Parts

Overview over our parts

Toolbox for bacterial microcompartments

With this toolbox, we are setting the foundation for the compartmentalization of bacteria. With three different compartments, with two having a rigid shell and one building liquid droplets. The rigid structures are wiffleballs and encapsulines. There are two versions of the wiffleball, the minimal-wiffleball containing only two (H-protein and T1-protein) proteins, and the full-wiffleball containing four proteins (H-protein, T1/T2/T3-protein). The second rigid compartment is built out of encapsulins, which are taken out of the organism M. xhantus. The liquid droplets are made out of SPD5 by liquid-liquid phase separation. It is possible to recruit proteins in all of those compartments. Into the wiffleball and SPD5 by fusing the snoop- or spyCatcher with the protein you wish to put inside those compartments. To put the protein inside of the encapsulines you would need to add a target peptide at the C-terminus of the protein.

ID	Name	Function
ID BBa_K4229029	H Protein of the BMC	Assembles hexameres and the basic structure of the BMCs
ID BBa_K4229030	T1 Protein of the BMCs	Assembles hexameres and a basic pore structure of the BMCs
ID BBa_K4229035	T2 Protein of the BMCs	Assembles together with the other T proteins a complex pore structure of the BMCs
ID BBa_K4229036	T3 Protein of the BMCs	Assembles together with the other T proteins a complex pore structure of the BMCs
ID BBa_K4229037	T1 with snoop/spyTag	T1 protein with a spyTag N-terminal and a snoopTag C-Terminal
ID BBa_K4229038	RBS of the T proteins	RBS of the T proteins
ID BBa_K4229039	RBS of the H protein	RBS of the H protein
ID BBa_K4229040	LambdapLhybdrid promotor	lacI regulated hybrid promotor, which regulates the whole BMC expression
ID BBa_K4229041	LacI promotor	regulated the Lambda pL promotor
ID BBa_K4229042	H and T protein (minimal wiffelball)	Builds the minimal wiffelball with a basic pore structure
ID BBa_K4229043	Minimal wiffelball with tags on the T1 protein	Builds the minimal wiffelball with a basic pore structure but T1 is able to recrute proteins with a snoop or spyCatcher
ID BBa_K4229044	H and T1/T2/T3 protein (full wiffelball)	Builds the full wiffelball with a more complex pore structure but T1 is able to recrute proteins with a snoop or spyCatcher
ID BBa_K4229045	full wiffelball with tags on the T1 protein	Builds the full wiffelball with a more complex pore structure
ID BBa_K4229046	Minimal wiffelball regulated	BBa_K4229042 under regulation of BBa_K4229040, BBa_K4229041
ID BBa_K4229047	Minimal wiffelball with tags, regulated	BBa_K4229043 under regulation of BBa_K4229040, BBa_K4229041
ID BBa_K4229048	Full wiffelball regulated	BBa_K4229044 under regulation of BBa_K4229040, BBa_K4229041
ID BBa_K4229049	Full wiffelball with tags regulated	BBa_K4229045 under regulation of BBa_K4229040, BBa_K4229041
ID BBa_K4229059	tetA/B promotor	a promotor wich regulates reporter Genes as mVenus2, mTurquoise2 and sfGFP
ID BBa_K4229062	mVenus2 with spyCatcher	Flourecent reporter, to see function spyTaged T1 protein
ID BBa_K4229063	RBS + mVenus2 with spyCatcher	RBS + mVenus2 with spyCatcher
ID BBa_K4229064	mTurquoise2 with snoopCatcher	Flourecent reporter, to see function snoopTaged T1 protein
ID BBa_K4229065	RBS + mTurquoise2 with snoopCatcher	RBS + mTurquoise2 with snoopCatcher
ID BBa_K4229066	mVenus2 with spyCatcher regulated by tetA/B promotor	BBa_K4229063 regulated by BBa_K4229059
ID BBa_K4229067	mTurquoise2 with snoopCatcher regulated by tetA/B promotor	BBa_K4229065 regulated by BBa_K4229059
ID BBa_K4229019	sfGFP with TargetPeptide	Through targetpeptid is possible to recruit into the encapsulines
ID BBa_K4229020	Encapsulines out of m. Xhantus	Aggregates together into encapsulines
ID BBa_K4229058	RBS for sfGFP with TargetPeptide	RBS for sfGFP with TargetPeptide
ID BBa_K4229060	sfGFP with TargetPeptide + RBS	BBa_K4229019 with RBS
ID BBa_K4229061	ssfGFP under T7 promotor	BBa_K4229060 under the regulation of BBa_K4229025
ID BBa_K4229068	araBad promotor	regulates one of the encapsuline plasmidse
ID BBa_K4229069	Encapsulin with RBS	BBa_K4229004 with the encapsuline
ID BBa_K4229076	SPD5	Builds liquid droplets by phase seperation
ID BBa_K4229077	SPD5 + RBS	BBa_K4229076 with BBa_K4229039
ID BBa_K4229078	SPD5 regulated by LambdaPl promoter abd LacI promotor	BBa_K4229077 regulated by BBa_K4229040, BBa_K4229041

Toolbox for Indigo/Indirubin & Trehalose pathway

This toolbox allows you to produce both indigo and indirubin by utilizing a new version of the indigo pathway. Our biggest change is the inclusion of the enzyme XiaI, which is a terpenoid cyclase replacing FMO in previously established pathways. We have stuck to TnaA as a PLP-dependent lyase which transforms L-tryptophan into indol. This molecule is then in turn converted into 2-hydroxyindol and 3-hydroxyindol by the previously mentioned XiaI. Additionally, we have added the flavin oxidoreductase Fre, which enhances the provision of reduced flavin that is required by XiaI to fulfil its function. Finally, TnaB was added, which is involved in the L-tryptophan transport across the cytoplasmatic membrane. In our toolbox, TnaA and XiaI were enhanced with the addition of a snoop- or spycatcher, allowing you to easily incorporate these core enzymes into our compartments!

