1.SUCCESSFUL CLONING
We did the transformation of our composite part (OmpA-linker-SpeB_Cleavage_Site-XylE) which is 1655 bp long. We applied our regular procedure (see procedures under the lab button) to E.coli colonies that we have prepared before. Electrophoresis results showed that our gene is produced by the colonies and it is in the correct range (1500-2000 bp). After the repetitions of the same procedure, we agreed that cloning was successful.
2.SDS PAGE EXPERIMENT
In order to check whether our enzyme is working or not and also to control if the proteins are producing, we made the SDS PAGE experiment. We showed that our enzyme is working and our construct is produced. After the experiment of lysate the band that we want to see is nearly 53 kda long.
1) IgG
2)IgG with PBS+Mercaptoethanol
3)IgG with SpeB+PBS+Mercaptoethanol
4) marker
5) Compatent E. Coli
6) Bacterial lysate (2,5 ul)
7)Bacterial lysate (5 ul)
8)Bacterial lysate (10 ul)
9)Bacterial lysate (20 ul)
10) Marker
Our system was working, we decided to do color change experiment.
3.COLOR CHANGE
Catechol-2,3-dioxygenase is the enzyme of reaction between catechol and oxygene. If there is not Catechol-2,3-dioxygenase in the medium, catechol reacts with oxygene and produces black color. In our construct Catechol-2,3-dioxygenase can work if it is liberated from the linker via SpeB, virulence factor secreted by S. Pyogenes which cleaves SpeB_Cleavage_Site.
SpeB enzyme works with a reducing agent in appropriate buffer. There are several buffers that SpeB can work in. We decided to use phosphate buffered saline (PBS), which is the most popular one. There are also several reducing agents that can be used with SpeB; we decided to use 2-Mercaptoethanol.
As we proved with SDS experiment that our protein is produced and the SpeB enzyme is working properly, we started our experiment to detect the existence of S. Pyogenes in the medium via color change. We used the different eppendorfs that include competent cell(1), colonies with our construct (2) SpeB added colonies with our construct (3); respectively. All mixtures that were in the eppendorfs dissolved in 2-Mercaptoethanol and PBS.
After incubating all the mixtures for a period of time, we observed a clear color difference. Sample of group 1 were black; sample of group 2 were a slightly brighter than the samples on group 1; sample of group 3 were fairly bright. When we measured of the absorbance values by using Varioskan Flash, we observed that between 300 nm and 350 nm, there was a peak value which was 330 nm. The biggest difference occurs in 330 nm. Therefore unique property of the reaction occurs in our project is the light emitting at 330 nm.
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