Coding

Part:BBa_K2091004

Designed by: I. Bariah, E. Zajfman, I. Segal   Group: iGEM16_BGU_ISRAEL   (2016-10-03)


LC-Cutinase Codon Optimized

An E. coli codon optimized version of the LC-Cutinase protein. (BBa_K936000)
The sequence also includes the pelB leader sequence.

This codon optimzed version was designed using the Genome Compiler software. 4 other variants of the protein were designed and thermodynamically stabilized using the PROSS algorithm developed by Dr. Sarel Fleishman from the Weizmann Institute of Science. We have characterized and tested all 5 LC-Cutinase variants using several methods.

Characterization

Expression and Secretion

In order to verify that our protein is indeed expressed and secreted we had to induce its expression, separate it from the bacterial cells and identify it in the supernatant. (see [http://2016.igem.org/Team:BGU_ISRAEL/Protocols Protocols])
We chose two methods:
1. Commasie Blue staining:

Figure 1: A SDS-PAGE analysis of a bacterial supernatant after induction with IPTG and protein purification, followed by Coomassie Blue staining. The LC-Cutinase codon-optimized protein is shown of the gel at ~27kDa.


2. Mass Spectrometry:

Figure 1: A MALDI analysis of a bacterial supernatant after induction with IPTG and protein purification. The spike corresponding to the LC-Cutinase codon-optimized protein is shown on the plot at 27,747Da.

As seen from both tests, our protein is being expressed and secreted and is present in the supernatant at a molecular weight of ~27.7kDa.

pNP-Butyrate degradation assay

In order to evaluate the activity of the codon optimized version of LC-Cutinase we used a pNP-Butyrate degradation assay (see [http://2016.igem.org/Team:BGU_ISRAEL/Protocols Protocols]).
We used different concentrations of the substrate - 50/125/250μM and measured the absorbance at 405nm from the moment the enzyme was added.

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


In all three concentrations of the substrate the codon optimized LC-Cutinase ("CO") has shown the highest activity compared to all other variants. We can conclude that the codon optimzed version is improved compared to the W.T. in terms of pNP-Butyrate degradation activity.

PET degradation assay

In order to test the PET degradation ability of the LC-Cutinase proteins, all variants were grown on M9 minimal medium plates with shredded PET pellets as a sole carbon source to test their ability to degrade PET. (for detailed instructions on the preparation of the media see [http://2016.igem.org/Team:BGU_ISRAEL/Protocols Protocols])

Figure 4: E. coli expressing the codon optimized LC-Cutinase grown on a M9 agar plate with shredded PET pellets as a sole carbon source. Colonies are marked in red.
Figure 5: Control. E. coli expressing the W.T. LC-Cutinase grown on a M9 agar plate no carbon source.


The colonies were isolated and re-grown on fresh agar plates to check for viability. All of them were viable, suggesting they are indeed bacterial colonies and the bacteria are utilizing the PET.

After examining the macroscopic aspect of the PET degradation ability of LC-Cutinase we wanted to explore the microscopic effects of the enzyme on PET.
We incubated PET pellets for 2 days in a liquid LB broth with our E. coli expressing the codon-optimized LC-Cutinase protein and examined the results under a scanning electron microscope.

Control:

Figure 6: A scanning electron microscope (SEM) image of a PET pellet after 2 days of incubation in a liquid LB broth with no bacteria. X1000 magnification.
Figure 7: A scanning electron microscope (SEM) image of a PET pellet after 2 days of incubation in a liquid LB broth with no bacteria. X1000 magnification.


Figure 8: A scanning electron microscope (SEM) image of a PET pellet after 2 days of incubation in a liquid LB broth with no bacteria. X2500 magnification.
Figure 9: A scanning electron microscope (SEM) image of a PET pellet after 2 days of incubation in a liquid LB broth with no bacteria. X5000 magnification.











































As seen from the images, the surface of the PET is relatively smooth. Also, the rod shaped, high crystallinity PET, is fairly covered.

After Degradation:

Figure 10: 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. X1000 magnification.
Figure 11: 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. X1000 magnification.


Figure 12: 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.


As seen above, after incubation with the LC-Cutinase expressing bacteria, the surface of the PET is rough. We notice a lot of holes in the PET surface and the rod shaped, high crystallinity PET, is more exposed than the control.

In order to verify that the changes between the PET incubated with bacteria and PET with no bacteria are in fact the result of the action of LC-Cutinase and not the action of the bacteria, we have incubated the PET pellets with E. coli transformed with the pACYC vector without the LC-Cutinase gene:

Figure 13: A scanning electron microscope (SEM) image of a PET pellet after 2 days of incubation in a liquid LB broth with E. coli transformed with the pACYC vector with no insert. X1000 magnification.
Figure 13: A scanning electron microscope (SEM) image of a PET pellet after 2 days of incubation in a liquid LB broth with E. coli transformed with the pACYC vector with no insert. X2500 magnification.

























As seen in the pACYC images, the surface of the PET is relatively smooth, similar to the PET with no bacteria, and is very different in texture than the PET incubated with the LC-Cutinase expressing bacteria.

We can conclude from these results that the change in texture of the PET pellets is in fact the result of the LC-Cutinase enzyme.





Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 550
  • 1000
    COMPATIBLE WITH RFC[1000]


[edit]
Categories
Parameters
None