Team:Aboa/Parts
Parts
Here we describe the parts we used to optimize our production and create the improved immunoassays. These parts were added to the part registry. We used a lot more parts, but these were the ones that worked. Links providing more information on each part can be found at the bottom of the page.
Parts for expression of Fab fragments
This is the codon harmonized sequence of a digoxigenin-recognizing Fab fragment, where the stop codon between the two chains have been changed into TAA stop codon to be able to express it in our system. Digoxigenin immunoassays are competitive immunoassays, which means that this Fab is immobilized onto a surface and labeled digoxigenin is added into the sample (1).
This part can be added to pAK vectors for expressing Fabs with non-canonical amino acids at the tail.
This part can be added to pAK vectors for expressing Fabs with non-canonical amino acids incorporated at a TAG codon in the heavy or light chain.
This vector backbone belongs to the pAK vectors and it can be used for expressing Fab fragments in Esherichia coli by ligating them after a SfiI digestion site. This vector has an ampicillin resistance gene and lac promoter following a pelB signal sequence upstream Fab ligation site.
Improvement to existing parts in registry"
This part is improved from the biobrick BBa_K567018 by removing the T7 promoter sequence. Since this part lacks the T7 promoter sequence, the biobrick can be directly used to test incorporation of noncanonical amino acids (ncaa) in amber stop codons in Escherichia coli hosts lacking T7 like the amberless C321.deltaA.exp (321.A) strain (2). The original iGEM team, STJU-BioX Shanghai have explained, that the idea of this part was to be able to report the functionality of their tRNA synthetases, but we used it to optimize our production.
References
1. Mayilo, S., Ehlers, B., Wunderlich, M., Klar, TA., Josel, HP., Heindl, D., Nichtl, A., Kürzinger, K. & Feldmann, J. (2009) Competitive homogeneous digoxigenin immunoassay based on fluorescence quenching by gold nanoparticles. Anal Chim Acta 646:119-122.
2. Escherichia coli C321.deltaA.exp from Addgene. [html document visited 10/09/2019] <groupparts>iGEM19 Aboa</groupparts>