Journal: bioRxiv

Article Title: Engineering orthogonal ribosomes for real-time monitoring using fluorescence

doi: 10.1101/2023.11.19.567736

Figure Lengend Snippet: ( a ) Candidate site identification. Candidate sites were identified by structural (red) and sequence analysis (blue) of the native E. coli ribosomal operons. In addition, the 5’ terminus of the TO-ribosome, and all three linker regions (tethers 1 and 2, and the connector; mustard-coloured) were also tested. Operon structures are coloured by ribosomal location: linker region, mustard; 23S rRNA, dark grey; 16S rRNA, light grey. ( b ) Location of identified sites on the 70S tethered ribosome structure (PDB: 8B0X). Tethers T1 and T2 are not modelling in this high-resolution structure and so a dashed mustard-coloured lines denote the points they would attach to in the 23S and 16S subunits. TO-, tethered orthogonal.

Article Snippet: For all plasmid cloning and propagation, Escherichia coli strain DH10B (Δ(ara-leu) 7697 araD139 fhuA ΔlacX74 galK16 galE15 e14-phi80dlacZΔM15 recA1 relA1 endA1 nupG rpsL (StrR) rph spoT1 Δ(mrr-hsdRMS-mcrBC) (New England Biolabs, C3019I) was used.

Techniques: Sequencing

Journal: bioRxiv

Article Title: Engineering orthogonal ribosomes for real-time monitoring using fluorescence

doi: 10.1101/2023.11.19.567736

Figure Lengend Snippet: ( a ) Schematics of the genetic constructs used to express the TO-ribosomes from the o-RiboT gene (top) and test their ability to express an oSD RBS (oRBS) driven mCherry reporter (bottom). ( b ) Performance of mutant TO-ribosomes in two E. coli strains. Cells were co-transformed with orthogonal ribosome and orthogonal mCherry reporter plasmids, and grown in M9 media, while oSD-mCherry was induced with 0.1% arabinose. mCherry production per cell was calculated from mCherry and OD readings, and results were compared to the readings from the ‘WT’ TO-ribosome variant at 720 min, which was set to 100%. Plotted data represents the mean and standard deviation of results over at least 2 independent repeats conducted in quadruplicate. Sites are coloured by ribosomal location: no insert/WT, red; insert in linker region, yellow; insert in 23S rRNA region, light blue; insert in 16S rRNA region, dark blue. ( c ) Structure of TO-ribosome, showing insertion sites by performance (red to dark grey, best to worst; PDB: 8B0X). Variants were classified as Excellent (>90% activity in BL21(DE3) and >85% activity in DH10B strains), Good (>80% activity in both strains), Moderate (>60% in BL21(DE3) and >70% in DH10B) and Poor (<60% in both). ( d ) Performance of WT and C888 insertion mutant when expressing mCherry and GFP orthogonal reporters in DH10B strain. Cells were grown and induced, and data was analysed, as in panel (b). ( e ) Permissiveness of C888 site for a range of insertions in DH10B strain. Cells were grown and induced, and data was analysed, as in panel (a). ( f ) Growth curve of WT and C888::Broccoli variants in DH10B strain over a standard assay. Representative of multiple experiments. WT, poRiboT2; C888, poRiboT2_C888::insertion. Insertions in panels (b)–(d) and f are Broccoli. In panel (d), the identity of the insertion is given on the x -axis.

Article Snippet: For all plasmid cloning and propagation, Escherichia coli strain DH10B (Δ(ara-leu) 7697 araD139 fhuA ΔlacX74 galK16 galE15 e14-phi80dlacZΔM15 recA1 relA1 endA1 nupG rpsL (StrR) rph spoT1 Δ(mrr-hsdRMS-mcrBC) (New England Biolabs, C3019I) was used.

Techniques: Construct, Mutagenesis, Transformation Assay, Variant Assay, Standard Deviation, Activity Assay, Expressing

Journal: bioRxiv

Article Title: Engineering orthogonal ribosomes for real-time monitoring using fluorescence

doi: 10.1101/2023.11.19.567736

Figure Lengend Snippet: ( a ) Diagram of Broccoli fluorescence with DFHBI-1T binding. ( b ) Broccoli fluorescence can be detected from cells expressing TO-ribosomes with C888::Broccoli insertion, from both a constitutive promoter (phage lambda pL promoter, without repressor) or an IPTG-induced P tac promoter. Cells (DH10B, left panel; DH10B-Marionette, middle and right panel) were transformed with orthogonal ribosome variants (poRiboT2: left panel, or pTac_oRiboT2, middle and right panel, with or without C888::Broccoli insertions), and grown in M9. Orthogonal ribosome expression was either not induced (left, middle panels) or induced with 1mM IPTG (right panel). Broccoli per cell was quantified by normalising green fluorescence per OD700 readings from Broccoli containing samples to matched controls. Plotted data represents the mean, standard deviation, and triplicate data points over time. WT, oRiboT2 construct without Broccoli insertion; C888, constructs containing C888::Broccoli.

