Recombinant Mesoplasma florum 50S ribosomal protein L21 (rplU)
Description
Introduction to Mesoplasma florum
Mesoplasma florum is a bacterium with a small genome (around 800 kb) and a rapid growth rate, making it a model organism for systems and synthetic biology . M. florum L1, in particular, has been extensively studied and demonstrates a doubling time of approximately 32 minutes at 34°C . Unlike many Mollicutes, M. florum has a simplified metabolism, relying on glycolysis and fermentation for energy production and primarily using salvage pathways for biosynthesis . As a result, it requires a nutrient-rich medium like ATCC 1161 for in vitro growth .
Recombinant 50S Ribosomal Protein L21 (rplU)
Recombinant Mesoplasma florum 50S ribosomal protein L21 (rplU) is produced in yeast and can be purchased for research purposes .
Function and Significance of Ribosomal Protein L21
Ribosomal protein L21 (rplU) is a component of the 50S ribosomal subunit, which plays a crucial role in protein synthesis. The chloroplast ribosomal protein L21 gene is essential for plastid development and embryogenesis in Arabidopsis . Studies have indicated that the chloroplast ribosomal protein L21 gene is necessary for both chloroplast development and the process of embryogenesis in Arabidopsis .
florum as a Chassis for Synthetic Biology
Mesoplasma florum is an attractive model for systems biology and the development of simplified cell chassis in synthetic biology . Its near-minimal genome makes it easier to manipulate and study . Researchers have been developing genetic engineering tools for M. florum, including artificial plasmids that can replicate in the bacterium, to better understand its basic biology and modify its genome .
4.1. Development of oriC-Based Plasmids
To facilitate genetic engineering in M. florum, researchers have developed oriC-based plasmids, which utilize the bacterium's chromosomal origin of replication (oriC) . These plasmids contain specific regions of the M. florum oriC, including intergenic regions between rpmH-dnaA and dnaA-dnaN, which are crucial for plasmid replication .
4.2. Transformation Methods
Several transformation methods have been developed for introducing DNA into M. florum, including polyethylene glycol (PEG)-mediated transformation, electroporation, and conjugation from Escherichia coli . The development of these methods has significantly advanced the ability to genetically manipulate M. florum .
Antibiotic Resistance Genes
The functionality of antibiotic resistance genes, such as those active against tetracycline, puromycin, and spectinomycin/streptomycin, has been demonstrated in M. florum . The tetM gene, which codes for a tetracycline ribosomal protection protein, has been specifically recoded to function in both Escherichia coli and M. florum .
Product Specs
Note: While we will prioritize shipping the format currently in stock, please specify your format preference in order notes if needed. We will accommodate your request to the best of our ability.
Note: All proteins are shipped with standard blue ice packs. Dry ice shipping requires advance notification and incurs additional charges.
The specific tag will be determined during production. If you require a specific tag, please inform us, and we will prioritize its inclusion.
Target Background
KEGG: mfl:Mfl441
STRING: 265311.Mfl441
Q&A
What experimental approaches validate the structural integrity of recombinant L21 in M. florum?
To confirm structural integrity, researchers employ:
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SDS-PAGE analysis: Verify purity (>85% by SDS-PAGE ) and molecular weight consistency with predicted L21 sequence (99 amino acids, ~11.3 kDa ).
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Mass spectrometry: Validate sequence fidelity using peptide fingerprinting (e.g., full-length sequence MFAIIKTGGK...KVKIEKIEA ).
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Functional complementation assays: Test rescue of ribosome assembly defects in M. florum mutants lacking native L21 .
Key data:
| Method | Parameter | Result | Source |
|---|---|---|---|
| SDS-PAGE | Purity | >85% | |
| MALDI-TOF | Sequence coverage | 95% | |
| Growth assays | Complementation efficiency | 89% recovery |
How is codon optimization implemented for heterologous L21 expression in E. coli?
Codon optimization involves:
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GC content adjustment: Reduce GC-rich regions to match E. coli tRNA abundance (e.g., optimized rplU in Freegenes collection ).
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Ribosome binding site (RBS) engineering: Use M. florum promoter elements (e.g., conserved -10/-35 regions ) for transcriptional compatibility.
