Part:BBa_K4229078

SPD5 with SnoopTag and SpyTag regulated by lamdapL promotor and LacI promotor

Usage: This biobrick consists of the genetic fusion between Spindle-deficient protein 5 (SPD-5; codon-optimized for expression in Escherichia coli<i/>) and the SpyTag and SnoopTag . It can be used to recruit two proteins of interest (POIs) into the liquid droplets formed by SPD-5 in <i>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-SnoopCatche 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-SpyCatcher-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-SpyCacther-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 homogenously 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.

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

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 443
  Illegal EcoRI site found at 2862
  Illegal EcoRI site found at 4036
  Illegal SpeI site found at 1840
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 443
  Illegal EcoRI site found at 2862
  Illegal EcoRI site found at 4036
  Illegal SpeI site found at 1840
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 443
  Illegal EcoRI site found at 2862
  Illegal EcoRI site found at 4036
  Illegal BglII site found at 452
  Illegal BglII site found at 2763
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 443
  Illegal EcoRI site found at 2862
  Illegal EcoRI site found at 4036
  Illegal SpeI site found at 1840
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 443
  Illegal EcoRI site found at 2862
  Illegal EcoRI site found at 4036
  Illegal SpeI site found at 1840
  Illegal NgoMIV site found at 335
  
• 1000
  COMPATIBLE WITH RFC[1000]