Positioning compositions, systems, and methods for the homeostasis of protein-based organelles

Abstract

Provided herein are compositions, systems, and methods for the assembly of a positioning system for protein-based organelles within a cell of an organism. In particular, the present invention relates to systems of homeostasis for both synthetic and natural bacterial protein-based organelles including bacterial microcompartments, encapsulin nanocompartments, and biomolecular condensates.

Description

[0001]UM-42106.601 POSITIONING COMPOSITIONS, SYSTEMS, AND METHODS FOR THE HOMEOSTASIS OF PROTEIN-BASED ORGANELLES The present application claims priority to United States Provisional Patent Application Serial Numbers 63,589,231, filed October 10, 2023, and 63,679,474, filed August 5, 2024, the disclosures of which are herein incorporated by reference in their entireties. SEQUENCE LISTING The text of the computer readable sequence listing filed herewith, titled “42106- 601_SEQUENCE_LISTING”, created October 10, 2024, having a file size of 36,932 bytes, is hereby incorporated by reference in its entirety. FIELD Provided herein are compositions, systems, and methods for the assembly of a positioning system for protein-based organelles within a cell of an organism. In particular, the present invention relates to systems of homeostasis for both synthetic and natural bacterial protein-based organelles including bacterial microcompartments, encapsulin nanocompartments, and biomolecular condensates. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH This invention was made with government support under 1941966 awarded by the National Science Foundation. The government has certain rights in the invention. BACKGROUND Recent studies have shown that many bacterial cells possess specialized protein-based organelles such as bacterial microcompartments (BMCs), encapsulin nanocompartments (encapsulins), and biomolecular condensates (condensates), for example, to functionally compartmentalize essential anabolic or sensitive catabolic reactions by encapsulating an agent(s) within the cell of an organism. Intracellular compartmentalization is essential for all life, promoting metabolic efficiency while limiting unwanted reactions. While eukaryotes use membrane-bound organelles, bacteria organize biochemical reactions using diverse protein-based organelles, including bacterial microcompartments (BMCs), encapsulin nanocompartments (encapsulins), and biomolecular condensates (condensates). BMCs are comprised of a polyhedral protein shell with diameters of 40 to 200 nm, forming a selectively permeable barrier that allows small molecules to pass while confining enzymes and intermediates (Kirst & Kerfeld. 2019). UM-42106.601 Encapsulins also form a protein shell, typically 24 to 42 nm in diameter, composed of one or fewer protein subunits compared to BMCs (Giessen. 2022). Unlike BMCs and encapsulins, condensates lack a protein shell as a physical barrier with the cytoplasm. Condensates form through the self-assembly of biopolymers like proteins, RNA, and DNA, increasing the local concentration of these components (Niu et al.2023). BMCs, encapsulins, and condensates are ubiquitous across bacterial and archaeal phyla and share some common features: modular structures, self-assembly ability, selective permeability, and cargo-loading capabilities. These features make them suitable for biomedical and biotechnological applications. BMC shell proteins have been repurposed for various biotechnological applications. They have been used to catalyze the formation of gold nanoparticles (Bari et al.2018), sequester toxic products like polyphosphate (Liang et al.2017), and compartmentalize non-native enzymes and catalytic cascades (Fan et al. 2010). Additionally, BMCs have been introduced into non-native hosts to enhance functions such as carbon fixation (Flamholz et al.2020) and developed into novel scaffolding systems for spatial organization of biosynthetic processes in cells (Zhang et al.2019). Encapsulins, like BMCs, can accumulate inorganic particles (Künzle et al. 2019) and function as bioreactors by encapsulating enzymes to produce desired products (Jenkins & Lutz. 2021), enabling the investigation of enzyme kinetics under natural conditions (Maity et al. 2015). Moreover, the small size and surface functionalization potential of encapsulins facilitate the development of diverse systems for targeted drug delivery or labeling (Lagoutte et al.2018; Moon et al.2014; Choi et al.2016). Condensates, on the other hand, have only recently been appreciated in bacteria (Azaldegui et al. 2021). Without a protein shell as a physical barrier, condensates are dynamic, liquid-like, and respond quickly to changes in the environment (Hoang et al. 2024). The reversible assembly and disassembly of synthetic condensates can mediate bistable cellular function, regulating the timing and efficiency of reactions for cellular control and metabolic engineering (Dai et al.2023). These diverse applications of bacterial protein-based compartments heavily rely on their reconstitution in alternative hosts, such as Escherichia coli, for large-scale industrial production. However, the reconstitution and engineering of these compartments currently lack spatial regulation, which is crucial for the proper distribution and partitioning of heterologous compartments in non-native hosts. Without spatial regulation, reconstituting these compartments in alternative hosts can lead to nucleoid-excluded aggregates at the bacterial cell pole, reducing reaction efficiency and causing asymmetric inheritance during cell division (Rillema et al. 2021). To date, the only known positioning system for any protein-based UM-42106.601 bacterial organelles is the McdAB system (MacCready et al. 2018; MacCready et al. 2021), which organizes carboxysomes, the model BMC. Carboxysomes enhance carbon fixation in cyanobacteria and some chemoautotrophs by encapsulating enzymes in a protein shell. Currently unknown, within the bioengineering field, is how this positioning system self- assembles and maintains its function within BMCs. SUMMARY Provided herein are compositions, systems, and methods related to positioning systems for the structuring and homeostasis of protein-based organelles including bacterial microcompartments (BMCs), encapsulin nanocompartments (encapsulins), and biomolecular condensates (condensates). In some embodiments, provided herein are compositions, systems, and methods for the reconstitution and positioning of protein-based organelles in heterologous cells using an McdAB system. For example, experiments conducted during the development of embodiments of the technology successfully reconstituted the McdAB system in E. coli to spatially organize carboxysomes. Further, it was surprisingly found that the McdAB system successfully led to the reconstitution of and controlled spatial distribution of condensates and encapsulins in heterologous cells. Further, in some embodiments, minimal autonomous positioning tags (MapTag) were designed. For example, a MapTag from the N-terminus of McdB was designed and shown to interact with McdA to spatially distribute protein-based organelles. This work provides minimal and controlled platforms for the stable maintenance of protein-based compartments in non-native hosts, enabling their efficient large-scale production for various applications in biotechnology and biomedicine. In some embodiments, the present disclosure provides a cell expressing a heterologous carboxysome distribution protein A (McdA), a heterologous carboxysome distribution protein B (McdB), and protein-based organelle (e.g., BCM, encapsulin, or condensate) proteins (e.g., heterologous protein-based organelles). In some embodiments, the cell comprises a protein- based organelle (e.g., BCM, encapsulin, or condensate) formed in the presence of such proteins. In some embodiments, the cell further comprises an agent encapsulated in the protein-based organelle (e.g., BCM, encapsulin, or condensate). In some embodiments, the agent is a nanomaterial and / or an enzyme. In some embodiments, the nanomaterial is an organic molecule, protein, inorganic molecule, or hybrid compound molecule. In some embodiments, the enzyme is ribulose-1,5-bisphosphate carboxylase / oxygenase (RuBisCo). In some embodiments, the McdA or McdB are fragments or variants of a natural McdA or McdB. For UM-42106.601 example, in some embodiments, a minimal autonomous positioning tag (MapTag) is generated from the McdA or McdB. For example, a N-terminus of McdB may be used as a MapTag that interacts with the McdA to spatially distribute a protein-based organelle in the cell. In some embodiments, heterologous McdA, McdB (e.g.,a MapTag at a N-terminus of McdB) and / or protein-based organelle (e.g., BCM, encapsulin, or condensate) proteins are expressed on one or more expression vectors (e.g., plasmids). In some embodiments, the cell is a bacterium, fungal cell, plant cell or an animal cell. In some embodiments, provided herein are compositions or systems comprising: one or more expression vectors encoding McdA, McdB, and / or one or more protein-based organelle (e.g., BCM, encapsulin, or condensate) proteins. In some embodiments, the compositions or systems comprises McdA, McdB, and / or one or more protein-based organelle (e.g., BCM, encapsulin, or condensate) proteins that are codon- optimized for expression in a heterologous host cell. In some embodiments, the compositions and systems comprise one or more expression vectors comprising a heterologous promoter in operable association with a sequence encoding McdA, McdB (e.g., a MapTag at a N-terminus of McdB) and / or one or more protein-based organelle (e.g., BCM, encapsulin, or condensate) t proteins. Further provided are methods for generating a protein-based organelle (e.g., BCM, encapsulin, or condensate) (e.g., a heterologous protein-based organelle (e.g., BCM, encapsulin, or condensate)) in a cell comprising: introducing a heterologous carboxysome distribution protein A (McdA), a heterologous carboxysome distribution protein B (McdB) (e.g., a minimal autonomous positioning tag (MapTag) of McdB) and one or more protein- based organelle (e.g., BCM, encapsulin, or condensate) proteins (e.g., heterologous protein- based organelle (e.g., BCM, encapsulin, or condensate) proteins) into a cell. In some embodiments, the method further comprises the steps of culturing the cell and selecting a cultured cell expressing said heterologous McdA, McdB (e.g., MapTag of a McdB) and protein-based organelle (e.g., BCM, encapsulin, or condensate) proteins. In some embodiments, the method further comprises selecting cells expressing an antibiotic resistance factor. In some embodiments, the protein-based organelle (e.g., BCM, encapsulin, or condensate) in the cell encapsulate factors that carry out a biological reaction. In some embodiments, the biological reaction is an enzymatic reaction. In some embodiments, the enzymatic reaction is a carbon fixing enzymatic reaction. Other aspects and embodiments of the disclosure will be apparent in light of the following detailed description and accompanying figures. UM-42106.601 BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows an McdAB system that equidistantly positions heterologous carboxysomes in