Our efforts to contribute to future iGEM projects.
Our contributions to future iGEM teams revolves around our novel AAVP vector and the protocols and sequences associated with it. Previous AAVPs were developed using the obsolete fUSE5 phage, in contrast to ours using the f3-55nm phage. We have reviewed the literature as well as worked directly with the developer of phage plasmid displays, Dr. George P. Smith, to troubleshoot various issues with engineering these plasmids. We have published all of our protocols and troubleshooting under the Protocols and Engineering Success sections as references on how to successfully grow, digest/ ligate, and transform these plasmids. Our design and protocols revise previous examples to be more relevant for current and future uses of the vector, and expand on the former literature to make the vector more intuitive for future teams to use.
Read more at Experiments and Engineering Success.
To further help other iGEM teams and researchers in the phage community, we have made the complete sequence of the f3-55nm-MCS plasmid available with annotations at http://parts.igem.org/Part:BBa_K4415000. In order to do this, we confirmed the sequence of the previously unsequenced fMCS plasmid via sanger sequencing. Moreover, we understand that these plasmids are incredibly difficult to manufacture from the sequence due to internal repeats, internal hairpin structures, and other engineering challenges. As such, we have grown the double-stranded forms of f3-55nm, fMCS, and f3-55nm-MCS plasmids and stored dozens of bacterial stocks. We have obtained permission and plan to make all of these samples available for other teams to use and experiment with.
Read more at Project Description.
We have created 7 new parts. These parts include our plasmid backbone, immune proteins granzyme B and TNF-α, and other sequences required for the phage to propagate and function, such as the GSG-E2A, the Kozak sequence and the anti-CD22 scFv. Our modifications to these parts from the original sources are examples of what to consider when creating AAVPs. Furthermore, these parts are also available for other uses by teams not working with AAVPs with only minor modifications in a few cases.
Read more at Parts.