Our product is made of parts and each has been well designed to fit together. Take a look at what we've made down below to get a deeper understanding of how we created our solution.
BBa_K3103001Name
pAM_f2Short Description
PrimerCategory
Devdhi Kasana and Sadie MeunierDesigners
Long DescriptionThis is the forward primer used to make fragment A from the pAM_4788 backbone of the superPETase gene.
Design ConsiderationsThe original forward primer was 18 bp long. However, after consulting with our adviser, Dr. Nick Menhart, the primer-dimer potential became apparent, as shown below:
To resolve this issue, an additional 4 base pairs were added at the 3’ end of the original primer.
Long DescriptionThis is the reverse primer used to make fragment B from the pAM_4788 backbone of the superPETase gene.
Design ConsiderationsThe sequence of this primer originally had high primer-dimer potential, as shown below:
However, this was resolved by adding an A at the 3’ end of the original primer before converting into the complement.
Long DescriptionCreates PET fragment from pCS_PET. It includes Gibson overlap regions used to join the pAM backbone and the PET gene into an entire vector.
Design ConsiderationsNone
Part SequenceTTA AGA AGG AGC CCT TCA CCA TGG CAA ACA ACG ACC TAT T
Long DescriptionAn internal primer used to split the pAM backbone into 2 regions. This reverse primer is used to fragment the backbone into fragment A.
Design Considerations Running PCR amplification for the pAM backbone would consist of >7k base pairs. This is too large to be efficient. To compensate for this, we divided the pAM backbone into 2 separate fragments and created primers. The primer's name "35K" refers to 3.5 kbp past pAM_f .
Long DescriptionA pAM-PET internal primer which is used to screen the potential vector. It is used with the pETseq_f primer to yield unique size fragments.
Design ConsiderationsWe did an initial screening with the sequencing primers that flank the gene insertion site in the vector backbone. Every plasmid backbone has a _f and_r sequencing primer which is used to sequencing interred genes, and can also be used to PCR the insert up. When screening by PCR we looked for a specific size (size of insert plus flanking sequence). To confirm OUR gene was inserted, we wanted a specific primer which only amplified our gene of interest. This is a lot more diagnostic. We did this by making primers specific to the gene, and using one of the _f or _r primers in conjunction with the sequencing _r and _f primers.
Long DescriptionA pAM-PET internal primer which is used to screen the potential vector. It is used with the pETseq_fr primer to yield a unique size fragments.
Design ConsiderationsWe did an initial screening with the sequencing primers that flank the gene insertion site in the vector backbone. Every plasmid backbone has a _f and_r sequencing primer which is used to sequencing interred genes, and can also be used to PCR the insert up. When screening by PCR we looked for a specific size (size of insert plus flanking sequence). To confirm OUR gene was inserted, we wanted a specific primer which only amplified our gene of interest. This is a lot more diagnostic. We did this by making primers specific to the gene, and using one of the _f or _r primers in conjunction with the sequencing _r and _f primers.
Nicholas Menhart, Thao Dang, Sarahi TrujilloDesigners
Long DescriptionThe purpose of the superPETase gene is to degrade PET plastic in the ocean through cyanobacteria.
Design ConsiderationsDuring the design one mutation was made to the wildtype PETase gene. An Isoleucine was changed to phenylalanine to enhance enzyme activity. Please see superPETase design. In addition the start codon of the wt PETase gene was removed and substituted by a ser.
