Team:AUC TURKEY/Project/Mechanism


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The most appropriate scale to deducing the qualitative chance in reporter systems relies on visual data.[i]The reporter system that we designed as an alternative to the current reporter systems uses the change factor in the current reporter systems as the response. Instead of observing change through the synthesis of color, the system is built around the observation of change through breaking down dye; the dye degradation plays a key role in the system, therefore for the system to function there is the requirement of using specific dye in experiments alongside the standard lab equipment. The bacteria will degrade the necessary dye to report the response.


Color-Based Reporter System

The dye that was designated and the enzyme that was to show activity in accordance to it was one of our concerns. The enzyme-substrate action in this novel system carried great importance and had to be designed with utmost care.

Peroxidases, which actively take part in the breakdown of lignin-based compounds thus contributing to the maintenance of the continuity of the existence of white rot fungi that we based our bleaching enzymes on ,were enzymes that we took into our prospect. We elected lignin peroxidase, an enzyme actively used by white rot fungi to continue their life , as the enzyme to be implemented into our system. To increase the variance and the richness in future application, we chose a secondary enzyme. The secondary enzyme was the horseradish peroxidase as it had a very strong structure and high activity.


Lignin peroxidase: Lignin is highly resistant to biodegradation and only higher fungi are capable of degrading the polymer via an oxidative process.Lignin is found to be degraded by an enzyme lignin peroxidases produced by some fungi like Phanerochaete chrysosporium.The mechanism by which lignin peroxidase (Lip) interacts with the lignin polymer which involves Veratryl alcohol (Valc), a secondary metabolite of white rot fungi, acts as a cofactor for the enzyme.

In enzymology, a lignin peroxidase (EC is an enzyme that catalyzes the chemical reaction

1,2-bis(3,4-dimethoxyphenyl)propane-1,3-diol + H2O2   3,4-dimethoxybenzaldehyde + 1-(3,4-dimethoxyphenyl)ethane-1,2-diol + H2O

Thus, the two substrates of this enzyme are 1,2-bis(3,4-dimethoxyphenyl)propane-1,3-diol and H2O2, whereas its 3 products are 3,4-dimethoxybenzaldehyde, 1-(3,4-dimethoxyphenyl)ethane-1,2-diol, and H2O.

This enzyme belongs to the family of oxidoreductases, specifically those acting on a peroxide as acceptor (peroxidases) and can be included in the broad category of ligninases. The systematic name of this enzyme class is 1,2-bis(3,4-dimethoxyphenyl)propane-1,3-diol:hydrogen-peroxide oxidoreductase. Other names in common use include diarylpropane oxygenase, ligninase I,diarylpropane peroxidase, LiP, diarylpropane:oxygen,hydrogen-peroxide oxidoreductase (C-C-bond-cleaving). It employs one cofactor, heme.

LiP catalyzes the H2O2-dependent oxidation of a variety of lignin model compounds in the following multistep reaction sequence:

LiP(Fe+3)P + H2O2 à LiP-I(Fe+4 – O)P’ + H2O

LiP-I(Fe+4 – O)P’ + R à LiP-II(Fe+4 – O)P + R’

LiP-II(Fe+4 – O)P + R + 2(H+) à LiP(Fe+3)P + R’ + H2O

Horseradish peroxidase: The enzyme horseradish peroxidase (HRP), found in the roots of plant horseradish, is used extensively in biochemistry applications primarily for its ability to amplify a weak signal and increase detectability of a target molecule. It is a metalloenzyme with many isoforms and the most studied type is C.

The structure of the enzyme was first solved by X-ray crystallography in 1997 and has since has been solved several times with various substrates. It is an all alpha-helical protein which binds heme as a cofactor.

Alone, the HRP enzyme, or conjugates thereof, is of little value; its presence must be made visible using a substrate that, when oxidized by HRP using hydrogen peroxide as the oxidizing agent, yields a characteristic change that is detectable by spectrophotometric methods.

Numerous substrates for the horseradish peroxidase enzyme have been described and commercialized to exploit the desirable features of HRP. These substrates fall into several distinct categories. HRP catalyzes the conversion of chromogenic substrates (e.g., TMB, DAB, ABTS) into coloured products, and produces light when acting on chemiluminescentsubstrates (e.g. ECL).

