Team:AUC TURKEY/Human Practices/Future Plan

 

1) Reporter Systems Overview:

a)  Fluorescent Proteins:  The green fluorescent protein (GFP) is a protein  that exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range.  Although many other marine organisms have similar green fluorescent proteins, GFP traditionally refers to the protein first isolated from the jellyfish Aequorea victoria. The GFP from A. victoria has a major excitation peak at a wavelength of 395 nm and a minor one at 475 nm. Its emission peak is at 509 nm, which is in the lower green portion of the visible spectrum. The fluorescence quantum yield (QY) of GFP is 0.79. The GFP from the sea pansy (Renilla reniformis) has a single major excitation peak at 498 nm.

In cell and molecular biology, the GFP gene is frequently used as a reporter of expression. In modified forms it has been used to make biosensors, and many animals have been created that express GFP as a proof-of-concept that a gene can be expressed throughout a given organism. The GFP gene can be introduced into organisms and maintained in their genome through breeding, injection with a viral vector, or cell transformation. To date, the GFP gene has been introduced and expressed in many Bacteria, Yeast and other Fungi, fish (such as zebrafish), plant, fly, and mammalian cells, including human. Martin Chalfie, Osamu Shimomura, and Roger Y. Tsien were awarded the 2008 Nobel Prize in Chemistry on 10 October 2008 for their discovery and development of the green fluorescent protein.

 

 

a)       Luciferin: Luciferin (from the Latin lucifer, "light-bringer") is a generic term for the light-emitting compound found in organisms that generate bioluminescence. Luciferins typically undergo an enzyme-catalysed oxidation and the resultingexcited state intermediate emits light upon decaying to its ground state. This may refer to molecules that are substrates for both luciferases and photoproteins. Luciferins are a class of small-molecule substrates that are oxidized in the presence of the enzyme luciferase to produce oxyluciferin and energy in the form of light. It is not known just how many types of luciferins there are, but some of the better-studied compounds are listed below. There are many types of luciferins, yet all share the use of reactive oxygen species to emit light.

 

b)      Xgal Reporter System: X-gal is an organic compound consisting ofgalactose linked to a substituted indole. The compound was synthesized by Jerome Horwitz and collaborators in Detroit, MI, in 1964. The formal chemical name is often shortened to less accurate but also less cumbersome phrases such as bromochloroindoxyl galactoside. The X from indoxyl may well be the source of the X in the X-gal contraction. X-gal is much used in molecular biology to test for the presence of an enzyme, β-galactosidase. It is also used to detect activity of this enzyme in histochemistry and bacteriology. X-gal is one of many indoxyl glycosides and esters that yield insoluble blue compounds similar to indigo as a result of enzyme-catalyzed hydrolysis.^[2] X-gal is an analog of lactose, and therefore may be hydrolyzed by the β-galactosidase enzyme which cleaves the β-glycosidic bond in D-lactose. X-gal, when cleaved by β-galactosidase, yields galactose and 5-bromo-4-chloro-3-hydroxyindole. The latter then spontaneously dimerizes and is oxidized into 5,5'-dibromo-4,4'-dichloro-indigo, an intensely blue product which is insoluble. X-gal itself is colorless, the presence of blue-colored product therefore may be used as a test for the presence of an active β-galactosidase. This easy identification of an active enzyme allows the gene for β-galactosidase (the lacZ gene) to be used as a reporter gene in various applications.

 

c)      GUS Reporter System: The GUS reporter system (GUS: β-glucuronidase) is a reporter gene system, particularly useful in plant molecular biology and microbiology. Several kinds of GUS reporter gene assay are available, depending on the substrate used. The term GUS staining refers to the most common of these, a histochemical technique. The purpose of this technique is to analyze the activity of a promoter (in terms of expression of a gene under that promoter) either in a quantitative way or through visualization of its activity in different tissues. The technique is based on β-glucuronidase, an enzyme from the bacterium Escherichia coli; this enzyme, when incubated with some specific colorless or non-fluorescent substrates, can transform them into coloured or fluorescent products.

 

d) Chloramphenicol acetyltransferase (CAT) System: Chloramphenicol acetyltransferase (or CAT) is a bacterial enzyme (EC 2.3.1.28) that detoxifies the antibioticchloramphenicol and is responsible for chloramphenicol resistance in bacteria. This enzyme covalently attaches anacetyl group from acetyl-CoA to chloramphenicol, which prevents chloramphenicol from binding to ribosomes. A histidine residue, located in the C-terminal section of the enzyme, plays a central role in its catalytic mechanism.