ID	Name	Function
ID BBa_K4229001	Fre	Reduces soluble flavins
ID BBa_K4229003	TnaB	transports L-tryptophan inside the bacteria
ID BBa_K4229004	RBS from pCDF-Duet-1	RBS from pCDF-Duet-1
ID BBa_K4229005	RBS for FRE	RBS for FRE
ID BBa_K4229006	RBS for TnaB	RBS for TnaB
ID BBa_K4229009	SnoopCatcher	Binds with the snooptag and makes localisation possible
ID BBa_K4229010	SpyCatcher	Binds with the SpyTag and makes localisation possible
ID BBa_K4229013	SnoopCatcher-TnaA N-terminal	TnaA fused with the snoopcatcher on the N-terminus
ID BBa_K4229015	spyCatcherXiaI N-terminal	XiaI fused with the snoopcatcher on the N-terminus
ID BBa_K4229016	RBS-snoopCatcherTnaA-RBS-FRE (snoTAF)	First half of the indigo-pathway with BBa_K4229013
ID BBa_K4229018	RBS-spyCatcherXiaI-RBS-TnaB (spyXTB)	Second half of the indigo-pathway with BBa_K4229015
ID BBa_K4229025	T7 promoter	is recognized by the T7 polymerase, is used to regulate genexpression
ID BBa_K4229026	Lac Operator	Operator of the lac operon, used to fix the leaky expression of the T7
ID BBa_K4229033	snoTAF under the T7 promotor and LacOperator	BBa_K4229016 under regulation of BBa_K4229025 and BBa_K4229026
ID BBa_K4229034	SpyXTB under the T7 promotor and LacOperator	BBa_K4229018 under regulation of BBa_K4229025 and BBa_K4229026
ID BBa_K4229050	OtsA	Enzyme that turns glycose-6-phosphate into trehalose-6-phosphate
ID BBa_K4229051	OtsB	Enzyme that turns trehalose-6-phosphate into trehalose
ID BBa_K4229052	OtsA + RBS	OtsA with BBa_K4229004
ID BBa_K4229053	OtsB + RBS	otsB with BBa_K4229004
ID BBa_K4229054	OtsA regulated by T7 promotor	OtsA regulated by T7 promotor
ID BBa_K4229055	OtsB regulated by T7 promotor	OtsB regulated by T7 promotor

Toolbox for amber suppression and ncAA incorporation

This collection of parts with amber stop codon mutatitions will serve as a tool to easily and precisely measure non-canonical amino acid incorportaion and utilize their potential. The different sfGFP mutants can be used as a model to test the efficiency of incorporation of non-canonical amino acids via amber stop codon suppression technology. We created 4 mutants with amber stop codon mutations at position G4, F8, Y74 and Y151. Fluoresce of the mutants can only be restored upon successful incorporation of non-canonical amino acids and therefore provides a direct way to measure amber stop codon suppression efficiency. The other part of our collection are bacterial microcompartments with amber stop codon mutations at functional positions. We created They can be used to strategically modify the compartments and try out the possibilities of non-canonical amino acids. The different mutations of the BMC-T1-spyt-snpt-6xHis protein were not randomly selected. We selected potential non-conserved amino acids using the consurf database. We followed the generally accepted view that the less conserved an amino acid is, the less relevant it is for the correct folding of the protein. The remaining non-conserved amino acids and their positions were considered and only amino acids that had a similar size and shape compared to the ncAAs were selected for site directed mutagenesis at their position. This resulted in the mutations: BMC-T1-spyt-snpt-6xHis F8, T35, R78 and Y96.

ID	Name	Function
ID BBa_K4229021	sfGFP_A4	Sequence for sfGFP (PDB: 2B3P) with an AMBER stop codon at the position of the 4th amino acid
ID BBa_K4229022	sfGFP_A8	The sequence for sfGFP (PDB: 2B3P) with an AMBER stop codon at the position of the 8th amino acid
ID BBa_K4229023	sfGFP_A74	The sequence for sfGFP (PDB: 2B3P) with an amber stop codon at the position of the 74th amino acid
ID BBa_K4229024	sfGFP_A151	The sequence for sfGFP (PDB: 2B3P) with an amber stop codon at the position of the 151st amino acid
ID BBa_K4229075	sfGFP	Original sequence of the sfGFP
ID BBa_K4229071	BMC-T1_F8X	The sequence for the T1 protein with an amber stop codon at the position of the 8th amino acid
ID BBa_K4229072	BMC-T1_T35X	The sequence for the T1 protein with an amber stop codon at the position of the 35th amino acid
ID BBa_K4229073	BMC-T1_R78X	The sequence for the T1 protein with an amber stop codon at the position of the 78th amino acid
ID BBa_K4229074	BMC-T1_Y96X	The sequence for the T1 protein with an amber stop codon at the position of the 96th amino acid

Improvement of an existing part

Usage: 

This biobrick 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-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:

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.

Improvement:

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