E. coli. A. Fluorescent microscopy shows the distribution of the carboxysome reporter CbbS-mTQ on the nucleoid when the McdAB system is present. The small subunit of Rubisco is labeled with mTQ (cyan), while the nucleoid is stained with DAPI (magenta). Phase contrast micrographs are displayed in black and white. Scale bar: 2 µm. FIG.2 shows electron micrographs demonstrating the localization of carboxysomes on the nucleoid in the presence of an exemplary McdAB system. Images show McdAB negative inclusion bodies (left image; grey arrows) and a heterologous carboxysome positioned on the nucleoid (right image; white arrows). Scale bar: 200 nm. FIG. 3A shows a representative images of mNG-McdA variants in E. coli at 2 hours post-induction without chloramphenicol (Cm) treatment. The phase contrast channel is merged with the mNG channel or the DAPI-stained nucleoid channel. Merges of all channels are also provided. Scale bar = 2 µm. FIG.3B shows a Pearson correlation coefficient of mNG-McdA variants against the DAPI-stained nucleoid in cells without Cm treatment. McdA [WT], n = 23; McdA[K15A], n = 25; McdA[K15R], n = 27. Dashed black lines represent the median of the data and dotted black lines represent the lower and upper quartiles. ns = not significant, ****p < 0.0001. FIG.3C shows representative images of mNG-McdA variants at 2 hours post- induction with Cm treatment to compact the nucleoid. The phase contrast channel (blue) is merged with the mNG channel or the DAPI-stained nucleoid channel. Merges of all channels are also provided. Scale bar = 2 µm. FIG.3D shows Pearson correlation coefficient of mNG- McdA variants against the DAPI-stained compacted nucleoid from cells treated with Cm. McdA [WT], n = 24; McdA[K15A], n = 25; McdA[K15R], n = 30;. Dashed black lines represent the median of the data and dotted black lines represent the lower and upper quartiles. ns=not significant; ****p < 0.0001. FIG. 4A shows representative images of E. coli cells coexpressing mNG-McdA (0.5 nM aTc) and mCherry-McdB (100 µM IPTG), without nucleoid compaction by chloramphenicol (Cm) treatment. Images of E. coli cells expressing mNG-McdA alone (5 nM aTc) are also provided (left column). All cells imaged 2 hours post induction. mNG-McdA, DAPI-stained nucleoid, mCherry-McdB channels are merged with the phase contrast channel. Merges of all channels are also provided for all conditions (bottom row). The middle column shows mNG-McdA and mCherry-McdB localization in cells with low mCherry-McdB expression and no condensate formation. The right column shows mNG-McdA and mCherry- McdB localization in cells with high mCherry-McdB expression and condensate formation. UM-42106.601 Scale bar = 2 µm. FIG. 4B shows a Pearson correlation coefficient analysis of mNG-McdA variants against the DAPI-stained nucleoid on cells expressing mNG-McdA and mCherry- McdB without Cm treatment. Data collected on cells 2 hours post induction with 5 nM aTc (mNG-McdA, n = 24) or with 10 nM aTc and 200 µM IPTG (mNG-McdA + mCherry-McdB, n = 24). Dashed black lines represent the median of the data and dotted black lines represent the lower and upper quartiles. ****p < 0.0001 by Welch’s t test. FIG.4C shows representative images of mNG-McdA E. coli cells and mNG-McdA + mCherry-McdB E. coli cells, with nucleoid compaction by Cm. Cells imaged 2 hours post induction, with 10nM aTc and 200 µM IPTG for mNG-McdA + mCherry-McdB conditions. Images of E. coli cells expressing McdA alone, induced with 5 nM aTc and Cm treatment are also provided. mNG-McdA, DAPI-stained nucleoid, mCherry-McdB channels are merged with the phase contrast channel. Merges of all channels are also provided for all conditions (bottom row). mNG-McdA + mCherry-McdB expression with no-foci phenotype in middle column. mNG-McdA + mCherry-McdB expression with foci phenotype in right column. Scale bar = 2 µm. FIG.4D shows a Pearson correlation coefficient analysis of mNG fusion proteins and the DAPI-stained nucleoid on cells expressing mNG-McdA and mCherry-McdB with Cm treatment. Data collected on cells 2 hours post induction with 5 nM aTc (mNG-McdA, N= 23) or with 10nM aTc and 200 µM IPTG (mNG-McdA + mCherry-McdB, N= 27). Dashed black lines represent the median of the data and dotted black lines represent the lower and upper quartiles. ****p < 0.0001 by Welch’s t test. FIG. 5A shows representative images of mCherry-McdBPopTaginduced with 25µM IPTG at 0, 1, and 3 hours post-induction. mCherry-McdB channels are merged with the phase contrast channel. Scale bar = 2 µm. FIG.5B shows percentages of E. coli cells with an McdB focus from 0 to 3 hours for both mCherry-McdB and mCherry-McdBPopTag. FIG. 5C shows normalized pixel intensity histograms of cells containing foci of mCherry-McdBPopTag(top) and mCherry-McdB (bottom). Each line represents individual cells. The y axis shows the frequency of pixels at that intensity (x axis). FIG.5D shows quantification of condensation coefficients of mCherry-McdBPopTagand mCherry-McdB foci. Condensation coefficient was calculated from cells with a detected focus at thresholds of 0.1 and 0.3 of the max intensity. Data points correspond to individual cells. Dashed line represents the median value of the sample, dotted lines represent the lower and upper quartiles. ****p < 0.0001 by Welch’s t test. FIG.5E shows a full Western blot for mCherry-McdBPopTagusing antibody targeting mCherry at 0, 2, and 4 hours post-induction. Lane 1: molecular weight marker, Lane 2: mCherry-McdBPopTag UM-42106.601 expression at 0 hours, Lane 3: expression at 2 hours, Lane 4: expression at 4 hours. The mCherry-McdBPopTagband (53.22 kDa) is shown in the black box. FIG.6A shows representative images of E. coli cells expressing mCherry-McdBPopTag(25 µM IPTG) in the absence and presence of McdA (1.5 nM aTc). DAPI-stained nucleoid and mCherry-McdB channels are merged with the phase contrast channel. Scale bar = 2 µm. FIG. 6B shows a graph indicating the percentage of cells with 0, 1, 2, 3, and ≥ 4 foci of mCherry- McdBPopTagin the absence (n = 359 cells) and presence (n = 878 cells) of McdA. FIG.6C shows a Pearson correlation coefficient analysis of mCherry-McdBPopTagand the DAPI-stained nucleoid in the presence (n = 25) and absence (n = 25) of mNG-McdA. Dashed black lines represent the median of the data and dotted black lines represent the lower and upper quartiles. ****p < 0.0001 by Welch’s T-test. FIG. 7A shows representative images of time-lapses where mCherry-McdBPopTagand McdA were co-expressed at t = 0. FIG.7B shows representative images of time-lapses where McdA was removed from the cells that showed positioned McdB-tagged condensates. FIG.7C shows representative images of time-lapses where McdA was induced after the large polar McdB-tagged condensates were already formed. All images were extracted from time-lapse movies where McdA and McdB-tagged condensates were co-expressed at different times. mCherry channel was merged with the phase contrast channel. Scale bar = 2 µm. FIG. 8 shows representative images of E. coli cells expressing synthetic condensates fused to the β- and α-MapTags (25 µM IPTG) in the absence and presence of their partner McdA proteins (1.5 nM aTc). mCherry-McdB channel are merged with the phase contrast channel. Scale bar = 2 µm. FIG. 9A shows a representative image of encapsulin expression in E. coli.. FIG. 9B shows representative images of encapsulins that are positioned by both the β- and α-MapTags and their partner McdA proteins in E. coli. All representative images show E. coli cells expressing encapsulins, and the encapsulins fused to the β- and α-MapTags (25 µM IPTG) in the absence and presence of their partner McdA proteins (1.5 nM aTc). mNG channel was shown in green. Scale bar = 1 µm. FIG. 10A shows representative images of time-lapses where encapsulinMapTagand McdA were co-expressed at t = 0. FIG.10B shows representative images of time-lapses where McdA was removed from the cells that showed positioned encapsulinMapTag. FIG.10C shows representative images of the time-lapses where McdA was induced after the large polar focus of encapsulinMapTagwas already formed. All images were extracted from time-lapse movies UM-42106.601 where McdA and MapTag-tagged encapsulins were co-expressed at different times. The mNG channel was merged with the phase contrast channel. Scale bar = 2 µm. DEFINITIONS To facilitate an understanding of the present technology, a number of terms and phrases are defined below. Additional definitions are set forth throughout the detailed description. The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not. For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6- 9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated. Unless otherwise defined herein, scientific, and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear; in the event, however of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The term “agent” is used in its broadest sense. Agents include chemical and biological agents. Chemical agents may be but are not limited to, any chemical element or compound, on their own or mixed, as it occurs in the natural state or as produced, used, or released. Chemical agents may be obtained from any source including animals, plants, and microorganisms and encompass fluids, solids, tissues, and gases. Biological agents may be but are not limited to, any molecules or materials obtained from bacteria, viruses, fungi, or other microorganisms (e.g., proteins, nucleic acids, lipids, metabolites, small molecules, etc.). Agents may be obtained from any source including animals, plants, and microorganisms and encompass fluids, solids, tissues, and gases. In some embodiments, the agent is from an environmental sample. Environmental samples include, but are not limited to, those obtained from lakes, streams, rivers, or any combination thereof. UM-42106.601 As used herein, a “microcompartment” is a structure comprising oligomer proteins that may include, for example, shell proteins from α- and β-carboxysomes, found in but not limited to, α- and β-cyanobacteria, respectively. The polymers or oligomers may be heterogenous or homogenous in composition and may be isolated from naturally occurring sources or may be artificially or synthetically produced. The term “heterologous” refers to the non-natural presence of or expression of a biomolecule (e.g., DNA, RNA, protein) in a cell, tissue, or organism. In some embodiments, a heterologous molecule comprises a molecule from a first organism present in or expressed in a second organism (e.g., a cyanobacteria protein expressed in E. coli is considered heterologous in E. coli). In some embodiments, a heterologous molecule comprises a molecule otherwise native to a particular organism, but synthetically provided or expressed in a manner not native to that organism (e.g., a cyanobacteria gene expressed on a synthetic plasmid in a cyanobacteria is heterologous in that cyanobacteria because it not native to that organism). Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting. DETAILED DESCRIPTION Bacterial microcompartments (BMCs) are large macromolecular complexes that function to compartmentalize essential anabolic or sensitive catabolic reactions by encapsulating key metabolic enzymes within a selectively permeable protein shell. Among BMCs studied to date, the carboxysome is the best characterized. Widely found in chemoautotrophic proteobacteria and among all photosynthetic cyanobacteria, the carboxysome is the key machinery responsible for sequestering and fixing almost 35% of the Earth's atmospheric CO2. Carbon fixation is required for phototrophic growth, and in the model cyanobacterium Synechococcus elongatus, carboxysomes are equidistantly spaced along the cell length. Bacterial microcompartments (BMCs) are organelles that segregate essential and sensitive metabolic pathways from the surrounding metabolic environment within an organism. Naturally occurring BMCs comprise a modular protein shell which surrounds an enzymatically active core, with the shell functioning as a semipermeable membrane for substrates and products that vary functionally amongst distinct BMCs. A key function of BMCs is their ability UM-42106.601 to self-assemble both their shell and enzymatic actions e.g., cargo from multiple protein oligomers to create complex protein structures. Specialized proteins are utilized to facilitate cargo aggregation as well as small encapsulation peptides that specifically associate proteins to the lumen of the shell. BMCs contribute to the metabolic versatility of bacteria, providing a competitive advantage in specific environmental niches. The present knowledge of native BMCs has resulted in an increase in the understanding of their role in microbial metabolism but also a burgeoning interest of their capabilities in a variety of applications including the field of synthetic biology. However, the ability to generate heterologous or synthetic BMCs in cells has been limited by the lack of understanding for how to assemble functional BMCs. Provided herein are compositions, systems, and methods that employ heterologous McdA and McdB proteins to facilitate formation and function of protein-based organelles (e.g., BCM, encapsulin, or condensate), including heterologous and synthetic protein-based organelles (e.g., BCM, encapsulin, or condensate). McdA and McdB (Maintenance of Carboxysome Distribution) are two recently identified proteins, which are essential for the inheritance and homeostasis of carboxysomes in cyanobacteria. These compositions, systems, and methods find use in the generation of non-natural protein-based organelles (e.g., BCM, encapsulin, or condensate), including the formation and use of protein-based organelles (e.g., BCM, encapsulin, or condensate) in heterologous cells. Prior to the present invention, it was not practical to form functional protein-based organelles such as carboxysomes in heterologous cells. Expression of carboxysome proteins, absent the McdA and McdB proteins provided herein, fail to generate functional intracellular carboxysomes. Specifically, without this two- component system, carboxysomes coalesce into a single aggregate, disrupting homeostasis and leading to cell death. In natural systems, the biogenesis of a single carboxysome takes time and requires the ordered assembly of numerous shell proteins and enzymes. Provided herein is a minimal and self-organizing two-component system that is both necessary and sufficient to position bacterial protein-based organelle (e.g., BCM (e.g., carboxysomes), encapsulin, or condensate) in heterologous hosts. McdA and McdB proteins McdA and McdB proteins employed in the systems, compositions, and methods provided herein may be obtained from any suitable organism and may be wild-type or variant proteins, including natural variants. In some embodiments, the McdA protein is from a cyanobacteria. In some embodiments, the McdA protein is from a β-cyanobacteria. In some embodiments, the McdA protein is a Type 1 or Type 2 McdA protein (see MacCready et al., Mol. Biol. Evol. UM-42106.601 37(5):1434–1451 (2020), herein incorporated by reference in its entirety). In some embodiments, the McdA protein has a sequence comprising any one or more of the sequences of (or fragments or variants thereof; e.g., having greater than 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity to): >Halothiobacillus neapolitanus MASNKAFTLAVANLKGGCGKTTISTNISAGLTQRGRVGLVDADPQGALKHWVDWG SKEADAQVPVLYSDHTDPVQNLKLAQPNHDFVVVDCPPSLDMAITCQLMIECDFILIP VLPSPLDLWASTQTIEMIESARKTNPKLKAALVLNQTEPRSAMTRAMQTTIERLGVP VLTTSVRRRAVYRNAVVEGVSVFQLGARGRSAADEINQILNEVIPS (SEQ ID NO:1) >Synechococcus elongatus PCC 7942 MLTVTCASLSGGQGKTTTALFLGRSLAARGKRVLMIDADPQSSLSFYLGCELSSEQA TLLEVLKKEVDVVDSLWNLDDRLALIPADDALDSAQDFLATSGMGAIVLRRRLSPL QDQFDFCVIDAPPQRSQLCMTSVGAADQLLIPAEASSKGLNSLLRTLDLVAEMSEVE AFQGQILGILPFRDRWLGRTQAKQSQKSLESMQEVAAGHPILPSILESEQFKKAIDQG VSLAALGYADLEYPFRTILEKLGC (SEQ ID NO:2) >Nostoc flagelliforme MLTLTCASLSGGQGKTTVAILLGRMLAQRGRRVLMIDADPQANLTFYLGHEVQQNQ PTLLEVLKKQINTEDGIYEIGENLWLIPADDGLDNAQEFLSGSGMGAIVLSKRLKEVS DLFQYCIVDAPPQRSQICLTSLGAADHIIIPVEASSKGVNSLIRTLSLVEELQEIDAFSGI VLGILPFRDKWVGNNQVAQSKASINAMRTIAEEIVVLPSILESEQFKKAIDKGVS LSSLGFEQLETPFQQIIERL (SEQ ID NO:3) >Gloeocapsa sp. MLTLTCTSLSGGQGKTTTSIFLGKMLAEEGHRILMVDADPQSSLTFYLGHEVQSNQP TLLEVLKKQVKVEDGIYEVGNNLWLIPSDDALDNAQDFLSGSGMGAVVLGKRLKEV SKLFEFCIIDAPPQRSQICLTTVGAADKVLIPVEASSKGLNSLIRTLDLVRELQDMDAF SGSVLGVLPFRDRWTGRTQANQSKNSIAAMKEIAHGIPILPSILESEQYKKAIDQGKT LLQLGHPQLEYPFIQIVDQL (SEQ ID NO:4) >Phormidesmis sp. MQTLTCTSLSGGQGKTTTSIFLGRALVQAGYRVLMIDADPQSSLTFYLGHEVQANQP TLLEVLKKLVPVEDGIYEVQENLWLIPSDDALDNAQDYLSGSGMGAIVLGKRLKEV ADLFQFCIIDAPPQRSQICLTTVGAANYVLIPAEASSKGVNSLIRTLDLIQELRDMDAF EGSVLGILPFRDRWTGRTQANQSKKSVQTMREIAGEIVILPSILESEQFKKAIDQGKTL RDLGYPNLEHPFQQIVQQL (SEQ ID NO:5) UM-42106.601 >Acidithiobacillus sp. S30A2 MASRRIVLFNAKGGCGKTTLAWNLAAGLAQRGSTLLLDADPQGSLGRWAEWSEAE GEGMTVAGSHLLDGDLATLKQPYVVVDCPPALEAQETQKALVQAHMVLVPVLPSP LDLWASQRSVEAIQAILQQRKSVRAALVLNQAEGRSALSRAAEHAIATLGLPVLSVQ VARRAIYRNAAVEGKSVYQMGKRGTAA (SEQ ID NO:6) >Sulfurimicrobium sp. GSVGLVDVDPQGALCHWAAWASGDGFPEVLAGGEYPLETVARAARKHHRVVVDC PPSLDMVITCRILQQVDTVLIPVLPSPLDLWACAETVEAVRQAREINPGLKAWLLVN QAEPSSALSRAMSEALTSLDVPAMKCVVRRRAAFRTAVVEGVSVYQMGARGREAV REIDQVIEEVL (SEQ ID NO:7) In some embodiments, the McdB protein is from a cyanobacteria. In some embodiments, the McdA protein is from a β-cyanobacteria. In some embodiments, the McdB protein is a Type 1 or Type 2 McdB protein (see MacCready et al., Mol. Biol. Evol.37(5):1434– 1451 (2020), herein incorporated by reference in its entirety). In some embodiments, the McdB protein has a sequence comprising any one or more of the sequences of (or fragments or variants thereof; e.g., having greater than 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity to): >Halothiobacillus neapolitanus MTNLEDKLSASIKTENKDTIAPASTPAKPAARSAAPETTKPASTGAKPATRTAAKKP VARATTTKPAVSKSSKPSADDIQVPAHDTSTHPRRVWPD (SEQ ID NO:8) >Synechococcus elongatus PCC 7942 MTDAFDRLKKRSRTPIAREGSLTTGPELSDRPLQLLPREFETFCDRYAVHAGDVIEAA LDLVLLDPDLQQRLLQRLRQGNGSDRVWLGTACPRSWQQQLQQQAQDQGLSEADL LQEAIAQRLDLVLGQTTLREEVTLLRQELDQLKRKLHGW (SEQ ID NO:9) >Anabaena_cylindrica_PCC_7122 MAKKNLSDLLQEEAQKFTPQVGDTAIEVTAQKVEEDNSSPLEEEPASTSNRRTTPTK ADLEITIKELTATLEKVQKKEVSLKEQISDFKTDLSVQKALAERLNTELHETKKTALQ LAESNSKLIAEINELKKVKEPVKETFIEPVKETSRSLSINPKKSHRSAERLQEMPNQSN DNFADNTWLYD (SEQ ID NO:10) >Cyanothece_sp._ATCC_51142 MTKKELSDLIREEAKNNSESTSETLQTPTVPSNRTRMTKAQLDELITQLNQALQQEK KQVDTLQHQVKTLETEINDERELSSRLQNELKEAEDYQSQLNEQKQLVEKLYTQLQ QKEELAVELAEKNELIATLQAKLQETPQPLATTESSQELLVKETALTRQARELEPFAA PASASKPLTNEDIGWFD (SEQ ID NO:11) UM-42106.601 >Filamentous_cyanobacterium_CCP2 MARKRLSDLVREEANKAQPEAEQPAVEQSAVEQSAVEQSADSQPDAETPPTPRAAV RSQSKAKSAQKPIPDASGDEPVDVKAEPVTADSSAAEDSSLSEAQTKIQELEATIADL QSNLKSAYETAHKNEAKLKQKITDLQTELDEKQETIDQLQEDGKQVRQLKAELEDA KKMILQLSQVNAQPAPPRPTPALNRAAEPELESSSSAMKPAAKGSPPPKQNQIALRQI LDHPTRPGKLPAMPSEKFSEKVEETDKIDIGWMD (SEQ ID NO:12) >Microcystis_aeruginosa_PCC_9717 MTKGNLNDLIRTEANKEVESQTSPSPKRSKETNATLQAKINDLTTELEKTEKNQQTL QEKVISLEKELQEHLELSVRLQQLQQQTEQLESVLLEKTTLVAQLSSQLSQSQTELTE KKQLIEKLSSQLSKTETELTEKKQLIEKLSSQLSQSQTELTEKKQLIEKLYNQIKTLENS PPPTPEPSPISKPAPKKPSLYNFEMATLARYIAPNPTPTELTDSEIGWFD (SEQ ID NO:13) >Acidithiobacillus_ferrivorans_SS3 MPVKKLGSKLAQGVRQVQAQQVDTPSPTPEACASAAPDDQAKGSSTAHPDEKPPLS PRASGDERSSTPRSEGHKNLHPRRVWPD (SEQ ID NO:14) >Nitrosomonas_eutropha_C91 MTNIKDKLTSSVRQAKSTTPTKAVAPRVAAEDTKQTNIQAKPVRQTAEPVSSAQALF PDRVWPD (SEQ ID NO:15) >Sulfuriferula_multivorans_TTN MSVKKMGSRLAQSVRDIKAQQTQEAVATPAAAAPERVTRPETTRPAAAPKAAKSD VQPFVHPDRVWPD (SEQ ID NO:16) Variant McdA and / or McdB proteins may include deletions (e.g., truncations), insertions, and / or one or more conservative or non-conservative amino acids substitutions relative to a wild-type McdA and / or McdB protein. A “conservative” amino acid substitution refers to the substitution of an amino acid in a peptide or polypeptide with another amino acid having similar chemical properties, such as size or charge. For purposes of the present disclosure, each of the following eight groups contains amino acids that are conservative substitutions for one another: 1) Alanine (A) and Glycine (G); 2) Aspartic acid (D) and Glutamic acid (E); 3) Asparagine (N) and Glutamine (Q); 4) Arginine (R) and Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), and Valine (V); 6) Phenylalanine (F), Tyrosine (Y), and Tryptophan (W); 7) Serine (S) and Threonine (T); and 8) Cysteine (C) and Methionine (M). Naturally occurring residues may be divided into classes based on common side chain properties, for example: polar positive (histidine (H), lysine (K), and arginine (R)); polar negative (aspartic acid (D), glutamic acid (E)); polar neutral (serine (S), threonine (T), UM-42106.601 asparagine (N), glutamine (Q)); non-polar aliphatic (alanine (A), valine (V), leucine (L), isoleucine (I), methionine (M)); non-polar aromatic (phenylalanine (F), tyrosine (Y), tryptophan (W)); proline and glycine; and cysteine. As used herein, a “semi-conservative” amino acid substitution refers to the substitution of an amino acid in a peptide or polypeptide with another amino acid within the same class. In some embodiments, unless otherwise specified, a conservative or semi-conservative amino acid substitution may also encompass non-naturally occurring amino acid residues that have similar chemical properties to the natural residue. These non-natural residues are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include, but are not limited to, peptidomimetics (e.g., chemically modified peptides, peptoids (side chains are appended to the nitrogen atom of the peptide backbone, rather than to the α-carbons), β-peptides (amino group bonded to the 0 carbon rather than the α carbon), etc.) and other reversed or inverted forms of amino acid moieties. Embodiments herein may, in some embodiments, be limited to natural amino acids, non-natural amino acids, and / or amino acid analogs. Non-conservative substitutions involve the exchange of a member of one class for a member from another class. Assembly The present disclosure provides methods for producing or assembling or modulating or expressing a positioning system for a protein-based organelle (e.g., BCM, encapsulin, or condensate) within a cell, tissue, or organism. In some embodiments, the method comprises, consists of, or consists essentially of: introducing a heterologous carboxysome distribution protein A (McdA), a heterologous carboxysome distribution protein B (McdB), and one or more heterologous protein-based organelle (e.g., BCM, encapsulin, or condensate) proteins into a cell. In some embodiments, the method utilizes a minimal autonomous positioning tag (MapTag) comprising