Coding

Part:BBa_K936000:Experience

Designed by: Nicholas Csicsery   Group: iGEM12_UC_Davis   (2012-07-17)


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Applications of BBa_K936000

User Reviews

UNIQe7499772b3105f62-partinfo-00000000-QINU UNIQe7499772b3105f62-partinfo-00000001-QINU

Contributions

Group: iGEM16_BGU_ISRAEL
Author: I. Bariah, E. Zajfman, I. Segal
Summary: We have created a codon optimized version of the LC-Cutinase protein for E. coli.
We have also conducted kinetic tests for the degradation of pNP-Butyrate and microbial PET degradation assays using this protein.
Uploads: BBa_K2091004, BBa_K2091005
Kinetic studies:
We have tested the LC-Cutinase W.T. protein and our variants with a pNP-Butyrate assay. We measured absorbance at 405nm as a function of time, from the addition of the substrate.

pNP-Butyrate degradation activity of all LC-Cutinate 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").
pNP-Butyrate degradation activity of all LC-Cutinate variants and W.T. at a substrate concentration of 125μM. For control we used E. coli strain BL-21 without any vector ("BL-21") with pACYC plasmid backbone only ("pACYC").






















pNP-Butyrate degradation activity of all LC-Cutinate variants and W.T. at a substrate concentration of 250μM. For control we used E. coli strain BL-21 without any vector ("BL-21") with pACYC plasmid backbone only ("pACYC").


Additional test were performed after purification with a cation exchange column with an enzyme concentration of 0.11mg/mL:

pNP-Butyrate degradation activity of 3 LC-Cutinase variants - CO, F4 and the W.T., at a substrate concentration of 125μM. Enzyme concentration is 0.11mg/mL.


PET bacterial degradation assay:

E. coli transformed with the LC-Cutinase (BBa_K936000) protein and grown on M9 soft agar plates with shredded PET pellets. Colonies marked in red.
Control. E. coli transformed with the LC-Cutinase (BBa_K936000) protein and grown on M9 soft agar plates with no carbon source.