Description

The collection described herein consists of parts, necessary to build a novel liposome-based platform for membrane protein (MP) synthesis, integration, and display. It is comprised of a cellular MP integration machinery, target membrane proteins and a unique chaperone. Guide RNAs introduce genetic modifications to nascent ribosome proteins for purification and further ribosome anchoring to nickel-chelating lipids of liposome membrane. Synthetic RNA thermometers act as a user-controllable mechanism for regulation of target protein expression and high-yield synthesis in an inducible temperature range. Thermoswitches elegantly complement the function of BAM complex which facilitates membrane-assembly of beta-barrel bearing proteins. Our target membrane proteins are fused with Mistic protein for a superior integration into the membrane. Additionally, MPs of our choice expose designated antibodies on the surface of the liposome. While all with different functions, together these parts establish a unique level of complexity and create a universal synthetic system for membrane protein research in liposomes.

Cellular MP integration machinery, the BAM complex, consists of five essential subunits, hence parts. They are well characterized and maintain their identical cellular functions when encapsulated inside liposomes. Parts are already labeled with His-tag, to ease the purification process of BamA-E proteins using the His-tag affinity chromatography.

Previous parts of BAM complex are essential for viable membrane insertion of beta-barrel bearing membrane proteins. There are two different membrane proteins in our collection to suit the needs of a user. OmpA is a member of OMP superfamily and one of the most well described and characterized proteins of gram-negative bacteria outer membrane. In this collection we use well characterized Lpp-OmpA (46-159) fusion protein. Another membrane protein in our collection is the beta-domain of Iga protease, which is attractive due to its autotransporter and pore-forming properties.

Our part collection also contains 11 different synthetic RNA thermometers. These parts act as thermosensors that regulate gene expression by temperature-induced changes in RNA conformation. Thermoswitches are the most universal feature of our part collection, as they can be easily applied not only in our system, but also any other in vivo or especially in vitro system that needs to incorporate chemical-free gene regulation. The applications of these parts range from RNA-ordered gene expression dynamics to tightly controlled modularization of genetic cascades. In this collection, however, RNA thermometers are needed to lock the initial translation of our target constructs bearing plasmids. It is needed to give enough time for BamA protein to correctly fold and insert into liposome membrane where it can accept and assemble our target beta-barrel bearing membrane proteins. It is one of many examples demonstrating the beautiful interplay between our parts.

A unique feature of this collection is Mistic. It is a fresh and still understudied protein, whose mechanism of action is unclear. However, for what is known, Mistic possesses functional ability to chaperone membrane proteins when fused to their N-terminus to membrane by associating with it. Its function is autonomous meaning not Sec-dependent. All these features make Mistic a perfect piece for our composite parts also containing membrane proteins and antibodies.

The part collection further features a camelid class antibody called GFP Nanobody and a single chain variable fragment (scFv) of anti-vaginolysin. These parts serve to prove the versatility and operation of our system. Comprising composite constructs, both GFP Nanobody and anti-vaginolysin can be displayed on liposome surface by membrane proteins which are fused both to the antibodies and protein Mistic. These parts prove that our system can display a full range of molecules: versatile antibodies, peptides, and other proteins, thus becoming a universal and smart novel exposition system.

Final feature of our part collection is parts bearing the sequences of guide RNAs for specific nascent ribosome protein modifications. It is extremely needed to ensure that an in vitro protein expression system will be high-yielding. We introduce a possibility to modify nascent ribosome proteins L23 and L24 by labelling their C’ terminal ends with a his-tag allowing ribosome anchoring to lipid membranes via an interaction between a his-tag and metal chelating lipids. Third part of this type has a sequence of gRNA that introduces a Strep-tag to the C’ terminus of protein L12 allowing to purify engineered ribosomes using Strep-tag affinity chromatography.

Although various and very different, when used right these parts elegantly interplay and orchestrate the assembly of novel synthetic membrane protein synthesis, display, and research system in liposomes.

Full Part Collection

 