a N-terminus of McdB as the McdB component. In some embodiments, the MapTag interacts with the McdA to spatially distribute a synthetic condensate and a encapsulin in the cell. In some embodiments, one or more of the proteins are introduced via introduction of an expression system into a cell, wherein the protein(s) are generated in the cell. In some embodiments, the expression system is introduced into the genome of a cell. In some embodiments, the expression system provides for extrachromosomal expression of the protein(s). In some embodiments, one or more of the proteins (McdA, McdB, or protein-based organelle (e.g., BCM, encapsulin, or condensate) proteins) are expressed from one or more expression vectors. In some embodiments, more than one (or all) proteins are expressed from a single expression vector. In some embodiments, two or more expression vectors are UM-42106.601 employed. In some embodiments, expression vectors comprise constitutive, regulatable, or inducible, cell type specific, tissue-specific, or species-specific regulatory sequences (e.g., promoters). For example, regulatory sequences may include constitutive promoters (Pcpc, PpsbA, Prbc), heterologous / inducible promoters (Plac, Ptrc, Ptet, PR), modified promoters (Pcpc (e.g. PcpcB, Pcpc560, Pcpt, PCP)), lac operons (T7, T3, Sp6), synthetic and hybrid promoters (J23-series, PconII, etc.) and native, inducible promoters (metal-inducible promoters, environmental sensor- or metabolic state-dependent promoters and cell type- specific promoters). In addition to the sequence sufficient to direct transcription, an expression vector can also include sequences of other regulatory elements that are involved in modulating transcription (e.g., enhancers, Kozak sequences, etc.). In some embodiments, the expression vector encodes McdA, McdB, or protein-based organelle (e.g., BCM, encapsulin, or condensate) proteins in combination with targeting molecules, detectable labels, enzymes, stable isotopes, antibodies, sequence tags, spacers, capture sequences, and the like. Any suitable expression vector or host cell may be used in the assembly methods. In some embodiments, the expression vector is a plasmid, virus, or nucleic acid fragment. In some embodiments, the expression vector is transformed or transfected into the cells. Suitable host organisms include, but are not limited to, bacteria such as E. coli, B. subtilis, S. cerevisiae, α- cyanobacteria (S. elongatus, Spirulina, Anabaena, Nostoc and Oscillatoria), β-cyanobacteria plants, algae, fungi, or other eukaryotic organisms. Cargo In some embodiments, one or more agents in included within or on the protein-based organelles (e.g., BCM, encapsulin, or condensate). Such agents are referred to herein as “cargo.” In some embodiments, agents include, but are not limited to proteins (e.g., fluorescent proteins, antigens, cytokines, macrophages, antioxidants antimicrobial proteins, signaling proteins, fibroins, albumins, gelatins, gliadines, legumins, 30Kc19, lipoproteins and / or ferritin proteins), organic agents (e.g., polymeric micelles, dendrimers, carbons and / or polymeric micelles), inorganic agents (e.g., metal, non-metal elements in the forms of an oxides, hydroxides, chalcogenides and / or phosphate compounds), hybrid compounds (e.g., hydrogels (polymeric nanogels and / or macromolecular micelles), materials made of complex composition (e.g., metals, oxides, chalcogenides) and / or coordination polymer), enzymes (e.g., lipases, amylases, invertase, glucose oxidases and / or proteases), antioxidant nanozymes (e.g., Superoxide Dimutas (SOD) nanozymes, catalase mimetics nanozymes) and / or pro-oxidant nanozymes (e.g., peroxidase mimetic nanozymes) and the like. Any of a wide variety of enzymes or multi-enzyme systems may be included in the microcompartments. Examples, UM-42106.601 include, but are not limited to, oxidoreductases, transferases, hydrolases, lyases, isomerases, and ligases (e.g., α-amylase, catalase, laccase, peroxidase, lysozyme, and reporter enzymes alkaline phosphatase, and luciferase). In some embodiments, enzyme cofactors are included within the protein-based organelle (e.g., BCM, encapsulin, or condensate) or are introduced into the protein-based organelle (e.g., BCM, encapsulin, or condensate) (via pores) after assembly. Such cofactors include, but are not limited to prosthetic groups, coenzymes, and metal ions (e.g., ascorbate, biotin, cadmium, calcium, cobalt, cobamide, copper, flavoproteins, heme, iron, magnesium, manganese, molybdenum, nickel, potassium, pyridoxal phosphate, pyrroloquinoline quinone, pyruvate, siroheme, sulfur, thiamin diphosphate, thiamine, tungsten, zinc, etc.). In some embodiments, the protein-based organelle (e.g., BCM, encapsulin, or condensate) encapsulation composition may comprise a system for carbon fixation of RuBisCo. In some embodiments, the protein-based organelle (e.g., BCM, encapsulin, or condensate) encapsulation composition may comprise a system for a biological reaction wherein the biological reaction is an enzymatic reaction. In some embodiments, the enzymatic reaction is a carbon fixing enzymatic reaction. Uses The assembled heterologous and / or synthetic protein-based organelles (e.g., BCM, encapsulin, or condensate) provided herein find use in a wide variety of research, diagnostic, industrial, agricultural, and clinical settings. For example, the protein-based organelles (e.g., BCM, encapsulin, or condensate) find use in any setting where it is desirable to enclose or isolate or segregate cargo. In some embodiments, the protein-based organelles (e.g., BCM, encapsulin, or condensate) provide a system for working with enzymes or other agents that are found naturally in endogenous protein-based organelle (e.g., BCM, encapsulin, or condensate) . However, with the ability to synthesize monomers and assemble the protein-based organelles (e.g., BCM, encapsulin, or condensate), a wide variety of additional agents may be employed on or in the protein-based organelles (e.g., BCM, encapsulin, or condensate). The protein-based organelles (e.g., BCM, encapsulin, or condensate) provide an alternative system to enclose, isolate, or segregate enzymes, reactions, or other agents that are currently worked with in organelles, liposomes, exosomes, micelles, nanoparticles, wells, channels, microfluidic chambers, and the like. Non-limiting examples of methods employing the protein-based organelles (e.g., BCM, encapsulin, or condensate) include: basic research, applied research, clinical research, drug screening, nanofactories, diagnostics, storage or stabilization of agents, synthetic biology, UM-42106.601 delivery of agents (e.g., ex vivo, in vivo, environmentally, etc.), sustain / controlled release, and bioremediation. EXAMPLES The following are examples of the present invention and are not to be construed as limiting. Example 1 The McdAB system equidistantly positions heterologous carboxysomes in E. coli. To co-express the McdAB system along with carboxysomes, the McdAB operon of H. neopolitaneus was cloned into the vector pBbA2k, resulting in the plasmid pYH85. pLT102 and pYH85 were then co-transformed into E. coli BL21 (AI). Exponentially growing E. coli cells harboring pLT102 and pYH85 were induced with 100 µM IPTG and 100 nM aTc (FIG. 1). In the presence of the McdAB system, small foci of RbcS-mTQ were observed over the nucleoid, as confirmed by DAPI staining. Example 2 Carboxysomes on the nucleoid localize in the presence of McdAB system as shown in electron micrographs. To further investigate the carboxysome distribution of carboxysomes by the McdAB system in E. coli, electron micrographs of cells with and without the McdAB were obtained (FIG. 2). Without the McdAB system, the majority of cells displayed large inclusion bodies, indicated by gray arrows, and no carboxysome-like compartments observed over the nucleoid. Conversely, when the McdAB system was co-expressed, carboxysome-like structures approximately 50 nm in diameter were distributed on the nucleoid, indicated by the white arrow. Example 3 McdA associates with the nucleoid in E. coli In its native host, McdA non-specifically binds nucleoid DNA in its ATP-bound form (MacCready et al.2018). The initial objective was to determine if McdA from Synechococcus elongatus, a model organism for the study of carboxysome biology, would also bind the nucleoid in E. coli. Plasmids were generated to expressed a fusion of McdA and the fluorescent protein mNeonGreen (mNG-McdA) under the control of an inducible promoter activated by anhydrotetracycline (aTc) (pYH81). The plasmid pYH81 was transformed into E. coli MG1665 and induced with 5 nM aTc for mNG-McdA expression. The E. coli nucleoid was stained with UM-42106.601 DAPI (2 µg / mL), and fluorescence microscopy images were captured to assess colocalization (Fig.3A). It was found that mNG-McdA associated with the E. coli nucleoid with a median Pearson correlation coefficient (PCC) of 0.973 (Fig. 3B). As a negative control for nucleoid binding, an ATP-binding deficient mutant of McdA (McdA[K15A]) was used in which that cannot bind to the nucleoid in S. elongatus (Hakim et al. 2021). Consistently, mNG- McdA[K15A] was diffuse and failed to associate with the E. coli nucleoid with a median PCC of 0.857, a PCC score notably lower compared to WT McdA (Fig.3A and Fig.3B). To further investigate the influence of McdA ATP-cycling on DNA binding activity in E. coli, an ATP-trap mutant of McdA (McdA[K15R]) was used, which was unable to undergo ATP hydrolysis. McdA[K15R] can bind and lock McdB-bound carboxysomes onto the nucleoid since ATP-hydrolysis of McdA is required to liberate this association (Hakim et al. 2021). When expressed alone, mNG-McdA[K15R] colocalized with the E. coli nucleoid (Fig. 3A), similar to that of WT McdA, with a median PCC of 0.974 (Fig. 3B). This result was consistent with the observation in S. elongatus - that ATP binding, but not hydrolysis, is required for nucleoid association. The small size of E.coli cells and the limited nucleoid-free cytoplasmic space made it difficult to definitively conclude the extent to which mNG-McdA variants were associating with the nucleoid. To resolve this issue, strains were treated with chloramphenicol (Cm, 100 µg / mL) to compact the E.coli nucleoid (Zimmerman and Murphy, 2001). Cells treated with Cm showed the same but more pronounced patterns of colocalization between the mNG-McdA variants and the compacted nucleoid (Fig. 3C). The PCC values of WT mNG-McdA (0.983) and mNG-McdA[K15R] (0.970) were significantly higher than mNG-McdA[K15A], with a median PCC of 0.712 (Fig. 3D). Altogether, these results show that S. elongatus McdA colocalizes with the nucleoid in E. coli and this interaction requires the ATP binding activity of McdA (Fig.3A, Fig.3B, Fig.3C, and Fig.3D). Example 4 McdB inhibits McdA association with the E. coli nucleoid In S. elongatus cells, McdB is a partner protein that stimulates the ATPase activity of McdA, resulting in the release of McdA from the nucleoid in the vicinity of carboxysome (MacCready et al.2018). It has been previously shown that McdB, when expressed alone, can undergo phase separation and form condensates; appearing as nucleoid-excluded fluorescent foci at the cell poles of E.coli (Hoang, Y. et al. 2024). The objective was to engineer spatial