Difference between revisions of "Part:BBa K5236006"

Revision as of 11:28, 2 October 2024

IsPETase M154L

IsPETase is friendly to enviorment and energy-saving to chemical recycling of PET. However, the temperature for it to react is even lower than glass transition temperature of PET. This basic part encodes mutated IsPETase M154L and constructed in Escherichia coli.

Usage and Biology

To insert our parts into plasmids, we’ve designed primers and performed PCRs. Then, our genes were recombined into plasmids and transformed into chassis. To test if our part codes for the mutated IsPETase M154L we want and whether the enzyme works, we've completed two large experimental processes. The first step is plasmid construction. And the second is to test the enzymatic activity.

By conducting colony PCR, we are able to test if our parts have been transformed into chassis successfully. The following result of electrophoresis proves that we’ve inserted genes into chassis since the sequence containing our mutated genes has a total of 891 base pairs and the results are in the right location.

After proving that our genes existed in chassis, we need to test if the bacteria can survive as usual with our genes. Thus, we’ve coated the bacteria on nutritional petri dish. And after a night, E. coli grew over the plate our plate, justifying that E. coli can survive with the gene of our part.

The result show that chasis carrying our PETase could survive.

Reference

Brott, S., Pfaff, L., Schuricht, J., Schwarz, J.-N., Böttcher, D., Badenhorst, C. P. S., Wei, R., & Bornscheuer, U. T. (2021, November 29). Engineering and evaluation of thermostable isPETASE variants for PET degradation. Engineering in life sciences. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8961046/

Sequence and Features