Article Snippet: For all plasmid cloning and propagation, Escherichia coli strain DH10B (Δ(ara-leu) 7697 araD139 fhuA ΔlacX74 galK16 galE15 e14-phi80dlacZΔM15 recA1 relA1 endA1 nupG rpsL (StrR) rph spoT1 Δ(mrr-hsdRMS-mcrBC) (New England Biolabs, C3019I) was used.

Techniques: Fluorescence, Binding Assay, Expressing, Transformation Assay, Standard Deviation, Construct

Journal: bioRxiv

Article Title: Engineering orthogonal ribosomes for real-time monitoring using fluorescence

doi: 10.1101/2023.11.19.567736

Figure Lengend Snippet: ( a ) Time course tracking of Broccoli fluorescence under a range of growth conditions suggests TO-ribosome abundance may be affected by bacterial strain, sugar and growth phase. DH10B cells were transformed with orthogonal ribosome variants (poRiboT2 with or without C888::Broccoli insertion), and grown in M9 supplemented with 0.8% fructose or glucose as a carbon source. Broccoli per cell was quantified by normalising green fluorescence per OD700 readings from Broccoli containing samples to matched controls. Plotted data represents the mean, standard deviation, and triplicate data points over time. Data is representative of at least three independent experiments. ( b ) DFHBI-1T tracking over extended time course assays in plate readers. OD426 was monitored over time, and DFHBI-1T concentration was calculated by normalising to media OD426, subtracting the cellular OD426 contribution, and converting the resultant DFHBI-1T OD426 from absorbance to concentration via its extinction coefficient (see Methods ). ( c ) Flow cytometric analysis of ribosome abundance. DH10B cells were grown as in panel (a) and aliquots were removed at 3 h (log phase) and 20 h (stationary phase) for flow cytometric analysis. DFHBI-1T was added to all samples (200 µM) and green fluorescence was quantified for cells with and without Broccoli, the latter used to normalise fluorescence for the former. Data represents the mean and standard deviation of a triplicate dataset. WT, poRiboT2; C888, poRiboT2_C888::Broccoli.

Article Snippet: For all plasmid cloning and propagation, Escherichia coli strain DH10B (Δ(ara-leu) 7697 araD139 fhuA ΔlacX74 galK16 galE15 e14-phi80dlacZΔM15 recA1 relA1 endA1 nupG rpsL (StrR) rph spoT1 Δ(mrr-hsdRMS-mcrBC) (New England Biolabs, C3019I) was used.

Techniques: Fluorescence, Transformation Assay, Standard Deviation, Concentration Assay

Journal:

Article Title: phnE and glpT Genes Enhance Utilization of Organophosphates in Escherichia coli K-12

doi:

Figure Lengend Snippet: Functional map of phnE and glpT genes. All clones and constructs were used to transform the wild-type strain E. coli JA221 and were tested for the ability to confer vigorous growth on plates containing 1 mM DIPP as described in Materials and Methods in order to determine the functional genes. The locations and directions of transcription of the ORFs are indicated by arrows. A plus sign indicates positive growth on DIPP-containing plates, corresponding to the growth of E. coli mutant DB. A minus sign indicates negative growth on DIPP-containing plates, equivalent to the growth of the wild-type strain E. coli JA221 transformed with the control vector. Restriction enzyme cutting sites are indicated as follows: B, BglI; P, PvuII; S, PstI; N, NsiI; H, HindIII; R, EcoRI; and V, EcoRV. (A) phnE: five overlapping clones of genomic DNA obtained from E. coli mutant DB (pB1, pB3, pB12, pE2, and pF1) and three constructs derived from the pF1 clone (pFΔRL, pFΔRR, pFΔF). No sites were found for BamHI, HindIII, and PstI. (B) glpT: genomic clone (pB9) obtained from the DB mutant, a derivative construct (pqTa), and PCR-amplified regions of the glpT gene (PCRpT). No sites were found for BamHI and EcoRI.

Article Snippet: E. coli K-12 strain JA221 (F − hsdM + hsdR lacY leuB6 ΔtrpE5 recA1 λ − ) (ATCC 33875) and the library of Alteromonas haloplanktis 214 variant 3 in E. coli K-12 strain JA221 (ATCC 37436) ( 32 ) were obtained from the American Type Culture Collection (Rockville, Md.).

