This is the LC Cutinase gene used to break PET into terephthalic acid and ethylene glycol. The enzyme Cutinase is a lypolytic/esterolytic enzyme that degrades cutin, which is found in most plant and fungi' cuticles. They enzyme itself is more commonly found in plants and bacteria. Cutinase also can degrades water soluble esters and insoluble triglycerides. The enzyme hydrolyzes these substrates by creating an acyl enzyme intermediate. A metagenomic analysis was done in order to find more novel enzymes such as lipases, esterases, proteases, and cellulases, as well as furthering our knowledge of protein sequence space in the environment. Naturally, compost samples would have enzymes that degrade cell walls and other compounds with potentially useful applications. A novel homolog of cutinase, known as Leaf Compost Cutinase (LC) was found to have a 57.4% genetic similarity to cutinase found in T. Fusca, meriting an experiment to overproduce the protein in E. Coli. The study found that it's applications could be most valuable in the textile industry, among other material fields.
It should be noted that ethylene glycol is moderately toxic to humans and other animals.
Through p-nitrophenyl butyrate (pNPB) assays, the UC Davis team gathered enough data to determine that the LC-Cutinase part (BBa_K936000) incorporated in this part most likely exhibits its intended function as an esterase. Additionally, the Cutinase part with a pelB tag (BBa_K936013) has been found to be secreted from the cells expressing it. A detailed description of these assays can be found on the Module Engineering Project page: http://2012.igem.org/Team:UC_Davis/Project/Catalyst.
Western data of media samples probed for a his-tag shows that a protein of the length corresponding to cutinase (~30 kDa) is being secreted from the cell.
Quantitative measure of protein secretion into the media at different points after induction.
The above plots show the results on pNPB assays in which esterase activity is measured by the absorbance at 405 nm. It is clearly shown that the activity of cells expressing cutinase is much higher the background (negative control).
Through 3-day cell growth assays in 24-well plates, Team DNHS_SanDiego gathered data to quantitatively measure the effectiveness with which the LC-Cutinase part (BBa_K936000) performs its function of breaking Polyethylene terephthalate (PET) into terephthalic acid and ethylene glycol when transformed into K-12 Escherichia coli. As a control group to measure the function of LC-Cutinase against, the team also created an E. coli strain transformed with an empty vector plasmid, lacking the capability to degrade PET.
The plate assays were incubated and shaken at 35 degrees Celsius and the strains were grown both in the presence and absence of ground PET pellets at a concentration of 8 mg/mL.
Mono-2-hydroxyethyl terephthalate (MHET) is an immediate product of PET degradation and can be detected at an absorbance of 260 nm. So, in order to evaluate cutinase activity, the team measured the absorbance of the cultures’ supernatant at a range of wavelengths from 200 to 300 nm. The following graph depicts this data, with the cultures being diluted 5-fold before relative absorbance was measured:
At 260 nm, the supernatant of the E. coli cultures transformed with LC-Cutinase and grown in the presence of PET had a relative absorbance of 2.90. The blank media had an absorbance of 0.201, the empty vector culture grown without PET had an absorbance of 2.245, the empty vector culture grown with PET had an absorbance of 2.214, and the cutinase culture grown without PET had an absorbance of 2.293.
This data illustrates that cutinase is clearly performing its function of degrading PET into MHET well. The absorbance of the blank media serves as a control value that would indicate the complete lack of molecules detected at 260 nm absorbance, including MHET. The relative absorbance values for the three middle strains (the empty vector strain without PET, the empty vector strain with PET, and the cutinase strain without PET) are all extremely close. These strains are also theoretically lacking in MHET, as they either cannot degrade PET or were cultured without it or both. This means that an average value of approximately 2.250 relative absorbance can be attributed to other molecules released from E. coli that can be detected at 260 nm absorbance. However, the relative absorbance of the LC-Cutinase strain with PET, at 2.9, is significantly greater than 2.250 and the presence of PET was the only difference in the environments of the two groups of LC-Cutinase bacteria, leading to the conclusion that this difference is a result of MHET produced and LC-Cutinase is effectively degrading PET.
This information can also be found on the DNHS_SanDiego team website: https://2019.igem.org/Team:DNHS_SanDiego/Contribution
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12COMPATIBLE WITH RFC
- 21COMPATIBLE WITH RFC
- 23COMPATIBLE WITH RFC
- 25Illegal AgeI site found at 600
- 1000COMPATIBLE WITH RFC
Functional Parameters: Austin_UTexas
Burden Imposed by this Part:
Burden is the percent reduction in the growth rate of E. coli cells transformed with a plasmid containing this BioBrick (± values are 95% confidence limits). This BioBrick did not exhibit a burden that was significantly greater than zero (i.e., it appears to have little to no impact on growth). Therefore, users can depend on this part to remain stable for many bacterial cell divisions and in large culture volumes. Refer to any one of the BBa_K3174002 - BBa_K3174007 pages for more information on the methods, an explanation of the sources of burden, and other conclusions from a large-scale measurement project conducted by the 2019 Austin_UTexas team.
This functional parameter was added by the 2020 Austin_UTexas team.