When constructing our BioBricks, we made use of the IDT synthesis offer. This way we were able to generate our required parts in a fast and efficient way. Additionally, we made use of PCR amplification with our constructed expression plasmids as template to construct several BioBricks. We created a part collection comprised of 6 basic parts encoding for different EPO coding sequences, 2 basic parts encoding for the required components for targeted sequencing and 1 composite part: our fusion protein. All BioBricks are submitted with elaborate characterisation so the user may choose the best protein for their project accordingly.
Our part collection establishes a platform for Targeted Next Generation Sequencing applicable for gene doping detection. Additionally, this part collection can easily be applied to any scientific case that requires analysis of specific sequences. The platform includes a transposase-dxCas9 fusion protein, transposon adapter sequence, sgRNA sequence, and multiple target and off-target EPO sequences. These components are required to perform RNA-guided adapter ligation needed for target specific sequencing. As a result, the technology can rapidly process the specific DNA sequence of interest, minimizing data output and sequencing time. The part collection also contains an adapter sequence containing an example barcode. Barcoding different samples allows for multiplexing, increasing the number of samples sequenced simultaneously. Our part collection sets the basis for targeted next generation sequencing by providing all necessary components, adjustable at will, and can be a solution in almost any sequencing case.
|♥||BBa_K2643000||Composite||dxCas9-linker-Tn5 fusion protein (HIS Tag)||TU Delft 2018|
|♥||BBa_K2643004||Basic||Coding region (CDS) of human erythropoietin (EPO) hormone||TU Delft 2018|
|BBa_K2643005||Basic||Human EPO gene with artificial intron (615 bp)||TU Delft 2018|
|BBa_K2643006||Basic||Human EPO gene with artificial intron (135 bp)||TU Delft 2018|
|BBa_K2643007||Basic||Human EPO gene with 2 artificial introns (615 + 135 bp)||TU Delft 2018|
|BBa_K2643008||Basic||Human EPO gene with mutation 1 (5 bp)||TU Delft 2018|
|BBa_K2643009||Basic||Human EPO gene with mutation 2 (12 bp)||TU Delft 2018|
|BBa_K2643011||Basic||Mosaic Ends-flanked Kanamycin cassette and RFP||TU Delft 2018|
|BBa_K2643012||Basic||T7p expressing sgRNA targeting the EPO coding sequence||TU Delft 2018|