Part:BBa_K936000
LC Cutinase
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:
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 600
- 1000COMPATIBLE WITH RFC[1000]
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