The crystal structure of the type III enzyme from Escherichia coli with chloramphenicol bound has been determined. CAT is a trimer of identical subunits (monomer Mr 25,000) and the trimeric structure is stabilised by a number of hydrogen bonds, some of which result in the extension of a beta-sheet across the subunit interface. Chloramphenicol binds in a deep pocket located at the boundary between adjacent subunits of the trimer, such that the majority of residues forming the binding pocket belong to one subunit while the catalytically essential histidine belongs to the adjacent subunit. His195 is appropriately positioned to act as a general base catalyst in the reaction, and the required tautomeric stabilisation is provided by an unusual interaction with a main-chain carbonyl oxygen. CAT is used as a reporter system to measure the level of a promoter or its tissue-specific expression. The CAT assay involves monitoring acetylation of radioactively labeled chloramphenicol on a TLC plate; CAT activity is determined by looking for the acetylated forms of chloramphenicol, which have a significantly increased migration rate as compared to the unacetylated form.

 

d)       Other Response Systems: Some fast response systems were for constructed based on traditional response systems like GFP or luciferin reporter system through protein engineering. Although these response systems give fast response, they have high costs and/or difficulty in implementation. The systems are devised by major genetics companies that synthesize these systems using high cost equipment thus reflecting the costs to the buyers resulting in very expensive prices making it almost impossible for most of the iGEM teams to obtain them.

 

2) Advantages of DegradEColor:

               

a) Our system aims to operate faster because it focuses on the prencipal which is making the produced enzyme effect more substrata in which are dyes instead of continuously producing new proteins.

       

Our system works in a way that is suitable for different dyes and peroxidase enzymes of the project it is going to be applied.

        

c)  Also our system ables scientists to; develop dyes suitable for their works, and chose the suitable enzyme for their subject living material

 

3) Disadvantages of DegradEColor:

 

a) Our protein is produced by HRP-C enzyme which is taken by horseradish plant, and the other one LiP-1 is taken by white rot fungus. Since our enzymes are taken from living material in nature it might make things harder for people who wishes to use them on different livings.

 

b) The dyes we use alongside of our enzymes origins are used in histologic and genetic studies in order to colour DNA, RNA and other parts of cell, this situation is the reason why  our dyes does not to include the molecules we need which will increase the efficiency of our project.

 

4) Planned Improvements of DegradEColor:

 The dyes and enzymes we used should be engineered to be suitable for our project if it is going to be used as a reliable reporter system which will be used in synthetic biology and genetics in future.

a) Dye Improvements:

 

One of the actions that we conducted to further develop our future plans was to meet up with organic chemistry experts specialized in the field of dye synthesis. As a result of the meetings, we learned that it would be possible to further develop the structure of our selected dyes in respect to the speed of degradation thus reducing the total time of the process. . Our stops were Prof. Dr. Cihangir Tanyeli from METU, and Doc. Dr. Zeynel Seferoğlu from Gazi University. We took their thoughts about the possibility and any point that can be advanced of our project. With this way, our project has been approved and developed by experts.

Prof. Dr. Cihangir Tanyeli

METU Faculty of Organic Chemistry

 

One of the first questions we asked to Mr Tanyeli was: What different technique or techniques can we increase the effectiveness of Horse radish peroxidase and lignin peroxidase or how can we make our dyes more sensitive and suitable through our enzymes and be able to improve the usage field of our project?

 

He focused on re-designing the dyes to make them suitable for enzymes which was his main field of study and he offered us 2 different methods.

 

1st Method: We can develop the dye matter by using 4-amino-TEMPO. This is a good redox system. By this way that we won’t need hydrogen peroxidase for Oxidation system. A polymer can be taken as a Matrix. 4-amino-TEMPO can be immobilized to biodegradable polymer. With the way that, sodium hypochlorite (NaClO) can be used as an alternative to hydrogen peroxide. Since this is a macro molecular system, it can be precipitated to water and regained this will provide us an ease of researching. When used in a bulk system in the nature, most usable metalloporphirins have silica like systems that has Si main structure, are more solid and hard to degrade, but still, if the biodegradable systems are used catalytic activity can be decreased and be a system that can fit to nature instead of being waste.

