Part:BBa_K2921150
6xHIS+LC Cutinase+GS+amilCP
This construct constitutively expresses Leaf Compost Cutinase (Basic part: BBa_K936000) linked with amilCP (Basic part: BBa_K592009). According to iGEM12_UC_Davis’s basic part page of BBa_K936000, cutinases are lypolytic/esterolytic enzymes that degrade and hydrolyze the ester bonds of cutin. The amilCP serves as a functional color reporter, allowing for convenient visible detection of the location of LC Cutinase.
Construct Design
This construct was created to constitutively express LC cutinase. Sequences used for the promoter, RBS, and double terminator came from parts included in the iGEM distribution kit. This construct consists of a strong promoter and strong RBS combination (BBa_K880005) to maximize protein production, the protein-coding gene LC cutinase (BBa_K936000), amilCP (BBa_K592009) and a double terminator (BBa_B0015) to end transcription.
To ensure that the binding protein and colored protein were fused, we inserted a flexible glycine-serine linker between the binding protein and the chromoprotein. For protein purification, in downstream applications, we added a hexahistidine tag (6xHis) after the RBS and before the open reading frame.
This entire construct was synthesized by Twist Bioscience.
PCR Check Results
The part was confirmed by PCR using the primers VF2 and VR, as well as sequencing by Tri-I Biotech.
We confirmed the size of K2921150 using the primers VF2 and VR, which resulted in the expected size of around 1.7 kb.
Functional Assay with Malathion
Our construct produces LC cutinase-GS-amilCP which is expected to degrade the organophosphate malathion into malathion monoacid and malathion diacid by ester hydrolysis (Kim, Ahn, Moon, & Lee, 2005). Theoretically, for our experimental group, we expect that the pH will decrease over time due to the production of acids. To test the functionality of this protein, we observed the change in pH over time in construct-expressing cells and negative-control cells. We had one experimental group: cells expressing the LC cutinase-GS-amilCP fusion protein. We had two negative control groups: cells expressing K88-amilCP protein and cells expressing the LC cutinase ORF-only plasmid; neither of these two constructs should produce proteins that would decrease the pH of the solution.
Overnight bacterial cultures were prepared and standardized to an OD of around 0.7. Then, the cultures were centrifuged and the pellets were resuspended in 2 mL of 1:1000 diluted malathion solution. The pH of each solution was measured every 5 minutes over 24 hours.
Preliminary analysis of the time-based data at room temperature suggested that the enzyme was either not functioning correctly due to being very inefficient because of a small Vm/Km ( see https://2019.igem.org/Team:TAS_Taipei/Model) or not present. Through more research, we discovered that the optimum condition for LC cutinase is at a pH of 8.5 and a temperature of 50°C (Sulaiman, et al., 2011). Even at optimal conditions, however, the Vm/Km of LC cutinase was only slightly increased. This led us to suspect that the enzyme was not actually present in the experiment, a fact that was later confirmed by our protein gel.
Characterization
We used SDS-PAGE to check for LC cutinase-GS-amilCP expression in E. coli carrying our construct. Bacterial cultures expressing the construct were grown overnight at 37°C, lysed and run on SDS-PAGE gels. We did not observe a signal for LC cutinase-GS-amilCP at the expected size of 58 kDa suggesting that LC cutinase-GS-amilCP was not being expressed. This result explains why our functional assay with malathion did not work.
To verify LC cutinase-GS-amilCP expression in E. coli, we subjected LC cutinase-GS-amilCP lysate to SDS-PAGE, expecting a signal at around 58 kDA. However we did not see a signal at the correct size which explains why our functional test did not work.
References
Kim, Y.-H., Ahn, J.-Y., Moon S.-H., Lee J., (2005). Biodegradation and detoxification of organophosphate insecticide, malathion by Fusarium oxysporum f. Sp. pisi cutinase. Chemosphere. doi: 10.1016/j.chemosphere.2005.02.023
Sulaiman, S., Yamato, S., Kanaya, E., Kim, J.-J., Koga, Y., Takano, K., & Kanaya, S. (2011). Isolation of a Novel Cutinase Homolog with Polyethylene Terephthalate-Degrading Activity from Leaf-Branch Compost by Using a Metagenomic Approach. Applied and Environmental Microbiology, 78(5). doi: 10.1128/aem.06725-11
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 716
- 1000COMPATIBLE WITH RFC[1000]
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