control of condensates based on insight from the McdAB system. Therefore, a determination UM-42106.601 was made to investigate how McdA and McdB proteins would interact with each other in E. coli cells, in the absence of carboxysomes. mNG-McdA and mCherry-McdB (Hoang, Y. et al. 2024) were co-expressed in E. coli and fluorescence microscopy was used to investigate their subcellular distribution. mNG-McdA remained under the control of the aTc inducible promoter on plasmid pYH81, whereas mCherry-McdB was expressed from a second plasmid (pYH71), under the control of an IPTG-inducible promoter (Ptrc). Both plasmids were co-transformed into E. coli MG1665 and protein expression was induced with aTc (5 nM) and IPTG (200 µM). Intriguingly, in the presence of McdB, mNG-McdA was no longer associated with the E. coli nucleoid (Fig.4A and Fig.4B). In cells where mCherry-McdB levels were too low for McdB condensation (below csat), both McdA and McdB were found to be diffuse in the cytoplasm (Fig. 4A, center column). In cells where mCherry-McdB underwent phase separation and formed polar foci, the majority of mNG-McdA colocalized with these McdB condensates, with a smaller fraction colocalized with the nucleoid (Fig.4A, right column). The median PCC of McdA and nucleoid in cells with or without a focus of mCherry-McdB, was 0.760, which is significantly lower than the median PCC of 0.973 in the absence of mCherry- McdB (Fig. 4B). The data suggests that McdB inhibits McdA association with the E.coli nucleoid via two concentration-dependent mechanisms: (1) At low McdB levels, cytoplasmic McdB stimulates McdA ATPase activity, releasing it from the nucleoid and preventing any detectable nucleoid association, and (2) at high McdB levels, McdB forms a condensate that sequesters McdA away from the nucleoid. For better visualization of the association between mNG-McdA and the nucleoid, the nucleoid was condensed by treating E. coli cells with Cm (100 µg / mL). As above, cells with compacted nucleoids showed no McdA colocalization with the nucleoid in cells with low levels of diffuse McdB (Fig. 4C, middle column) or in cells with mCherry-McdB condensates that sequestered mNG-McdA (Fig. 4C, right column). In these McdB-expressing cells with compacted nucleoids, the median PCC of McdA with the nucleoid was 0.836, which is significantly lower than the median PCC of 0.983 in the absence of McdB (Fig.4D). The data once again suggests McdB releases McdA from the nucleoid in E. coli. UM-42106.601 Example 5 Increased partitioning of McdB into the condensate prevents global McdA release from the nucleoid In the native host, the majority of McdB is bound to the carboxysome (MacCready et al.2018) However, in the absence of carboxysomes, a large fraction of McdB remains diffuse in the E. coli cytoplasm (i.e., the dilute phase), which in turn actively stimulated McdA release from the nucleoid. This findings suggests McdB must be concentrated to prevent off-target McdA release from the nucleoid. The objective was to reduce the cytoplasmic fraction of McdB by fusing it to the PopTag. The PopTag has been shown to endow phase separation activity to the fused protein of interest (Hoang et al.2024; Lasker et al. 2022). Furthermore, the linker length influences the csatand material state of the condensate. For example, a short linker lowers the csat of the fusion protein and provides a more viscous or gel-like condensate. mCherry-McdB was fused to the PopTag (mCherry-McdBPopTag) using a short GSGSGS linker, which was expressed from the IPTG inducible plasmid pYH82. pYH71 or pYH82 were transformed into MG1665 E. coli cells for the expression of mCherry-McdB or mCherry-McdBPopTag, respectively. mCherry-McdB formed foci at 3 hours post-induction with 100 µM of IPTG (Hoang et al.2024). However, mCherry-McdBPopTagformed foci at much lower IPTG concentrations (25 µM) (Fig. 5A, top row). At 1 hour of induction, more than 60% of cells exhibited polar foci of mCherry-McdBPopTag, which increased to 89% at 3 hours (Fig. 5B). mCherry-McdB, on the other hand, did not undergo phase separation and no foci were observed under the same induction conditions (Fig.5A and Fig.5B). To determine if the dilute phase of mCherry-McdBPopTagwas significantly reduced compared to that of mCherry-McdB, he condensation coefficients were calculated as described previously (Hoang et al.2024). Briefly, the fluorescence intensity for each pixel in a cell was corrected for background, normalized, and binned to generate histograms that represent the localization pattern for each pixel in cells with a focus (Fig.5C). The majority of the pixels in mCherry-McdBPopTagexhibited low intensity (Fig. 5C, top), while more pixels in mCherry- McdB showed higher intensity (Fig. 5C, bottom). To quantify the differences between the intensity profiles of mCherry-McdBPopTagand mCherry-McdB, the condensation coefficient were calculated, which represents the fraction of pixels with a normalized intensity below selected threshold values (I < 0.1 and I < 0.3). These thresholds were chosen for better visualization of the differences in condensation coefficients, as the majority of mCherry- McdBPopTagand mCherry-McdB proteins are localized to a focus. The average condensation UM-42106.601 coefficients of mCherry-McdBPopTagare 0.69±0.19 and 0.80±0.21 with a threshold of 0.1 and 0.3 respectively, significantly greater than those of mCherry-McdB whose average condensation coefficients were 0.09±0.05 and 0.30±0.17 for thresholds 0.1 and 0.3 respectively (Fig 5D). To determine the stability of the mCherry-McdBPopTagfusion protein, samples from 0 to 4 hours post-induction were collected for Western Blot analysis. A dominant band was visualized at the expected size of mCherry-McdBPopTag(53 kDa). There was minimal leaky expression of mCherry-McdBPopTagand some smaller protein products present at 2 and 4 h post- induction. However, the data suggests that mCherry-McdB can be stably expressed for imaging in E. coli with minimal degradation of the fusion protein (Fig.5E). Together, the results show that McdB phase separation activity was enhanced when fused with the PopTag, resulting in an increased proportion of McdB in the condensate. Example 6 McdA positions McdB-tagged condensates on the E. coli nucleoid It was predicted that enhancing the partitioning of McdB into condensates using the PopTag could prevent the global release of McdA from the nucleoid. To determine if McdA can spatially regulate mCherry-McdBPopTagcondensates over the E. coli nucleoid, experiments were conducted where mCherry-McdBPopTagwas expressed alone or in the presence of McdA to investigate the spatial distribution of both proteins in E. coli. The plasmids pYH81 and pYH82 were transformed into E. coli MG1665 for expression of McdA and mCherry-McdBPopTag, respectively. mCherry-McdBPopTagalone formed polar foci in the majority of cells (Fig. 6A, top). Strikingly, in the presence of McdA, mCherry- McdBPopTagformed multiple condensates that were distributed across the cell length (Fig.6A, bottom). Two or more foci of mCherry-McdBPopTagwere visualized in 77% of cells expressing both McdA and mCherry-McdBPopTag(n= 573), whereas mCherry-McdBPopTagfoci were visualized in only 47% of cells expressing mCherry-McdBPopTagalone (n= 878) (Fig. 6B). In the absence of McdA, the mCherry-McdBPopTagcondensates were nucleoid-excluded (Fig.6A, top), as shown by a median PCC of 0.163 (Fig. 6C). However, mCherry-McdBPopTagcondensates were distributed over the E. coli nucleoid when co-expressed with McdA (Fig.6A, bottom), shown by a significantly higher median PCC of 0.585 (Fig.6C). To further characterize the distribution of mCherry-McdBPopTagcondensates by McdA, time-lapse videos of their co-expression were recorded. When both mCherry-McdBPopTagand McdA were induced at the start of the time-lapse videos, mCherry-McdBPopTaggradually localized to the nucleoid (Fig.7A). Once the mCherry-McdBPopTagconcentration reached csat, UM-42106.601 it began to form condensates on the E. coli nucleoid. For E. coli cells with McdB-tagged condensates positioned by McdA, cells were washed and replaced the supernatant with fresh media to remove the inducer aTc, thereby halting McdA expression. Over time, the previously positioned condensates merged into large polar condensates (Fig.7B). At 6 hours, many cells no longer had condensates due to unequal inheritance. Testing was conducted to determine if McdA could dissolve large polar McdB condensates and redistribute them onto the E. coli nucleoid. McdA was induced in cells where large polar McdB-tagged condensates had already formed. Over time, mCherry-McdBPopTagwas extracted from the condensates and redistributed onto the E. coli nucleoid (Fig.7C). The results demonstrated that McdA can spatially distribute mCherry-McdBPopTagcondensates across the E. coli nucleoid. This represents the first ATP-driven positioning system for synthetic condensates in bacteria. Example 7 A minimal autonomous positioning tag (MapTag) from the N-terminus of McdB Previous studies have shown that the C-terminus of McdB is important for associating with the carboxysome cargo, while the N-terminus is crucial for interaction with McdA (Basalla et al.2022). A short peptide derived from the N-terminus of McdB was designed to be sufficient for the positioning reaction when co-expressed with McdA. This tag is called MapTag (Minimal Autonomous Positioning Tag). According to previous AlphaFold docking models, the first 20 amino acids of the N-terminus of McdB are critical for interaction with McdA (Pulianmackal et al.2023). Therefore, the pJAB39 construct was created that produces the mCherryPopTagcondensate, tagged with the first 20 amino acids of S. elongatus McdB. There are two subclasses of carboxysomes, alpha and beta, which differ in their assembly and components. S. elongatus contains β-carboxysomes. Therefore, the tag derived from S. elongatus McdB was named the β-MapTag. Strikingly, it was observed that the distribution of multiple β-MapTagged condensates upon co-expression with S. elongatus McdA (Fig.8, left). The α-carboxysome positioning system in chemoautotrophs was distinct from that in β-cyanobacteria, highlighting the potential of engineering diverse positioning systems for the positioning of multiple cargos within the same cell population. To explore this, an α-MapTag derived from Halothiobacillus neapolitanus was engineered. AlphaFold docking models once again indicate that the first 20 amino acids of H. neapolitanus McdB are crucial for its interaction with its partner ATPase (Pulianmackal et al. 2023). Therefore, similar to the β- system from S. elongatus, the mCherryPopTagcondensate was tagged with the α-MapTag UM-42106.601 (plasmid pRED2). This α-MapTag was sufficient to interact with H. neapolitanus McdA, resulting in the positioning of multiple condensates along the cell length (Fig.8, right). Example 8 McdA and the MapTag position encapsulins on the E. coli nucleoid Another major type of protein-based compartment in bacteria is the encapsulin. Similar to carboxysomes, encapsulins are composed of a shell protein that encapsulates specific cargos. Encapsulins are significantly smaller than carboxysomes, making them challenging to visualize in live cells. To address this, a method was developed to observe encapsulin nanocompartments