "Decolorization of textile dyes by laccases from a newly isolated strain of Trametes modesta", Water Research, 36(6), pp.1449-1456.
- [4] Vršanská M., Voběrková S., Jiménez Jiménez A., Strmiska V. ,and Adam V. (2017), Preparation and Optimisation of Cross-Linked Enzyme Aggregates Using Native Isolate White Rot Fungi Trametes versicolor and Fomes fomentarius for the Decolourisation of Synthetic Dyes, International Journal of Environmental Research and Public Health, 15(1), p.23.
- [5] Guo H., Zheng B., Jiang D. ,and Qin W. (2017) Overexpression of a Laccase with Dye Decolorization Activity from Bacillus sp. Induced in Escherichia coli Journal of Molecular Microbiology and Biotechnology, 27(4), pp.217-227.
- [6] Hildén K, Hakala TK, Lundell T, Thermotolerantand thermostable laccases, Biotechnol Lett 2009, 31:1117.
- [7] Witze A. (2019), Will people have enough water to live? [online] Science News. Available at: https://www.sciencenews.org/article/future-will-people-have-enough-water-live [Accessed 11 Apr. 2019].
- [8] Low O.K., Mahadi M.N, Illias Rosli Md. (2013), Optimization of signal peptide for recombinant protein secretion in bacterial hosts. Appl Microbiol Biotechnol, 97:3811-3826