Difference between revisions of "Part:BBa K4229076"

Revision as of 15:16, 12 October 2022

SPD5 with SnoopTag and SpyTag

This Biobrick shows a microcompartment, which is build through liquid-liquid phase seperation, SPD5.

Usage: Phase separation droplets can function as membrane-less organelles, found to be related to e.g. microtubule nucleation, stress granules genome organization, etc. in vivo [1]. Moreover, phase-separation has attracted attention in the field of synthetic biology due to its spatial localization and separation properties. SPD-5 is such a liquid-droplet-forming protein and fused to spy-and snoop-tag this allows for the targeting of any catcher-containing protein to the liquid droplets.

Biology Liquid droplet formation is driven by a manifold of interactions between the molecules involved. Multivalency, describes a molecule capable of interacting with other molecules at many different sites. Therefore, the formation of liquid droplets is depending on the concentration of molecules. Liquid droplets may form from one single type of proteins or multiple different types. Liquid droplets are expected to be dynamic in vivo. However, over time it is known that phase separating proteins transition from a dynamic state, where the droplets can move inside the cell to a more static gel-like phase, also known as molecular aging. Section based on [1] and [2].

SPD-5 is a protein natively expressed in C.elegans which spontaneously self-assemble dynamic organelles in vitro and in vivo [3]. Not only does SPD5 show the advantageous property of forming droplet-like structures, but it also has been shown to naturally recruit enzymes and related molecules into the dynamic formation [4]. Even though SPD5 liquid droplets are dynamic and do not permit exclusive entry and exit of specific molecules, it has been successfully used to enhance the efficiency of reactions, for example, by improving non-canonical amino acid incorporation with an orthogonal translation system [5].

Experimental Results:

Disclaimer: These experiments presented are performed with a version of SPD-5 where silent mutations were introduced through site-directed mutagenesis to make it compatible with the registries assemblies. However, experiments performed on our wiki (iGEM Freiburg 2022) are performed with a SPD-5 with the same aminoacid sequence, but with the slight difference in the DNA sequence. With these results presented on this page, we argue that the silent mutations introduced does not change the function of the protein and that the two versions of SPD-5 are comparable. Aim: The biobrick contains SPD-5 fused to spy- and snoop-catcher, which allows for the catching of mVenus and mTurquoise. This is expected cause formation of fluorescent “foci” representing the liquid droplets. Catching via spy- and snoop-tag was further characterized with western blot. Experimental setup: Strains are prepared in MG1655 and are co-transformed with either pBbE6a containing SPD-5 or SPD-5 with spy- and snoop-tag on N- and C-term, respectively and pBbA2c containing either mVenus or mTurqouise. Strains were induced at OD: 0.6-0.8 and then incubated for 24h at 18°C. IPTG and Doxycyclin induces the expression of SPD-5 and mVenus/mTurquoise, respectively. Samples were induced with 10 µM IPTG and 25 ng/ml Doxycycline for mVenus and 10 ng/ml for mTurquoise.

Figure 1: Fluorescent microscopy of cells expressing SPD-5 and mVenus. MG1655 was co-transformed with SPD-5 with spy- and snoop-tag on N- and C-term as well as a plasmid expressing A mVenus or B mTurquoise. Bacteria expressing SPD-5 and mVenus or mTurqouise form dots with strong fluorescent intensity (scale bar 5µm).

Figure 2: Western blot of catching between SPD-5 and mVenus or mTurquoise. MG1655 was co-transformed with a plasmid with SPD-5 with spy- and snoop-tag on N- and C-term and either a plasmid expressing mVenus (lane 2, 3, 4, 5) or a plasmid expressing mTurquoise (lane 6,7, 8, 9). There is a shift in the band for mVenus and mTurquoise when expressed in the presence of SPD-5

Figure 1 shows fluorescent microscopy of the same samples presented in Figure 2. When expressed alone mVenus and mTurqouise are located throuout the cytoplasm and do not show any presence of liquid droplets. However, when expressed in the presence of SPD-5 fused with spy- and snoop-tag, there are a clear localization of the fluorescence to the poles of the cells.

Figure 2 shows the western blot of the same samples as above, for SPD-5 and mTurquoise or mVenus. For both the spy-catcher containing mVenus and the snoop-catcher containing mTurquoise, the western blot shows a clear shift in their bands, when expressed in the presence of SPD-5_spy/snoop. These results are in agreement with previous western blot performed with not mutated SPD-5. These results therefore show that the SPD-5 with spy-and snoop-tag is capable of catching the respective cargo proteins, which the SPD-5 alone, according to our previous results, is not capable of. 

Conclusion: From the fluorescent microscopy and the western blots comparing the previous not mutated SPD5-5 we can conclude that the results obtained of these two constructs are highly similar and it is fair to assume that the silent mutations we performed with site-directed mutagenesis, do not influence the function of the SPD-5 and also not its ability to catch cargo through the spy- and snoop-tag.

[1] S. Alberti, “Phase separation in biology,” 2017, doi: 10.1111/pbi.1280. [2] M. C. Huber et al., “Designer amphiphilic proteins as building blocks for the intracellular formation of organelle-like compartments,” Nat Mater, vol. 14, no. 1, pp. 125–132, Jan. 2015, doi: 10.1038/nmat4118. [3] D. R. Hamill, A. F. Severson, J. C. Carter, and B. Bowerman, “Centrosome maturation and mitotic spindle assembly in C. elegans require SPD-5, a protein with multiple coiled-coil domains,” Dev. Cell, vol. 3, no. 5, pp. 673–684, 2002, doi: 10.1016/S1534-5807(02)00327-1. [4] A. K. Tiwary and Y. Zheng, “Protein phase separation in mitosis,” Curr. Opin. Cell Biol., vol. 60, no. 1, pp. 92–98, Oct. 2019, doi: 10.1016/j.ceb.2019.04.011. [5] C. D. Reinkemeier, G. E. Girona, and E. A. Lemke, “Designer membraneless organelles enable codon reassignment of selected mRNAs in eukaryotes,” Science (80-. )., vol. 363, no. 6434, 2019, doi: 10.1126/science.aaw2644.

Sequence and Features