 

In order to block the degrading ability of the 4-amino-TEMPO system, antioxidants like ascorbic acid can be used this happens by radical group gets silenced by ascorbic acid which goes into a reaction with Bleach. He also stated that we can use other antioxidants that alternatives of ascorbic acid. In the other hand,. NaClO(Bleach instrument) is could be harmful for the environment. 4 amino tempo system we suggested has an incredible balance with bleach insturemuer so we for the nature we can chose the most envorment insrtie , destroying its harms for environment because bleach + tempo system  In this method we focus on 4 amino tempos.. The reason we use amino instead of hydroxide, we want our TEMPO molecule (oxidation instruments) can easily interact with some organic molecules that can be produce by organisms with amino group..

2^nd method: Some functional groups can be placed on Carbon Nanotubes. Carboxylic acid can be placed on Carbon Nanotubes with desiccation. Also 4-amino-TEMPO added on Carboxylic acid functional group with direct covalent immobilization approach. If the 4-amino-TEMPO stated gets inserted on and bleach instruments like NaClO added the environment dye degradation will be happen. Big advantages of Carbon nanotubes that can be regained with filtration and precipitation and this make this system more efficient and economic and using over and over.

 

Both of this methods are using with switch systems that working with pH or temperature control by an enzymes, or a bacteria or a bio systems.

 

We stated that time used by the systems used currently used around the world takes 16 hours is decreased to a few hours by our system while we were talking to our precious scholar. He gave us valuable information. We thank to him for giving us time.

 

Doc. Dr. Zeynel SEFEROĞLU

Gazi University Faculty of Organic Chemistry

 

We started our conservation with our scholar how we can make our dyes more suitable for our dyes and make it fit right in to our project. He suggested us to make a research on quantum efficient dyes and increase our knowledge on functional fluorescence dyes. When we first mentioned our project he liked it and stated that this could be used easily and efficiently in genetic and synthetic biology world. He gave us some articles that might help. He also stated that it is important to introduce to science to the world since iGEM is already doing that we made someone else like iGEM. We thank our scholar for giving us opportunity to have this conservation.

 

 

 

 

 

 

 

 

 

 

ALFA Dye and  Chemistry

We wanted to inform the local companies about iGEM and our project. Our first target was a professional corporation Alfa Dye. Here we talked about the possibility and usage in daily life and received some information on industrial dyes. We learned the dyes used in histologic dying are an addition to pigments as connecting molecules. So if we want to add other activities other than bioremediation of dye wastes we should add some small systems which will cleave such connections. With this visit we had new a perspective through the future. The company got happy for us to be in such competition. They sacrificed their time in order to give us directions we thank them for that.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

b) Enzyme and Genetic Dyes Improvements:

 

Another activity to further develop our future plans was to consult genetic, biology and biochemistry experts and genetics companies to assist in the development of the theory of our project. The feedback that we received was indicating that through the development of the binding sites of the enzymes with protein engineering the project could be improved and that the project beared great potential for the improvision of synthetic biology.

 

Prof. Dr. Sadık Çiğdem

Tsukuba University Genetic Faculty Japan

 

He is one of the guest scientist of Turgut Ozal University Genetic Department. When we mentioned him about our project DegradEColor he is really impressed about the idea behind our project and also he gave us some advice and information about our HRP enzyme. He informed us that one of our enzyme HRP is used by ELISA and Western-Blot experiments. HRP is added on antibodies in Western Blot and also ELISA and cause the substrate ECL give response. We thanked to him for this opportunity.

 

 

 

 

 

 

 

 

 

 

 

Prof. Dr. Esra Gündüz

Turgut Özal University Faculty of Medicine

 

We informed her about our iGEM HS Project DegradEColor, she really interested on our project. She was very pleased when she saw us, doing such work at this young age. She stated that she will provide any support she can.   

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Burak Yılmaz

The founder of SENTEGEN and its CEO

 

We impressed Mr. Yılmaz with our project and aim to reduce the time of response systems. He stated managing time is a very big problem for companies. The company which will use our system will manage their time 14~15 times more efficient. He said “This advantage can’t be ignored”. He congratulated us for coming up with such a wonderful idea we thank him for this.