Team:BGU ISRAEL/ImprovedParts

PlastiCure

Improved Parts

Our team has chosen to improve the LC-Cutinase eznyme using a rational mutagenesis approach.

We have chosen to test our improved variants' activity using a pNP-Butyrate degradation assay with different substrate and enzyme concentrations.

Our experiments support that our designed proteins are significantly improved compared to the existing part BBa_K936000 due to the following reasons:

First, our experiments showed a significant improvement of pNP-B degradation activity in two of our LC-Cutinase variants:

  1. The codon optimized version (BBa_K2091004)
  2. The F4 variant (BBa_K2091005)

Our conclusion is based on kinetic test results which showed faster rise in O.D levels compared to the W.T. protein in three different substrate concentration (Fig. 1,2,3):

Figure 1: pNP-Butyrate degradation activity of all LC-Cutinase variants and W.T. at a substrate concentration of 50μM. For negative control we used E. coli strain BL-21 without any vector ("BL-21") and also E. coli strain BL-21 with only the pACYC plasmid backbone ("pACYC").

Figure 2: pNP-Butyrate degradation activity of all LC-Cutinase variants and W.T. at a substrate concentration of 125μM. Controls are the same as with 50μM concentration.

Figure 3: pNP-Butyrate degradation activity of all LC-Cutinase variants and W.T. at a substrate concentration of 250μM. Controls are the same as with 50μM concentration.

These tests were conducted using a bacterial supernatant, without measurement of enzyme concentrations, which might suggest that the improvement in activity is accomplished by an increase of enzyme expression/concentration. Although it suggests an improvement in expression levels and not efficiency, this hypothesis is still considered an improvement over the W.T. as it increases the degradation rate of the substrate.

In order to minimize the effect of expression levels on activity, we isolated and cleaned the enzymes and performed the experiment again, with equal enzyme concentrations.

Figure 4: pNP-Butyrate degradation activity of 2 LC-Cutinase variants - CO, F4 and the W.T., at a substrate concentration of 125μM. Enzyme concentration is 0.11mg/ml.

As seen from the results above, even in equal enzyme concentrations our LC-Cutinase variants display a significant improvement in pNP-B degradation in comparison to the W.T. LC-Cutinase.

Secondly, We have improved the characterization of the LC-Cutinase protein with regards to:

  1. pNP-Butyrate degradation - Previous data does not include kinetic tests, only final O.D values. We have also measured activity with different substrate concentrations at known enzyme concentrations, also, not performed previously.
  2. PET degradation - We have shown evidence of LC-Cutinase PET degradation activity using two different experiments - Agar plates containing shredded PET and Electron microscopy of PET samples incubated with LC-Cutinase. All previous data in the registry does not include records of PET degradation activity, only pNP-B.
Figure 5: E. coli expressing the W.T LC-Cutinase grown on a M9 soft agar plate with shredded PET pellets as a sole carbon source. Colonies are marked in red.
Figure 6: A scanning electron microscope (SEM) image of a PET pellet after 2 days of incubation in a liquid LB broth with E. coli expressing the codon-optimized LC-Cutinase protein. X5000 magnification.

For more results see our parts' pages - BBa_K2091004, BBa_K2091005.

Address:

Ben-Gurion University of the Negev
Ben Gurion 1, Beer Sheva 8410501, Israel

Mail: igembgu2016@gmail.com

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