"
Team:AUC TURKEY/Results/Functional Essay
From 2014hs.igem.org
Spectrophotometric Absorption Measurements: We used Thermo Scientific Varioskan Flash Multimode Reader for our absorption measurements.
1) Measurements of Dyes:
Graphic 1: This graphic shows our absorption rate of our dye (Methylene Blue) in different concentrations. ( 1X=8 uM, 10X=80uM, 100X=800uM, 1000X=uM)
In the Graphic 1, we have got the correct absorption rates for every concentration of Methylene Blue dyes that correlates with the same results in the literature.
2)
Preparations of Lysates :
The verified plasmids were then cultivated in liquid culture for isolation. The 50 mL’s of bacteria containing the parts BBa_K1291071 and BBa_K1291299 were centrifuged at 4100 rpm for 5 minutes, the supernatant was discarded and then lysis solution was added. After 10 minutes of incubation in ice, the lysates were sonicated in the minimum activity level. After a spin of 15000 rpm for 10 minutes, the supernatant was transferred to eppendorphs and dye was added; the solution was boiled at 95 °C for 5 minutes. The lysate were stored at -20 °C.
3) Decolorization Assay:
In this assay we mixed our liquid cell culture and also our lysates with dyes. All the concentrations of the solutions are adjusted according to the literature. We measured the first absorptions of the samples and compared it with the 2nd measurement in the end of 1st hour.
Graphic 2: This graphic shows our absorption rate of our mixed sample that includes Lysates of our BL-21 Bacteria that have LiP gene and Methylene Blue (80uM) dyes and for the activate the LiP we also added Hydrogen Peroxide (800uM) measured in the 1 hour.
The result of Graphic-2 is that our Lysates of BL-21 Bacteria that have LiP gene degraded the Methylene Blue (80uM) dye with approximately %83,3 efficiency.
Graphic 3: This graphic shows our
absorption rate of our mixed samples that includes Lysates of our BL-21
Bacteria that have HRP gene and other Bacteria that have LiP gene and Methyl
Green (150uM) dyes and for the activate the HRP and LiP we also added Hydrogen
Peroxide (8.8 uM-HRP), (0,176 uM-LiP) concentrations that measured in the
different time periods.
The result of Graphic-3 is that our Lysates of BL-21 Bacteria that have HRP gene and other Bacteria that have LiP gene degraded the Methyl Green (8.8 uM) dye with approximately %71,4 efficiency in the end of the 7 hours.
.
Graphic 4: This graphic shows our absorption rate of our mixed samples that includes Lysates of our BL-21 Bacteria that have HRP gene and other Bacteria that have LiP gene and Methyl Green (150uM) dyes and for the activate the HRP and LiP we also added Hydrogen Peroxide (8.8 uM-HRP), (0,176 uM-LiP) concentrations that measured in the beginning of the experiment and in the 1st hour.
The result of Graphic-4 is that our Lysates of BL-21 Bacteria that have HRP gene and other Bacteria that have LiP gene degraded the Methyl Green (8.8 uM) dye with approximately %71,4 efficiency. Also in this graphic the most important point that our control group BL-21 bacteria that don’t include our genes of enzymes(HRP or LiP) couldn’t degrade the Methyl Green (8.8 uM) dye.
Graphic 5: This graphic shows our absorption rate of our mixed sample that includes liquid culture of our BL-21 Bacteria that have LiP gene and Methylene Blue (80uM) dyes and for the activate the LiP we also added Hydrogen Peroxide (800uM) measured in the 1 hour.
The result of Graphic-5 is that our Liquid cultures of BL-21 Bacteria that have LiP gene also degraded the Methylene Blue (80uM) dye with approximately %70 efficiency.
Graphic 6: This graphic shows our absorption rate of our mixed sample that includes liquid culture of our BL-21 Bacteria that have HRP gene and Methylene Blue (80uM) dyes and for the activate the HRP we also added Hydrogen Peroxide (800uM) measured in the 1 hour.
The result of Graphic-6 is that our Liquid cultures of BL-21 Bacteria that have LiP gene also degraded the Methylene Blue (80uM) dye with approximately %65 efficiency.
The Results of Graphic 5 and 6 show that our liquid culture of our BL-21 Bacteria that have HRP gene and other Bacteria that have LiP gene could degraded the Methylene Blue (80uM) dye but our Lysates of our BL-21 Bacteria that have HRP gene and other Bacteria that have LiP gene degraded the Methylene Blue (80uM) dye more efficiently. This results gave us an opinion that when our enzymes have further interaction with dyes they could degrade much more.
Graphic 7: This graphic shows our absorption rate of our mixed sample that includes liquid culture of our Control BL-21 bacteria that don’t have any HRP or LiP gene and Methylene Blue (80uM) dyes and for the activate the any peroxidases, Control BL-21 bacteria have, we also added Hydrogen Peroxide (800uM) measured in the 1 hour.
The result of Graphic-7 is that our Liquid culture of BL-21 Bacteria that don’t have any HRP or LiP gene could degrade the Methylene Blue (80uM) dye with approximately % 18 efficiency. This result show that Control bacteria can degrade the the Methylene Blue (80uM) dye but at the very low percentage when compared with our liquid culture BL-21 Bacteria that have HRP gene and other Bacteria that have LiP gene so we thought that some peroxidases also can degrade the dyes but our enzymes HRP and LiP have very high catalytic ability for the our dyes(Methlylene Blue and Methyl Green). These results and graphics show that our enzymes are functional and working properly.
