Difference between revisions of "Part:BBa K3009033"

Latest revision as of 22:38, 13 October 2022

SPD5 Group: Freiburg Author: Alisa Summary: Spindle-deficient protein 5 derived from C. elegans is able to assemble into condensates that built a separate phase from the surronding cytosol, therefore emulating a reaction compartment. Fusion proteins will also be confined to this organelle. Documentation:

Usage

SPD 5 proteins assemble into phase-separated condensates in the cytosol, building a dynamic artificial organelle. Fusion proteins of SPD5 will be confined inside the organelle and separated from the all other components inside the cell. Due to the lack of a membrane, all other molecules can freely move in and out of the organelle.[1]

Biology

In C. elegans, SPD5 , forms condensates that aid the organization of microtubule arrays in the centrosome. [2]

Characterization

By expressing a SPD5:sfGFP fusion in E.coli, we have proven droplet formation in the E.coli cytosol.

Fig. 1: E.coli cells expressing a SPD5:sfGFP fusion verify droplet formation

To confirm that the constructs were were seeing were in fact dynamic droplets and not dead aggregates, we bleached several regions of interest with a 475 nm laser beam. Due to the dynamic nature of the droplets, flurescent should be recovered by diffusion of unbleached proteins

File:BBa K3009033 FRAP.mp4

References

[1]] Reinkemeier et. al 2019): Designer membraneless organelles enable codon reassignment of selected mRNAs in eukaryotes. In: Science (New York, N.Y.) 363 (6434). [2]Woodruff et. al (2017): The Centrosome Is a Selective Condensate that Nucleates Microtubules by Concentrating Tubulin. In: Cell 169 (6), 1066-1077

Improvement: iGEM2022_Freiburg

Biobrick for SPD5

Usage: The improved biobrick BBa_K42290076 consists of the genetic fusion between Spindle-deficient protein 5 (SPD-5; codon-optimized for expression in Escherichia coli) and the SpyTag and SnoopTag . It can be used to recruit two proteins (POIs) of interest into the liquid droplets formed by SPD-5 in E. coli. The POIs should be fused to the SpyCatcher (BBa_K42290009) and SnoopCatcher (BBa_K4229010), respectively.

Biology: Liquid droplets are membraneless organelles which form by liquid-liquid phase separation. Typically, proteins forming liquid droplets are multivalent, that is, they can bind to many other molecules at many different sites. Therefore, the formation of liquid droplets depends on the concentration of molecules. Liquid droplets may form from one single type of protein or multiple ones. Liquid droplets are expected to be dynamic in vivo. However, it has been observed that the droplets transition from a dynamic, liquid state, to a gel-like, more static one [2]. Liquid droplets have been functionally related for instance to microtubule nucleation [3], and stress granule formation [1]. Recently, the process of phase separation has attracted attention in the field of synthetic biology due to the possibility to exploit it to perform spatial localization of proteins of interest.

Spindle-deficient protein 5 (SPD-5) is a protein naturally found in Caenorhabditis elegans that spontaneously self-assembles liquid droplets in vitro and in vivo [3]. Not only does SPD-5 show the advantageous property of forming liquid droplets in cells, it also has been shown to naturally recruit enzymes and related molecules into them [4]. SPD-5-mediated liquid droplets have been successfully used to enhance the efficiency of reactions, for example improve non-canonical amino acid (ncAA) incorporation with an orthogonal translation system [5].

The iGEM team Freiburg 2019 showed that SPD-5 forms liquid droplets in E. coli. They used it to recruit specific mRNAs into the droplets to improve ncAA incorporation as done by C. D. Reinkemeier et al. and colleagues in mammalian cells [5]. For this reason, they genetically fused SPD-5 to the Ms2 coat protein (MCP).

Experimental Results:

Aim: Show with fluorescence microscopy that mVenus2 and mTurquoise2 localize into liquid droplets by means of the interaction with SPD-5. Additionally, prove with Western blotting that a peptide bond is formed between the SpyTag and the SpyCatcher and the SnoopTag and the SnoopCatcher, leading to the covalent bond of SPD-5, mVenus2 and mTurquoise2.

Experimental setup: SPD-5 is N-terminally fused to the SpyCatcher and C-terminally fused to the SnoopCatcher. mVenus2 is C-terminally fused to the SpyTag, while mTurquoise2 is C-terminally fused to the SnoopTag. The plasmids used are the following:

MG1655 cells were co-transformed with either pBbE6a containing untagged SPD-5 (original biobrick BBa_K3009033) or SpyCacther-SPD-5-SnoopCatcher (improved biobrick) and pBbA2c containing either mVenus2-SpyTag or mTurqouise2-mTurquoise2-SnoopTag. Cells were induced at OD600 ~ 0.6-0.8 and then incubated for 24 h at 18°C. 10 µM IPTG and 25 ng/ml doxycycline were added to induce the expression of SPD-5/SpyCacther-SPD-5-SnoopCatcher and mVenus2-SpyTag/mTurquoise2-SnoopTag, respectively. For the microscopy, the images were taken after 24 h of induction with IPTG and doxycycline in an inverse Zeiss Axio Observer Z1/7 fluorescence microscope equipped with a Pecon light tight incubator, an alpha Plan-Apochromat 100x/1.46 Oil DIC (UV) M27 objective with Zeiss Immersol 518 F immersion oil and an Axiocam 506 mono camera. The selected channel in Zeiss Zen 3.0 (blue edition) for images was GFP and brightfield. Excitation was done automatically using the EGFP channel (475 nm LED, 5-20 % intensity, 150 ms exposure time) and filters for excitation wavelength at 488 nm and emission wavelength at 509 nm.

Results:

Figure 1: Representative fluorescence microscopy images of MG1655 cells co-transformed with pBbE6a-SpyCacther-SPD-5-SnoopCatcher and either pBbA2c-mVenus2-SpyTag or pBbA2c-mTurqouise2. Scale bar, 5µm.

Figure 2: Western blot showing the formation of the peptide bond between SPD-5 and mVenus2. MG1655 cells were either co-transformed with pBbE6a-SPD-5 and pBbA2c-mVenus2-SpyTag or with pBbE6a-SpyCatcher-SPD-5-SnoopCatcher and pBbA2c-mVenus2-SpyTag. (-/+) refers to whether the sample was induced or not.

Discussion: Figure 1 shows fluorescent microscopy of the same samples presented in Figure 2. When expressed alone, mVenus2-SpyTag and mTurqouise2-SnoopTag are homogeneously distributed in the cytoplasm of the bacterial cells. When expressed in the presence of SpyCacther-SPD-5-SnoopCatcher, we observe the appearance of fluorescent foci towards the poles of the cells. Figure 2 shows the Western blot of the same samples shown in Figure 1. When expressed with untagged SPD-5, mVenus2-SpyTag runs at its expected size. When expressed in the presence of SpyCatcher-SPD-5-SnoopCatcher, we observe the appearance of higher molecular weight band corresponding to the fusion of the protein to SpyCacther-SPD-5-SnoopCatcher.

Conclusions: From the fluorescent microscopy, we conclude that SPD-5 fused to the catchers still forms liquid droplets in E. coli. From the western blot comparing the untagged SPD-5 (old biobrick) to the version with the catchers (improved biobrick), we can conclude that the protein of interest (mVenus in this example) are fused to SPD-5, thus co-localizing with it into the liquid droplets.

Improvement: This new biobrick can now be used to localize proteins of interest into liquid droplets in E. coli.

References:

[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