Team:UCL/Parts
Parts
Part Highlights
Thanks to IDT and Twist's generous DNA synthesis offers, we were able to create and test a large number of parts. Some of our most important parts are detailed and described in the text and diagram below. For our full list of parts see the Part Table
Characterize
BBa_K2842690 - GFP intein
We considered using inteins as part of our modular drug delivery system to join binding peptides to our delivery vesicles (encapsulins) post-expression since one of our main concerns about our engineered encapsulin vehicle was that the targeting peptides loaded onto the encapsulin monomers’ surface may hinder proper encapsulin assembly. We thought rather than fusing the targeting peptide directly to the monomers, we could fuse the relatively small intein unit to the monomers (not hindering assembly), then subsequently add the targeting peptide with a matching intein and have it splice onto the surface of the already assembled encapsulin shells. Therefore we characterized UCL iGEM 2018's part, GFP intein.
Validate
BBa_K3111001 - Myxococcus Xanthus encapsulin monomer
BBa_K3111001 has been used to explore the modularity of our drug delivery platform in terms of the delivery nanoparticle. It encodes for Myxococcus xanthus encapsulin monomer which once expressed self-assembles into an 180-mer to form a hollow 30-32 nm encapsulin shell. We wanted to study its expression in E. coli and the possibility of purification to get an insight of its production feasibility. Moreover, using transmission electron microscopy and dynamic light scattering we studied its morphological properties as well as its physicochemical stability to different pH and temperature conditions.
BBa_K3111011, BBa_K3111201, BBa_K3111202- DARPin929 Constructs
This part was used as a binding and uptake facilitator for our drug delivery platform target, HER2+ breast adenocarcinoma cells. To observe this, we fused the DARPin with mScarlet, a red fluorescent protein, confirming binding with confocal microscopy and flow cytometry. We demonstrated that DARPin929 retains its specificity when fused on the C-terminus of another protein in BBa_K3111201 and BBa_K3111202, as structural modeling showed concerns on the binding site getting buried. This property was then used to join DARPin929 to the outer shell of T. maritima encapsulin, where it was shown to still exhibit specificity to HER2 receptor.
Improve
BBa_K3111103 - T. maritima encapsulin (6-His) with a StrepII-tag expressed under T7 promoter
This part encodes for the Thermotoga maritima encapsulin monomer, which, once expressed, self-assembles with additional monomers into a 60-mer encapsulin shell with a diameter of approximately 20-24 nm. This BioBrick constitutes a modification to BBa_K2686002, which was designed by the EPFL 2018 iGEM team. Like its previously existing counterpart, our BioBrick has a HexaHistidine linker insert (GGGGGGHHHHHHGGGGG) between residues 43 and 44, a sequence that has been shown to increase the heat stability of the encapsulin multimer. However, unlike BBa_K2686002, our construct encodes an inserted StrepII-tag to purify assembled encapsulins without heat treatment. Although this modification was originally expected to only expand the variety of chromatographic methods that could be applied to purify T. maritima encapsulin monomers, a comparative characterisation of the unstudied in vivo expression of the existing part and our modified version of the part concluded that the insertion of the StrepII-tag also resulted in in greater purity and solubility.
BBa_K3111031 - sfGFP with Encapsulin targeting tag
This part encodes sfGFP with a targeting peptide sequence that loads it into the T.maritima encapsulin. This part was created in order to ensure we could indeed load cargo into the T.maritima encapsulin, as well as to estimate loading efficiency of the encapsulin.
Demonstrate
BBa_K3111502 - T. maritima encapsulin monomer fused to DARPin929 and co-expressed along miniSOG with a targeting peptide.
This composite part formed our drug delivery vehicle and was used to demonstrate its proof of concept application in the potential treatment of HER2 positive cancer. This involved cloning and expression of the assembled encapsulins with surface displayed DARPin929, followed by downstream cloning of the photosensitiser miniSOG with a C-terminal tag (1) which allows cargo loading. We proceeded to study targeted delivery and cytotoxicity on SK-BR-3 cells which overexpress HER2 receptors, to which the fused DARPin929 is specific (2,3). This involved, incubating the sample with cultured SK-BR-3 cells and illuminating them in order to activate miniSOG's generation of singlet oxygen. Taking advantage of the fluorescent properties of miniSOG the cells were observed under the confocal microscope to observe uptake. After imaging imaged the cells subjected to flow cytometric analysis to determine the pattern of cell death and overall loss of viability. These experiments have shown that our drug delivery platform successfully targets HER2 overexpressing SK-BR-3 cells and reduces their viability at a significantly higher level than MSCs once illuminated with light, thus demonstrating double selectivity in terms of cytotoxicity.
Part Table