Techniques: Functional Assay, Clone Assay, Construct, Mutagenesis, Transformation Assay, Plasmid Preparation, Derivative Assay, Amplification

Journal:

Article Title: phnE and glpT Genes Enhance Utilization of Organophosphates in Escherichia coli K-12

doi:

Figure Lengend Snippet: E. coli growth with different DIPP concentrations as the sole P sources. Plates were photographed following 40 h of incubation at 34°C. (A and B) Enhanced growth of OPU mutants with DIPP as the sole P source. Five mutant strains (DB, DC, FB, FC, and FL) and the wild-type strain E. coli JA221 were used. (A) Medium containing 1 mM DIPP. (B) P-deficient and Pi-rich media. The tiny colonies of the wild-type strain on DIPP-containing plates (A) and of both the wild-type strain and mutant DB on P-deficient plates (B) could not be reproduced by photography. (C) OPU phenotype conferred by the multicopy glpT gene is independent of the gene source. The wild-type strain was transformed with the pUC19 vector carrying the PCR-amplified glpT gene from the wild-type JA221 genome (JA), the DB mutant genome (DB), the pB9 clone (pB9), or the pUC19 vector alone (control). Following transformations cells were plated onto medium containing 1 mM DIPP.

Article Snippet: E. coli K-12 strain JA221 (F − hsdM + hsdR lacY leuB6 ΔtrpE5 recA1 λ − ) (ATCC 33875) and the library of Alteromonas haloplanktis 214 variant 3 in E. coli K-12 strain JA221 (ATCC 37436) ( 32 ) were obtained from the American Type Culture Collection (Rockville, Md.).

Techniques: Incubation, Mutagenesis, Transformation Assay, Plasmid Preparation, Amplification

Journal:

Article Title: The Stringent Response of Mycobacterium tuberculosis Is Required for Long-Term Survival

doi:

Figure Lengend Snippet: Strains and plasmids used in this study

Article Snippet: Analysis of cell wall composition and mycolic acid subclass distribution was conducted as previously described ( 80 ). table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Strain or plasmid Characteristics Source or reference Strains E. coli DH5α supE44 ΔlacU169 (Δ80 lacZ ΔM15) hsdR17 recA1 endA1 gyr A96 thi-1 relA1 Laboratory collection M. tuberculosis H37Rv Virulent laboratory strain (ATCC 27294) Laboratory collection SJA14 Single-crossover (SCO) recombinant with pSA1; relA ΔrelA :: hyg lacZ Hyg r This work SJA16 Double-crossover (DCO) recombinant with pSA1 containing integrated copy of pSA1; Δ relA :: hyg lacZ Hyg r This work SJA32 SCO recombinant with pSA3; relA + sacB Hyg r This work SJA33 DCO recombinant obtained with pSA3; Δ relA :: hyg Hyg r This work CDC1551 Virulent M. tuberculosis clinical isolate (CSU93) 68 Plasmids pGEM3Z(+)f E. coli cloning vector; Ap r Promega Pac6 Cosmid from pYUB328::H37Rv carrying relA This work pIJ963 E. coli cloning vector; Ap r Hyg r 29 pSMT3 E. coli-Mycobacterium shuttle vector carrying mycobacterial hsp60 promoter; Hyg r 55 pSMT3 lacZ pSMT3 derivative carrying lacZ cloned in Bam HI site; hsp60-lacZ This work p2NIL E. coli cloning vector with unique Pac I site; Km r 56 pGOAL13 E. coli cloning vector with hsp60-sacB cassette carried on Pac I fragment; Ap r 56 pRelΔS pGEM3Z(+)f derivative carrying Δ relA allele lacking internal Sph I fragment This work pRelΔSH pRelΔS derivative carrying Δ relA marked with hyg gene from pIJ963 inserted in Bgl II site of Δ relA (Δ relA :: hyg ) This work pSA1 pRelΔSH carrying hsp60-lacZ This work pSA3 p2NIL derivative carrying Δ relA :: hyg allele from pRelΔSH and Pac I cassette from pGOAL13; hsp60-sacB ; Km r Hyg r This work pMV306K E. coli-Mycobacterium shuttle vector; integrates into the L5 attB site in Mycobacteria ; Km r 41 pMV306K- rel pMV306K derivative carrying rel Mtb This work pMV306K- hsp60-luc pMV306K carrying the Not I- Sal I luc fragment from pGL3 (Promega) and the Kpn I- Hin dIII groEL promoter from pMV261 16 Open in a separate window Strains and plasmids used in this study Construction of the Δ rel Mtb :: hyg allele.

Techniques: Plasmid Preparation, Recombinant, Cloning, Clone Assay