in E. coli. The fluorescent protein mNG was used as encapsulin cargo by fusing it to an encapsulin-targeting peptide. A degron tag was also fused to mNG to degrade unencapsulated mNG, thereby enhancing the visibility of encapsulins packed with mNG. When the encapsulin shell protein and mNG cargo were expressed from plasmid pSK3 in E. coli, fluorescent focus at the cell pole was observed, indicating the clustering and aggregation of encapsulins, likely due to nucleoid exclusion (Fig.9A). Encapsulins were then fused with both the β- and α-MapTags (construct pSK1 and pSK2, respectively). In the absence of the partner McdA, the MapTagged encapsulins once again appeared as nucleoid-excluded fluorescent foci at the cell pole, similar to encapsulins without the MapTag (Fig. 9B, top). When β- or α-McdA were co-expressed with β- or α- MapTagged encapsulins, respectively, multiple fluorescent foci were observed distributed along the cell length (Fig. 9B, bottom). The faint fluorescent signals are consistent with the small size of encapsulins, which are capable of packing only a few molecules of mNG. To further validate the positioning of encapsulins by the MapTag and McdA, time-lapse movies of their expression were recorded. Upon induction of both MapTagged-encapsulins and their partner McdA proteins at t = 0, it was observed that the formation of small fluorescent foci along the cell length over time, starting from 3 h (Fig. 10A). For cells with positioned encapsulinMapTag, McdA expression was halted by replacing the supernatant with fresh media supplemented only with IPTG (excluding aTc). After 4 hours, most foci had fused together, with the merging more pronounced at 6 hours (Fig. 10B). Moreover, many cells lacked encapsulin compartments due to unequal inheritance. To assess the effect of McdA on the nucleoid-excluded encapsulin clusters at the cell pole, aTc was introduced to induce McdA expression in cells previously induced only with IPTG, which exhibited a large polar fluorescent focus. Within 5 minutes, encapsulins migrated towards the nucleoid, and dimmer fluorescent foci formed on the nucleoid, suggesting that McdA was redistributing encapsulins along the E. coli nucleoid (Fig.10C). UM-42106.601 MATERIALS AND METHODS Plasmids The plasmid pLT102 was used to express carboxysomes in E. coli and the plasmid pYH85 was used to express the McdAB system. At least one construct was made using Gibson assembly from PCR fragments and verified by Sanger sequencing. The plasmid pLT102 was generated from the plasmid pHnCB2by inserting mTurquoise2 to the C-terminus of CbbS, the small subunit of the enzyme Rubisco: atggctgaaatgcaggattacaagcaaagcctcaaatatgagactttctcttatcttccacccatgaacgcggaacgcatccg cgctcaaatcaagtacgcaattgctcaaggctggagccccggcattgagcacgtagaagtgaaaaactccatgaaccaatattggtac atgtggaaacttcccttcttcggcgaacaaaatgtcgacaacgtgttggctgaaattgaagcgtgtcgtagtgcgtatccaacacaccag gtcaaactggtggcttatgacaactatgcgcaaagcttaggtctggccttcgtggtctaccgcggcaacggatctgggagtgggagtat ggtgtccaaaggggaagaattattcacgggcgtcgtcccaatcctggtagagctcgatggagacgtgaatgggcataaattcagtgta agcggagagggcgaaggcgacgcaacatacggaaagctgaccctcaagttcatttgcacaacgggcaaattgcctgtgccatggcc gacattggtcacaacactgagttggggtgttcaatgcttcgcccggtatcccgatcacatgaagcaacatgacttttttaagtcggctatg ccggaaggatatgtacaagaacgtactattttttttaaagacgatggcaactataaaacacgggctgaggtaaaattcgaaggtgacac gctcgtgaaccgtattgaattaaaaggcatcgactttaaggaggatggaaacattctgggccataagctggagtataactatttctcggat aacgtgtatattaccgcagataagcaaaaaaatggtatcaaggcaaattttaaaatccggcataacatcgaggacgggggggtgcagt tggctgaccactaccagcagaacacgcctattggggatggccctgtattgctgccagacaaccattatctcagcacgcagtccaagct gagcaaagatccaaacgaaaaacgtgaccacatggttttattggagttcgtcacagccgctggcattactctgggcatggatgagttata caaataa (SEQ ID NO:17) MAEMQDYKQSLKYETFSYLPPMNAERIRAQIKYAIAQGWSPGIEHVEVKNSM NQYWYMWKLPFFGEQNVDNVLAEIEACRSAYPTHQVKLVAYDNYAQSLGLAFVV YRGNGSGSGSMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK FICTTGKLPVPWPTLVTTLSWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKD DGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYFSDNVYITADKQKN GIKANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRD HMVLLEFVTAAGITLGMDELYK (SEQ ID NO:18) The pYH85 was constructed by amplifying the McdAB operon from genomic DNA of H. neapolitanus and cloned into the multiple cloning sites of the vector pBbA2. Plasmids were introduced into their respective host strains by chemical transformation and selection for antibiotic resistance encoded by the plasmid. Growth conditions Lysogeny broth (LB) and AB media were used as either broth and / or solid for culturing bacterial strain growth. LB medium was inoculated with one colony and bacteria were grown UM-42106.601 overnight in a 15 mL tube in 5 mL of LB broth at 37 °C on an orbital shaker at 200–225 rpm for 12-16 hours. When appropriate, the following chemicals were added to the LB medium at the indicated final concentrations: chloramphenicol (25 μg / ml), kanamycin (50 or 100 µg / mL) for selecting plasmids in culture, IPTG (0.1–1 mM or 20 - 200 µM) and L-arabinose (0.2%) for protein induction, anhydrotetracycline (aTc, 0.5 - 10 nM or 100 nM) for McdA induction. AB medium was used for inducing protein expression in E. coli MG1665 to ensure reproducibility, as all components are defined. It was supplemented with 0.2% of a carbon source (glycerol for growth or glucose to inhibit basal protein expression from the Ptrc promoter), 0.2% casamino acids, 10 μg / ml thiamine, and 25 μg / ml uracil. Exponential phase cultures were created by diluting the overnight LB culture into AB media (1:100). Induction The plasmids pLT102 and pYH85 were transformed into BL21 (AI) cells and a 5 ml overnight culture containing 20 μg / ml of chloramphenicol and 50 μg / ml of kanamycin in LB medium was grown at 37 °C with shaking at 225 rpm. The overnight culture (50 μl) was used to inoculate 5 ml of LB supplemented with 20 μg / ml of chloramphenicol and 50 μg / ml of kanamycin in a 15-ml tube. The cells were grown at 37 °C and shaken at 225 rpm to an OD600 of 0.2–0.6. Protein expression was then induced by the addition of 100 μM IPTG, 0.2% Arabinose solution, and 100 nM of aTc to the tube. The cultures were incubated at 37 °C with shaking at 225 rpm for at least 3 hours. Imaging Cells (2 µl) were spotted on a 1 cm diameter pad made of 1.5% UltraPure agarose in LB and supplemented with 100 μM IPTG, 0.2% Arabinose, and 100 nM of aTc. After at least two minutes, the cell-containing side of the pad was flipped onto a 35-mm glass-bottom dish and mounted onto the stage of a Nikon Ti2-E motorized inverted microscope controlled by NIS Elements software with a SOLA 365 LED light source, a 100x objective lens (Oil CFI Plan Apochromat DM Lambda Series for Phase Contrast), and a Photometrics Prime 95B back- illuminated sCMOS camera. mTQ was imaged using the “CFP” filter set (C-FL CFP, Hard Coat, High Signal-to-Noise, Zero Shift, Excitation: 436±10 nm [426–446 nm], Emission: 480±20 nm, Dichroic Mirror: 455 nm). Fusions to mNG were imaged using a “YFP” filter set (C-FL YFP, Hard Coat, High Signal-to-Noise, Zero Shift, Excitation: 500±10 nm, Emission: 535±15 nm, Dichroic Mirror: 515 nm. mCherry was imaged using a “Texas Red” filter set (C- FL Texas Red, Hard Coat, High Signal-to-Noise, Zero Shift, Excitation: 560±20 nm, Emission: 630±37.5 nm; Dichroic Mirror: 585 nm). Exponential phase cultures (prepared as stated above) were induced with aTc and / or IPTG as the experiment required. Samples from the induced UM-42106.601 cultures were collected at specified time points and remained on an orbital shaker (200 - 225 rpm) at 37℃ until imaging. DAPI Staining in BL21 (AI) cells E. coli BL21 (AI) cells with plasmid pLT102 and pYH85 were induced with 100 μM IPTG, 0.2% Arabinose, and 100 nM of aTc. DAPI was added to the exponentially growing culture at a final concentration of 2 μM. Cells were incubated in DAPI for 15 min at 25oC before imaging, without rinsing. DAPI was imaged using the “DAPI” filter set (C-FL DAPI, Hard Coat, High Signal-to-Noise, Zero Shift, Excitation: 350±25 nm, Emission: 460±25 nm, Dichroic Mirror: 400 nm). DAPI staining in MG1665 cells and Chloramphenicol treatment E. coli MG1665 cells with our plasmids of interest were induced as specified in each experiment. DAPI was added to the exponentially growing cultures at a final concentration of 2 μM and the culture was then incubated for 15 min at 37°C before imaging, without rinsing. DAPI was imaged using the “DAPI” filter set (C-FL DAPI, Hard Coat, High Signal-to-Noise, Zero Shift, Excitation: 350±25 nm, Emission: 460±25 nm, Dichroic Mirror: 400 nm).For nucleoid compaction, Chloramphenicol was added to the exponentially growing cultures at a final concentration of 100 μg / mL. The treated cultures were incubated at 37°C for 30 min before imaging. Time-lapse videos of protein expression in E. coli MG1665 E. coli MG1665 with plasmids pTrc99A-mCherrymcdB (pYH71) and / or Ptrc-mCherry- poptag-mcdB (pYH82) were induced with 25 µM IPTG and 5 nM aTc and then immediately spotted on an AB agarose pad. The pad was prepared at a final concentration of 1.5% UltraPure agarose in AB media and supplemented with 0.2% glycerol, 0.2% casamino acids, 10 μg / ml thiamine, and 25 μg / ml uracil, as well as 100 μg / ml of carbenicillin and 25 μM IPTG in a 35- mm glass-bottom dish. Once cooled and hardened, a 14 mm section was punched out, and 2 μl of the cell culture was spotted onto the bottom before flipping the pad back into the dish. A series of images were taken with the “Texas Red” filter set (see the “Wide-field fluorescence and phase-contrast imaging” section) every 10 min for 5 hours. Cell segmentation Cell segmentation was performed with the Cellpose51 package in Python. To train the model optimally for bacterial cell morphology, 26 raw phase contrast images of cells were manually annotated. To segment the cells using the trained model, a Gaussian blur (standard deviation of Gaussian = 0.066 µm) was applied to the bacterial cell phase-contrast images and the blurred cells were segmented. Cells touching the borders of the image were ignored. UM-42106.601 Erroneous segmentations were manually corrected using the Cellpose GUI or excluded from further analysis. Condensation analysis The fluorescence intensity for each pixel in a cell was corrected for background by subtracting the median value of all pixels in an image outside of the cell regions and normalized by the minimum and maximum pixel intensity values in the corresponding cell: Next, cells were classified by the presence or lack of a focus. An ROI was defined for each cell using the segmentation phase mask. Each ROI was applied a Gaussian blur and normalized using the above equation. Foci were detected by generating a binary image where only pixels above a specified intensity threshold (0.05) were assigned a non-zero value. Normalized intensity pixel histogram: The normalized pixel intensities were then binned to generate histograms that represent the localization pattern for each protein in cells with a focus. Homogeneously distributed protein would display a flat distribution while a strongly left-skewed distribution indicates more clustered proteins in the cell. Erroneous foci or cell identifications were excluded from further analysis. Condensation coefficient: To quantify the differences in the clustering between the proteins