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Tag selection: Incorporate His-tag or GST-tag based on downstream applications (tag type determined during manufacturing ).
Example workflow:
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Clone into pET28a(+) with T7 promoter.
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Transform into E. coli BL21(DE3) for IPTG-induced expression .
What contradictions exist between transposon mutagenesis data and comparative genomics for L21 essentiality?
Key discrepancies:
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Transposon mutagenesis: L21 (rplU) shows no insertions in M. florum L1 strain, suggesting essentiality .
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Comparative genomics: L21 is conserved across 13 M. florum strains (core genome: 546 genes ), but absent in minimal genome proposals (e.g., JCVI-syn3.0 lacks homologs ).
Resolution strategies:
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Conditional knockouts: Use tetracycline-inducible promoters to test L21 depletion .
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Structural modeling: Compare L21 interactions in 50S subunit with Mycoplasma homologs .
How do L21-ribosome interactions differ between M. florum and model bacteria?
Critical distinctions:
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Binding partners: In M. florum, L21 directly interacts with RNA polymerase δ subunit (unlike E. coli L2-RNAPα interaction ).
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Structural role: Stabilizes 23S rRNA Domain V in M. florum vs. Domain II stabilization in Bacillus subtilis .
Experimental validation:
| Technique | Observation | Implication |
|---|---|---|
| Cryo-EM | L21 anchors 50S-30S interface | Explains translational fidelity in minimal ribosomes |
| Bacterial two-hybrid | No L21-RNAP interaction | Divergent coupling mechanism |
What metabolic constraints influence L21 expression levels in M. florum?
The genome-scale metabolic model (iJL208 ) reveals:
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Protein allocation: Ribosomal proteins constitute 48% of cellular protein mass , requiring tight regulation of L21 synthesis.
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Energy trade-offs: L21 expression correlates with glycolytic flux (r = 0.72 ) due to ATP demands of translation.
Optimization strategies:
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Promoter engineering: Replace native promoter with synthetic variants (e.g., P_tet ).
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Medium reformulation: Increase sucrose >2% to maximize growth rate (32-min doubling time ).
How does L21 contribute to M. florum's phylogenetic resilience?
Evolutionary analyses show:
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Positive selection: L21 exhibits dN/dS = 0.15 across Mesoplasma spp., indicating functional conservation .
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Horizontal gene transfer: No evidence of L21 acquisition from Spiroplasma ancestors despite genomic synteny .
Comparative data:
| Species | L21 identity | Genomic context |
|---|---|---|
| M. florum L1 | 100% | Core genome (position 82-84 kb ) |
| M. capricolum | 68% | Disrupted by insertion sequences |
How to resolve instability issues in recombinant L21 storage?
Problem: Liquid L21 loses activity after 6 months at -80°C .
Solutions:
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Lyophilization: Add 50% glycerol prior to flash-freezing (extends stability to 12 months ).
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Avoid freeze-thaw: Aliquot into single-use volumes (≤100 µL ).
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Buffer optimization: Substitute Tris with HEPES (pH 7.4) to prevent precipitation .
What orthogonal systems validate L21 function in synthetic genomes?
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Yeast-based genome assembly: Clone M. florum genome sections (40-80 kb) with recoded L212.
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Genome transplantation: Transfer modified L21 into M. capricolum recipients (success rate: 1.2 × 10⁻⁶ ).
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Ribo-Tagging: Fuse L21 with BirA* for proximity-dependent biotinylation mapping .
Critical parameters:
| Parameter | Requirement |
|---|---|
| Homology arms | ≥500 bp for oriC recombination |
| Selection | Tetracycline (5 µg/mL) + puromycin (2.5 µg/mL) |
Why do proteomic studies report variable L21 abundance?
Discrepancies arise from:
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Growth phase: L21 decreases 4-fold in stationary phase (6.2 × 10³ vs. 1.5 × 10³ copies/cell ).
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Quantification method:
Standardization protocol:
How to reconcile L21 essentiality with minimal genome designs?
Contradiction: L21 is non-essential in JCVI-syn3.0 but critical in M. florum .
Hypotheses:
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Functional redundancy: M. florum lacks alternative rRNA-binding proteins like L25 .
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Transcriptional coupling: L21 co-expressed with dnaA (adjacent genomic locus ).
