Difference between revisions of "Part:BBa K1602020"
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<h2>Production of HiC in E.coli</h2> | <h2>Production of HiC in E.coli</h2> | ||
| − | + | You can easily produce HiC by inoculation of 20 mL LB 100 mL flask with E.coli BL21(DE3) containing Bba_K1602020. After incubation at 180 repulsion per minute (rpm) at 37°C until an OD of 0.6 is reached you can start induction with IPTG (conc. 0.5 mM). The final incubation of the cell suspension take place over night at 180 rpm at 28°C. | |
| − | + | The SDS-Page reveals protein expression before and after induction. HiC can be detected with an molecular mass of 22-24 kDa. | |
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4-MU shows fluorescence at an excitation wavelength of 360 nm and an emission wavelength of 449 nm. | 4-MU shows fluorescence at an excitation wavelength of 360 nm and an emission wavelength of 449 nm. | ||
As increasing fluorescence intensity directly correlates with increasing concentration of 4-MU, this assay is perfect for studying enzyme kinetics of HiC in real time and high resolution. | As increasing fluorescence intensity directly correlates with increasing concentration of 4-MU, this assay is perfect for studying enzyme kinetics of HiC in real time and high resolution. | ||
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| + | <h3>Results</h3> | ||
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<html><div class="contentSection"> | <html><div class="contentSection"> | ||
<figure class="centerFig"> | <figure class="centerFig"> | ||
| − | <img src="https://static.igem.org/mediawiki/2015/8/80/Da15_hic.png"> | + | <img style="width: 565px; height: 110px; margin-left: 15px; margin-right: 15px;" src="https://static.igem.org/mediawiki/2015/8/80/Da15_hic.png"> |
<figcaption><b>Figure 4</b> This graph show the activity of the HiC at 25°C.</figcaption> | <figcaption><b>Figure 4</b> This graph show the activity of the HiC at 25°C.</figcaption> | ||
</figure> | </figure> | ||
</div></html> | </div></html> | ||
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<html><div class="contentSection"> | <html><div class="contentSection"> | ||
<figure class="centerFig"> | <figure class="centerFig"> | ||
| − | <img src="https://static.igem.org/mediawiki/2015/c/c7/Da15_hic_heat_test.png"> | + | <img style="width: 565px; height: 110px; margin-left: 15px; margin-right: 15px;" src="https://static.igem.org/mediawiki/2015/c/c7/Da15_hic_heat_test.png"> |
<figcaption><b>Figure 5</b> Heat activity test with rising temperature from 45°C to 70°C..</figcaption> | <figcaption><b>Figure 5</b> Heat activity test with rising temperature from 45°C to 70°C..</figcaption> | ||
</figure> | </figure> | ||
Revision as of 20:49, 24 September 2015
Contents
Inducible generator of Humicola insolens cutinase
This generator overexpress Humicola insolens cutinase (HIC) by using a T7 promoter system. For more information about the Humicola insolens cutinase (HIC) have a look on the coding sequence.
Production of HiC in E.coli
You can easily produce HiC by inoculation of 20 mL LB 100 mL flask with E.coli BL21(DE3) containing Bba_K1602020. After incubation at 180 repulsion per minute (rpm) at 37°C until an OD of 0.6 is reached you can start induction with IPTG (conc. 0.5 mM). The final incubation of the cell suspension take place over night at 180 rpm at 28°C. The SDS-Page reveals protein expression before and after induction. HiC can be detected with an molecular mass of 22-24 kDa.
Activity with 4-Methylumbelliferyl butyrate (4-MUB)
To test for hydrolysis activity on aromatic esters, a fluorescence assay using 4-MUB, which was originally developed for screening lipase activity, can be used. HiC converts 4-MUB to the fluorescent product 4-methylumbelliferole (4-MU) and butyric acid. 4-MU shows fluorescence at an excitation wavelength of 360 nm and an emission wavelength of 449 nm. As increasing fluorescence intensity directly correlates with increasing concentration of 4-MU, this assay is perfect for studying enzyme kinetics of HiC in real time and high resolution.
Results
Large polyester dergradation
Final volume of the plate was 200µl containig:
- 142µl Buffer (Na2HPO4 pH 7.0)
- bromothymol blue 10% (v/v)
- TES protein fraction HiC (ranging from 5µl to 30µl; concentration unknown)
- 8µl prepolymer (dissolved in Triton100 and DMSO 1:50)
- TES protein fraction (from BL21 cells ranging from 5µl to 30µl; as a control)
The negative control was done by just adding buffer to the well.
The 96 well microplate was loaded as depicted in the picture below: </p>
The assay was run in a in a TECAN® Infinite 200 PRO multi plate reader for 100 kinetic cycles, each 5 mins long and with 25 photo pulses per cycle. The reader was heated to the appropriate temperature of 42° celsius. Absorbance was measured at the absorption maximum of BTB which in this case is 620nm.
Pictures of the multi plate