measured, the fraction of pixels were calculated with a normalized intensity below a threshold value, I < 0.1 and 0.3, for each cell. Foci Count Analysis Images were captured from multiple fields of view using methods described above, these images were then analyzed with a McdB foci detection and analysis script written in Python. Results were then filtered by incorrect identification of foci which were manually removed from further analysis. The sum of cells with 0, 1, 2, 3, and 4+ foci was then calculated and graphed using GraphPad Prism (GraphPad Software, San Diego, CA). Western Blot E. coli cells expressing mNG-McdA or mCherry-McdBPopTagwere collected for Western Blot analysis as described below. E. coli cultures were grown until exponential phase (O.D600~ 0.2) and were then induced with 1.5 nM aTc or 25 µM IPTG. Samples were standardized by spinning down 500 µl of the culture and resuspended in AB media to a final OD of 0.5: UM-42106.601 Then, 150 µl of the resuspended cells were then combined with 50 µl of 40X Laemelli buffer. Finally, the samples were boiled at 95°C prior to storage at -20°C. These steps were repeated at 0, 2, and 4 h post-induction. Between sample collection timepoints, the culture continued to grow at 37°C and shake at 200-225 rpm. Prior to gel electrophoresis, all samples were denatured at 95°C for 10 minutes. Then, the gel electrophoresis cassette tank is prepared with 1X SDS Loading Buffer and loaded with 5ul of the molecular weight protein ladder Precision Plus Protein All Blue Standards. Next, the samples were loaded into the NUPAGE Bis Tris gel wells and ran at 110V for 70 minutes. Next, the PVDF membrane was soaked in 70% ethanol for activation and the completed gel was trimmed for loading into the transfer sandwich. The transfer stack included: transfer buffer reservoirs soaked in transfer buffer, the PVDF membrane, the gel containing the protein samples, and another stack of transfer buffer reservoirs. The case was then inserted into the Turbo Transfer Machine and ran on the MixedMW setting for 7 minutes. After transfer, the membrane was blocked with 5% Bovine Serum Albumin (BSA) in TBST (100mM Tris-HCLpH 7.4, 3 mM KCl, 180 mM NaCl, .05% Tween-20) for 20 minutes, and then was introduced to the Primary antibody solution (1:1000 dilution in TBST+BSA) . The primary antibody used for the mCherry-McdBPopTagwestern was Rabbit Anti-mCherry (Hoang, et. al. 2023, Preprint) and the primary antibody used for the mNG-McdA western was Rabbit Anti-mNG. The membranes were blocked with the primary antibody solution between 2 hours or overnight in the cold room at 4°C. The primary antibody was then washed with TBST for 10 minutes 3X prior to incubation with the secondary antibody. The secondary antibody used for both protein westerns was IRDye 800CW Goat anti- Mouse. The antibody was diluted in TBST+BSA in a 1:5000 ratio and inoculated the membrane for 1 - 2 hours. Prior to imaging, the secondary antibody was replaced with TBST. Imaging was completed with the LiCOR software using fluorescence based detection of our protein in the 800 nm channel and detection of our molecular weight ladder in the 365 nm channel. Fluorescence microscopy Cells (2 µl) were spotted on a 1 cm diameter pad made of 1.5% UltraPure agarose in LB and supplemented with 100 μM IPTG, 0.2% Arabinose, and 100 nM of aTc. After two minutes, the cell-containing side of the pad was flipped onto a 35-mm glass-bottom dish and mounted onto the stage of a Nikon Ti2-E motorized inverted microscope controlled by NIS Elements software with a SOLA 365 LED light source, a 100x objective lens (Oil CFI Plan UM-42106.601 Apochromat DM Lambda Series for Phase Contrast), and a Photometrics Prime 95B back- illuminated sCMOS camera. mTQ was imaged using the “CFP” filter set (C-FL CFP, Hard Coat, High Signal-to-Noise, Zero Shift, Excitation: 436±10 nm [426–446 nm], Emission: 480±20 nm, Dichroic Mirror: 455 nm). Transmission electron microscopy E. coli cells expressing carboxysomes and McdAB were pelleted and washed twice with 0.1M Cacodylate Buffer (CB, pH 7.2). The samples were then fixed with 3% glutaraldehyde and 3% paraformaldehyde for 2 hours at room temperature followed by overnight storage at 4°C. On the following day, the samples were washed with 0.1M CB and then incubated with 1.5% K4Fe(CN)6and 2% OsO4 one hour. Post-fixation, samples underwent three washes with 0.1M CB and then incubated with 0.1M Na2+ Acetate buffer (pH 5.2) for one hour. Dehydration was performed on the third day using ethanol and acetone, followed by infiltration with Spurr’s resin. On the fourth day, the resin is embedded and placed in an oven set at 70°C for 24 hours. On the fifth day, the samples were sectioned to obtain 70 nm-thick sections using the Leica EM UC7 Ultramicrotome. The sections were stained with 4% uranyl acetate and Reynold's lead citrate before visualization using a JEOL 1400-plus transmission electron microscope equipped with an XR401 AMT sCMOS camera. Bacterial strains, plasmids, and primers Strains, plasmids, and primers used in this study are listed in Table 1 and Table 2. All constructs were made using Gibson assembly from PCR fragments or synthesized dsDNA (Integrated DNA Technologies) and verified by Sanger sequencing. Plasmids were introduced into their respective host strains by chemical or electrical transformation and selection for antibiotic resistance encoded by the plasmid. The plasmid pYH81 for mNG-McdA expression was generated from plasmid pBbA2k- RFP by replacing RFP with mNG-McdA. The plasmid pBbA2k-RFP was amplified using primers pBbA2k F and pBbA2k R to generate the first fragment. The mNG-McdA fragment was synthesized, and codon optimized for E. coli. The two fragments were then added to a Gibson assembly reaction to enzymatically join the overlapping DNA sequences. A construct was generated, designated pYH91, in which the McdA protein from S. elongatus was not fused to mNG, utilizing the KLD kit (New England BioLabs). Initially, the plasmid pYH81 was amplified using Q5 Hot Start High-Fidelity DNA Polymerase and the primers Se_McdA F and Se_McdA R. Subsequently, the PCR product was incubated with an enzyme mix containing a kinase, a ligase, and DpnI, facilitating the rapid circularization of the PCR product and the removal of template DNA. Finally, the incubated KLD mix was transformed into E. coli UM-42106.601 competent cells. The plasmid pYH84, used for the expression of the McdA protein from H. neapolitanus, was constructed analogously to pYH81. The McdA gene was amplified from the H. neapolitanus genome using primers HnA F and HnA R, and subsequently ligated into the pBbA2k vector using Gibson assembly. To generate variants of McdA, site directed mutagenesis was preformed using the KLD kit (New England BioLabs). Primers K15R F and K15R R were used to generate pYH90 for mNG-McdA [K15R] expression, and primers K15A F and K15R R were used to generate pYH91 for mNG-McdA [K15A] expression. The plasmid pYH71 was generated by adding the PopTag and McdB protein sequence to the plasmid pYH75, which expressed mCherryPopTagunder the control of an IPTG-inducible promoter on a pTrc99A expression vector (PTrc99A-mCherry-poptag). The plasmid pYH75 was amplified using primers pop_AB F and pop_AB R. The McdB fragment was amplified using primers mcdB_AB F and mcdB_AB R. The fragment containing PopTag was synthesized, and codon optimized for E. coli. The three fragments were then added to a Gibson to generate the plasmid pYH71 (PTrc99A-mCherry-poptag-mcdB). To construct pJAB39, Map2 R and Map1 F primers were used to amplify the truncation of the N-terminus of S. elongatus McdB from the plasmid pYH71 using the KLD kit (New England BioLabs). The plasmid pRED2 was generated by first amplifying the plasmid pYH75 using the primers Hn_20aa F and Hn_20aa R. A nucleotide sequence encoding the codon-optimized first 20 amino acids of H. neapolitanus McdB (MTNLEDKLSASIKTENKDTI) was synthesized. The two fragments were then joined using Gibson assembly. The plasmid pSMK1 for the expression of mNEON encapsulating encapsulin with alpha-McdB tag was generated by Gibson assembly between pTrc99A backbone vector and the synthesized and E. coli. codon optimized gene fragment encoding mNG and alpha-McdB tagged encapsulin. The plasmid pSMK2 was prepared by replacing alpha-McdB in pSMK1 with beta-McdB and plasmid pSMK3 was prepared by truncating alpha-McdB in pSMK1, using the overhang PCR with primers pSMK23-F / pSMK2-R and pSMK23-F / pSMK3-R. UM-42106.601 Table 1: Bacterial Strains and Plasmids Table 2: Primers UM-42106.601 UM-42106.601 Making chemical and electroporation competent cells of E. coli Chemically competent cells. E. coli cells were grown from an overnight culture in Lysogeny broth (LB, 1:100 dilution) at 37℃ until they reached an OD600 of 0.4. Next, the culture was incubated on ice for 10 minutes before a 10 minute centrifugation at 5,000 rpm at 4℃. The cell pellet was then resuspended in 100 mM MgCl2 prior to another 10-minute centrifugation at 4,000 rpm and 4℃. The pellet was then resuspended in 100mM CaCl2. A 20- minute incubation was completed on ice followed by one more 10-minute centrifugation at 4,000 rpm and 4℃. The pellets are then resuspended in the 100mM CaCl2 and combined with a 1:3 dilution of 80% glycerol (2mL glycerol in 6mL cell mixture with CaCl2) before storage at -80℃. Electroporation competent cells. E. coli cells were grown from a 1:100 dilution of an overnight culture in LB media at 37℃ until they reached an OD600 of 0.4 - 0.6. They were then harvested via centrifugation and washed with sterile, cold water (4 times). Next, a 20% (w / v) glycerol and 1.5% (w / v) mannitol solution was slowly dispensed to the bottom of the cell suspension so that the high-density glycerol-mannitol solution forms an underlayer, resulting in an upward displacement of the E. coli culture. Lastly, the culture was centrifuged to force the bacteria through the high density layer, removed the supernatant, and added cold glycerol- mannitol solution to resuspend the pellet prior to storage at -80℃. Transformation of plasmids into chemical and electroporation competent E. coli cells 50 - 100 µL of competent E. coli cells were thawed on ice and mixed with 5 µL of plasmid DNA. Depending on the method of transformation - heat shock or electroporation - the mixture is submerged into a water bath maintained at 42oC for 20 seconds or placed into the electroporator at 1700 V, respectively. Transformations were recovered in 500 µL of LB broth and shaken at 200-225 rpm at 37℃ for one hour. Clones were selected by plating on selective medium with antibiotics. All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein. Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred UM-42106.601 embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context. REFERENCES 1. Lee, T. S. et al. BglBrick vectors and datasheets: A synthetic biology platform for gene expression. J Biol Eng 5, 12 (2011). 2. Lasker, K. et al. The material properties of a bacterial-derived biomolecular condensate tune biological function in natural and synthetic systems. Nat Commun 13, 5643 (2022). 3. Hoang, Y. et al. An experimental framework to assess biomolecular condensates in bacteria. Nat Commun 15, 3222 (2024). 4. 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Claims