Horseradish peroxidase is a 44,173.9-dalton glycoprotein with 6 lysine residues which can be conjugated to a labeled molecule. It produces a coloured, fluorimetric, or luminescent derivative of the labeled molecule when incubated with a proper substrate, allowing it to be detected and quantified. HRP is often used in conjugates (molecules that have been joined genetically or chemically) to determine the presence of a molecular target. For example, an antibody conjugated to HRP may be used to detect a small amount of a specific protein in a western blot. Here, the antibody provides the specificity to locate the protein of interest, and the HRP enzyme, in the presence of a substrate, produces a detectable signal.Horseradish peroxidase is also commonly used in techniques such as ELISA and Immunohistochemistry due to its monomeric nature and the ease with which it produces coloured products. Peroxidase, a heme-containing oxidoreductase, is a commercially important enzyme which catalyses the reductive cleavage of hydrogen peroxide by an electron donor.

Horseradish peroxidase is ideal in many respects for these applications because it is smaller, more stable, and less expensive than other popular alternatives such as alkaline phosphatase. It also has a high turnover rate that allows generation of strong signals in a relatively short time span.

Moreover, "In recent years the technique of marking neurons with the enzyme horseradish peroxidase has become a major tool. In its brief history, this method has probably been used by more neurobiologists than have used the Golgi stain since its discovery in 1870."



In order to verify that the horseradish peroxidase and lignin peroxidase enzymes, which were not transformated and synthesized in bacteria in the past, worked; we decided to use a constitutive promoter. We chose the constitutive promoter as J23100, one of the strongest.  We wanted to be certain that our protein is surely produced due to the J23100 constitutive promoter that is 35 bp and coming after the prefix of 22 base pairs. The exhibition of our results in Western Blotting through the addition of a 6 histidin long his-tag at the end of the horseradish peroxidase C and lignin peroxidase 1 enzyme sequences, which contain BamH-I restriction cites that are followed by our ‘AAAGAGGAGAAA’ sequenced RBS, was added to our lab plans. This would in turn give us the safest experimental procedure to validate the presence of the proteins syntesized.

The accruity of our design was to be tested through getting the dye to react with the culture of bacteria containing enzymes that were to be rapidly synthesized through the constitutive promoter. The changes ranging from the visually observable changes in the color concentration to the shifts in the wavelenght absorbance which can only be detected through Varioskan approve the validity of our project. The degradation of the specified dyes by the lignin peroxidase and horsereadish peroxidase, enzymes we selected to breakdown these dyes, is enough to complete the rudimentary part of our project. The addition of the enzymes as a composite at the end of another part would enable the production of the protein in the sequence as well as the the enzymes enabling the response through an accelerated breakdown process.

Aside of the affirmation of the synthesis of the enzymes with the Western Blott results, to confirm the functionality of the proteins carries great importance. We came to the conclusion that working with several methods to verify the results would be more professional and certain:

-The confirmation of the enzyme activity can be done through collecting the data acquired through the observation of the interaction between the interaction of the specified dyes and DegradEcolor bacteria. For this feat to be accomplished, the selection of a dye that both lignin peroxidase and horseradish peroxidase can degrade in equivalent levels would be more reasonable. We chose Methylene blue as the dye to be used as it was degraded by the two enzymes in similar amounts during trials. The trials were consisting of the observation of the changes in the absorbance and color through the reactions between methylene blue and the two enzymes. The degradation of the dye plays a keyrole in the determination of the enzyme activity..

-HRP activity was assayed using 2,4-dichlorophenol and 4-aminoantipyrine as the substrates, substrates used to assay the activity of enzymes. LiP activity was assayed using veratryl alcohol as the substrate.

