Difference between revisions of "Part:BBa K1602020"
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<h1>Inducible generator of Humicola insolens cutinase</h1> | <h1>Inducible generator of Humicola insolens cutinase</h1> | ||
| − | This generator overexpress Humicola insolens cutinase (HIC) by using a [https://parts.igem.org/Part:BBa_I719005 T7 promoter system]. For more information about the Humicola insolens cutinase (HIC) have a look on the [https://parts.igem.org/Part:BBa_K1602018 coding sequence]. | + | This generator overexpress Humicola insolens cutinase (HIC) by using a [https://parts.igem.org/Part:BBa_I719005 T7 promoter system]. |
| + | For more information about the Humicola insolens cutinase (HIC) have a look on the [https://parts.igem.org/Part:BBa_K1602018 coding sequence]. | ||
| + | The Cutinase as an α/β-hydrolase posseses two very interesting properties. It can connect as well as cleave ester bindings which makes it possible | ||
| + | to conduct composition as well as degradation of a varaiety of substrates. HiC is a monomeric enzyme with its active side at its surface. Due to this unique feature | ||
| + | HiC is capable to hydrolyze large polymers. | ||
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| + | <html><div class="contentSection"> | ||
| + | <figure class="centerFig"> | ||
| + | <img style="width: 400px; margin-left: 15px; margin-right: 15px;" src="https://static.igem.org/mediawiki/parts/c/cf/Hic.png"> | ||
| + | <figcaption><b>Figure 1</b> X-Ray structure of HiC with a resolution of 3 Angström (PDB: 4OYY).</figcaption> | ||
| + | </figure> | ||
| + | </div></html> | ||
<h2>Production of HiC in E.coli</h2> | <h2>Production of HiC in E.coli</h2> | ||
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<html><div class="contentSection"> | <html><div class="contentSection"> | ||
<figure class="centerFig"> | <figure class="centerFig"> | ||
| − | <img style="width: | + | <img style="width: 565px; margin-left: 15px; margin-right: 15px;" src="https://static.igem.org/mediawiki/parts/e/ee/Page_hic_a_v2.png"> |
| − | <figcaption><b>Figure | + | <figcaption><b>Figure 2</b> SDS Page of HiC expression.</figcaption> |
</figure> | </figure> | ||
</div></html> | </div></html> | ||
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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> | <h3>Results</h3> | ||
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</figure> | </figure> | ||
| − | The HiC is capable of degrading the prepolymer and in this process releases acidic compounds which acidify the buffer solution in the wells. This made visible via the indicator BTB. The temperature optimum was characterized as 45 | + | The HiC is capable of degrading the prepolymer and in this process releases acidic compounds which acidify the buffer solution in the wells. This made visible via the indicator BTB. The temperature optimum was characterized as 45%deg;C. We decided on such long kinetic cycles because the metabolic rate was proven very low. |
Revision as of 22:32, 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. The Cutinase as an α/β-hydrolase posseses two very interesting properties. It can connect as well as cleave ester bindings which makes it possible to conduct composition as well as degradation of a varaiety of substrates. HiC is a monomeric enzyme with its active side at its surface. Due to this unique feature HiC is capable to hydrolyze large polymers.
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