UM-42106.601 CLAIMS What is claimed is:

1. A cell expressing: a) a heterologous carboxysome distribution protein A (McdA), b) a heterologous carboxysome distribution protein B (McdB), and c) one or more proteins of a heterologous protein-based organelle.

2. The cell of claim 1, wherein the protein-based organelle is a bacterial microcompartment.

3. The cell of claim 1, wherein the protein-based organelle is an encapsulin nanocompartment.

4. The cell of claim 1, wherein the protein-based organelle is a biomolecular condensate.

5. The cell of claim 1, further comprising an agent encapsulated in said protein-based organelle.

6. The cell of claim 2, wherein said agent is a nanomaterial.

7. The cell of claim 3, wherein said nanomaterial is an organic molecule.

8. The cell of claim 3, wherein said nanomaterial is a protein.

9. The cell of claim 3, wherein said nanomaterial is an inorganic molecule.

10. The cell of claim 3, wherein said nanomaterial is a hybrid compound molecule.

11. The cell of claim 5, wherein said protein is an enzyme.

12. The cell of claim 8, wherein said enzyme is ribulose-1,5-bisphosphate carboxylase / oxygenase (RuBisCo).

13. The cell of claim 1, wherein said McdB is a minimal autonomous positioning tag (MapTag) comprising a N-terminus of McdB.

14. The cell of claim 1, wherein said cell is a bacterium.

15. The cell of claim 13, wherein said bacterium is a gram-negative bacterium.

16. The cell of claim 14, wherein said gram-negative bacterium is an Escherichia coli (E. coli).

17. The cell of claim 14, wherein said gram-negative bacterium is a cyanobacteria.

18. The cell of claim 1, wherein said cell is a fungal cell.

19. The cell of claim 1, wherein said cell is a plant cell.

20. The cell of claim 1, wherein said cell is an animal cell.

21. A composition comprising the cell of any of claims 1-20.UM-42106.601 22. A system comprising: one or more expression vectors encoding McdA, McdB, and / or one or more protein-based organelle proteins.

23. The system of claim 22, wherein said McdA, McdB, and / or one or more protein-based organelle proteins are codon-optimized for expression in a heterologous host cell.

24. The system of claim 22, wherein one or more expression vectors comprises a heterologous promoter.

25. A method of expressing a heterologous bacterial microcompartment in a cell comprising: introducing a heterologous carboxysome distribution protein A (McdA), a heterologous carboxysome distribution protein B (McdB), and a one or more heterologous protein-based organelle proteins into a cell.

26. The method of claim 25, further comprising the step of culturing the cell.

27. The method of claim 26, further comprising the step of selecting a cultured cell expressing said heterologous McdA, McdB, and protein-based organelle protein.

28. The method of claim 27, wherein said selecting comprises selecting cells expressing an antibiotic resistance factor.

29. Use of a cell of any of the claims 1-20.

30. Use of a cell of any of claims 1-20 for encapsulating a biological reaction.

31. The use of claim 30, wherein the biological reaction is an enzymatic reaction.

32. The use of claim 31, wherein enzymatic reaction is a carbon fixing enzymatic reaction.