-While examining the general properties of the two designated enzymes, we found out that the horseradish peroxidase enzyme had the ability to determine the presence of a molecular target. If we are to recall the Western blott procedure we see the following:

The proteins in the lysate that were planted in the jel first run and transferred to the membrane afterwards. After being left to interact with the primary antibodies causing the immuglobins to bind with the specific binding site located on the protein.In the case of contamination or the absence of protein synthesis, no binding will be observed. After the completion of the binding of the primary antibodies, the secondary antibody binding process begins. While the secondary antibodies bind to the primary antibodies, they bring with them the horseradish peroxidase enzymes that are attached to them. The enhanced chemiluminescence solution does emission under the Western blotting device. The luminol in the enhanced chemiluminescence solution is oxidised by hydrogen peroxide, however this reaction can only occur in the presence of the horseradish peroxidase; as explained, if the proteins are correctly synthesized the horseradish peroxidase will cause oxygenation to take place causing emission. The same effect can be acquired through the horseradish peroxidase that is synthesized by DegradEcolor. In thıs case, the functionality of the enzyme could be proven after seeing emission from the interaction between the enhanced chemiluminescence solution and DegradEcolor or lysate prepared from DegradEcolor. As horseradish peroxidase was able to catalise the reaction of hydrogen peroxide, lignin peroxidase, another peroxidase enzyme, would most likely be able to have the same effect. We are expecting to see emission from the bacteria or lysate prepared from the bacteria that produce lignin peroxidase.

Dye Degradation

Methylene blue, the dye which the horseradish peroxidase and lignin peroxidase exhibited equivalent levels of degradation on, will show that DegradEcolor produced the functional proteins. Addition of the horseradish peroxidase or lignin peroxidase enzyme sequence to the composite part would make the part include a reporter system. If the part is coded and the enzyme is translated, the enzyme will be synthesized and change in methylene blue will be observed. Many deductions can be obtained with the results of the methylene blue degradation including the following:

-The bleeching of the dye color, which can be understood through qualitative observation, allowsus to reach certain conclusions. The bleeching of the color primarily indicates that the enzymes were synthesized. therefore no contamination took place. Apart from the synthesis factor, the conclusion that  the conformational structure is functional can be reached. This will in turn provide insight that the other desired proteins will also be synthesized. The system will also enable the ability to evaluate the efficiency of the system through the gradient of color concentration.

-The changes in absorbance will ease the acquirement of the certain results. The absorbance data can be used to prepare a professional and highly efficient scale for examination and use. The prepared scales can be used for comparison between the future experiments and the current experiments. It would be hard and even erronous to make judgement on strains that do not have high protein synthesis but the comparison between the acquired absorbance values and the scales not only  removes the risk factor but also gives the opportunity of making precise judgement in the absence of visual data.






Advanced System

The main principle of our advanced system that has brought a new dimension to reporter systems lies in the variance in the activity levels of the lignin peroxidase and horseradish peroxidase enzymes. The enzymes to have effect on one specific dye at the same time would not bring us any experimental gain. For this reason, the advanced system contains two different dyes alongside the two enzymes. The system is built around the inability of one enzyme in degrading the one dye while the other can, while the opposite is true for the other dye. Experimental systems containing a complex unison of enzymes and dyes can be constructed.

To further enhance our project, we formed such a complex system. The bacteria were given two parts, one containing horseradish peroxidase while the other contains lignin peroxidase. The dyes that were selected were Methyl green and Azure-b. While methyl green is, as the name implies, green colored, azure-b has a color in between pink and purple. Horseradish peroxidase can effectively degrade methyl green but lacks the ability to effectively degrade azure-b meanwhile lignin peroxidase is active in the degradation of azure-b and lackluster in degrading methyl green.

If parts starting off with different inducible promoters were transformed to the same bacteria, different data from different conditions could be acquired. Think of a system in which the bacteria have inducible promoters that are oxygen sensitive and the other carbon dioxide sensitive. If methyl green and azure-b was to be added to cause interaction with the bacteria, the varying levels of oxygen and carbon dioxide would therefore result in different levels of enzymatic activity. Let’s say that the oxygen inducible bacteria contain genes that code for horseradish peroxidase and the carbon dioxide inducible bacteria contain genes that code for lignin peroxidase. The variation in the level of oxygen and carbon dioxide would change the rate of specific enzyme activity hence leading to a difference in the degradation levels of methyl green and azure-b. This would change the color of the resultant substrate thus allowing the identification of the activity of the inducible promoters. In this way, scales could be prepared for many different types of inducible promoters for countless possibilities.

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