Part Number	Type	Name	About	Length, nt
BBa_2622001	Coding	BamB	Beta-barrel assembly protein B (BamB)	1261
BBa_2622002	Coding	BamA	Beta-barrel assembly protein A (BamA)	2520
BBa_2622003	Coding	IgA	Immunoglobulin protease A (IgA) from Neisseria gonorrhoeae	1398
BBa_2622004	Coding	scFv_antiVLY	Single chain fragment variable antibody anti-vaginolysin (scFv_antiVGY)	733
BBa_26220005	Coding	GFP Nanobody	Camelid antibody agains green fluorescent protein (GFP Nanobody)	352
BBa_2622006	Coding	Mistic (Mstx)	Membrane-integrating protein Mistic (MstX) from Bacillus subtilis	330
BBa_2622007	RNA	gRNA for ribosome protein L12	Guide RNA for modification of ribosome protein L12 using CRISPR-Cas9 system	174
BBa_2622008	RNA	gRNA for ribosome protein L23	Guide RNA for modification of ribosome protein L23 using CRISPR-Cas9 system	174
BBa_2622009	RNA	gRNA for ribosome protein L24	Guide RNA for modification of ribosome protein L24 using CRISPR-Cas9 system	174
BBa_2622010	Regulatory	RNA thermoswitch1 (GJ2)	Temperature-sensitive non-coding RNA 1	39
BBa_2622011	Regulatory	RNA thermoswitch2 (GJ3)	Temperature-sensitive non-coding RNA 2	38
BBa_2622012	Regulatory	RNA thermoswitch3 (GJ6)	Temperature-sensitive non-coding RNA 3	53
BBa_2622013	Regulatory	RNA thermoswitch4 (GJ9)	Temperature-sensitive non-coding RNA 4	32
BBa_2622014	Regulatory	RNA thermoswitch5 (GJ10)	Temperature-sensitive non-coding RNA 5	33
BBa_2622015	Coding	BamC	Beta-barrel assembly protein C (BamC)	1057
BBa_2622016	Regulatory	RNA thermoswitch 6 (Sw2)	Temperature-sensitive non-coding RNA 6	33
BBa_2622017	Regulatory	RNA thermoswitch 7 (Sw3)	Temperature-sensitive non-coding RNA 7	33
BBa_2622018	Regulatory	RNA thermoswitch 8 (Sw6)	Temperature-sensitive non-coding RNA 8	33
BBa_2622019	Regulatory	RNA thermoswitch 9 (Sw7)	Temperature-sensitive non-coding RNA 9	33
BBa_2622020	Regulatory	RNA thermoswitch 10 (Sw9)	Temperature-sensitive non-coding RNA 10	33
BBa_2622021	Regulatory	RNA thermoswitch 11 (Sw11)	Temperature-sensitive non-coding RNA 11	33
BBa_2622022	Coding	BamD	Beta-barrel assembly protein D (BamD)	757
BBa_2622023	Coding	BamE	Beta-barrel assembly protein E (BamE)	448
BBa_2622024	Coding	SurA	Survival protein A (SurA)	1374
BBa_2622025	Composite	GJ6-His-GFP	Recombinant N-terminus histidine tagged GFP with temperature-sensitive non-coding RNA 3	809
BBa_2622026	Composite	Lpp-OmpA-His	Recombinant C-terminus histidine tagged OmpA fused with Lpp	597
BBa_2622027	Composite	Mstx-Lpp-OmpA-His	Recombinant C-terminus histidine tagged OmpA fused with Mistic and Lpp	933
BBa_2622028	Composite	Mstx-Lpp-OmpA-GFPN	Recombinant protein OmpA fused with Mistic, Lpp and GFP Nanobody	1272
BBa_2622029	Composite	Lpp-OmpA-GFPN	Recombinant protein OmpA fused with Lpp and GFP Nanobody	936
BBa_2622030	Composite	Lpp-OmpA-scFv_antiVLY	Recombinant protein OmpA fused with Lpp and scFv_antiVLY	1317
BBa_2622031	Composite	Mstx-Lpp-OmpA-scFv_antiVLY	Recombinant protein OmpA fused with Mistic, Lpp and scFv_antiVLY	1653
BBa_2622032	Composite	His-IgA-Mstx	Recombinant N-terminus histidine tagged IgA protein fused with Mistic	2001
BBa_2622033	Composite	His-scFv_antiVLY	N-terminus histidine tagged scFv_antiVLY	927
BBa_2622034	Composite	GFPN-IgA-Mstx	Recombinant protein IgA fused with GFP Nanobody and Mistic	2286
BBa_2622035	Composite	scFv_antiVGY-IgA-Mstx	Recombinant protein IgA fused with scFv_antiVLY and Mistic	2667
BBa_2622036	Composite	Sw11-OmpA-His	C-terminus histidine tagged OmpA with temperature-sensitive non-coding RNA 11	598
BBa_2622037	Composite	Sw11-Mstx-OmpA-His	Recombinant C-terminus histidine tagged OmpA fused with temperature-sensitive non-coding RNA 11 and Mistic	1082
BBa_2622038	Composite	Mstx-scFv_antiVLY	Recombinant protein scFv_antiVLY fused with Mistic	1266
BBa_2622039	Composite	BamA-His composite	C-terminus histidine tagged BamA	2569
BBa_2622040	Composite	BamA composite	C-terminus histidine tagged BamA controlled by T7 promoter and terminator	2580
BBa_2622041	Composite	BamA composite	BamA controlled by T7 promoter and terminator	2628
BBa_2622042	Composite	BamB composite	BamB controlled by T7 promoter and terminator	1369
BBa_2622043	Composite	BamC composite	BamC controlled by T7 promoter and terminator	1165
BBa_2622044	Composite	BamD composite	BamD controlled by T7 promoter and terminator	865
BBa_2622045	Composite	BamE composite	BamE controlled by T7 promoter and terminator	556
BBa_2622046	Composite	SurA composite	SurA controlled by T7 promoter and terminator	1482
BBa_2622047	Composite	Mstx-OmpA_full-His	Full length recombinant C-terminus histidine tagged protein OmpA fused with Mistic	1340
BBa_2622048	Coding	OmpA full	Full lenght outer transmembrane protein A (OmpA)	1041