Journal:

Article Title: Cloning and Expression of the Lactococcus lactis purDEK Genes, Required for Growth in Milk

doi:

Figure Lengend Snippet: Strain and plasmid list

Article Snippet: As purine sources the following were added at the indicated final concentrations: adenine (15 mg/liter) hypoxanthine (15 mg/liter), and guanosine (30 mg/liter). table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Strain or plasmid Genotype and/or relevant feature(s) Source or reference L. lactis CHCC285 Wild type 21 MG1363 Plasmid free 9 MG1614 MG1363 Rif r Str r 9 E. coli DH5α Φ80 lacZΔM15 Δ( lacZYA-argF ) U169 recA1 endA1 hsdR17 supE44 thi-1 gyrA96 relA1 10 LN3 SØ609/λZAPII- purDEK This study SØ609 ara thi rpsL Δ( pro-gpt-lac ) hpt deoD purD 13 Plasmids pBluescript KS(+/−) Cloning vector Ap r Stratagene pIC19H Cloning vector Ap r ATCC 37408 b pAK80 Ery r ; promoterless lacLM 11 pLN48 7.2-kb Kpn I fragment from LN3 inserted into pBluescript KS(−) This study pLN51 3.2-kb Kpn I- Spe I fragment from pLN48 inserted into pBluescript KS(+) This study pLN67 0.9-kb Eco RI fragment from pLN51 inserted into pBluescript KS(+) This study pLN71 0.9-kb Hin dIII fragment from pLN67 ( purD ′) fused to lacLM in pAK80 This study pLN72 0.9-kb Hin dIII fragment from pLN67 ( orf1 ′) fused to lacLM in pAK80 This study p10 a Exonuclease III deletion derivative of pLN71 This study pLN82 0.9-kb Eco RI fragment from pLN67 inserted in pIC19H This study pLN84 to pLN90 Exonuclease III deletion derivatives of pLN82 This study pLN93 to pLN99 Hin dIII- Bam HI fragments from pLN84-90 fused to lacLM in pAK80 This study Open in a separate window a p19, p22, p27, and p30 are also exonuclease III deletion derivatives of pLN71 (this study). b American Type Culture Collection, Rockville, Md.

Techniques: Plasmid Preparation, Cloning

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: Infection and Immunity

Article Title: Cj1136 Is Required for Lipooligosaccharide Biosynthesis, Hyperinvasion, and Chick Colonization by Campylobacter jejuni

doi: 10.1128/IAI.00151-12

Figure Lengend Snippet: Bacterial strains and plasmids

Article Snippet: Culture media were also supplemented with antibiotics, including ampicillin (100 μg ml −1 ), kanamycin (50 μg ml −1 ), and chloramphenicol (20 μg ml −1 ), where appropriate. table ft1 table-wrap mode="anchored" t5 caption a7 Strain or plasmid Description Source or reference Strains C. jejuni 01/51 Hyperinvasive wild-type strain 01/51 11 01/51 Δ cj1136 cj1136 mutation generated in strain 01/51 This study 01/51 Δ cj1136 :: cj1136 cj1136 mutation complemented by wild-type copy of the gene in strain 01/51 This study 81116 Δ( flaA flaB ) Double mutation in flaA and flaB genes in strain 81116 52 E. coli TOP10F′ F′ lacI q Tn10(Tet r ) mcrA Δ( mrr-hsdRMS-mcrBC ) φ80 lacZ ΔM15 Δ lacX74 recA1 araD139 Δ( ara-leu ) 7697 galU galK rpsL endA1 nupG Invitrogen Plasmids pGEM-T Easy Cloning vector encoding resistance to ampicillin Promega pGEM:1136::kan pGEM-T Easy vector carrying 1.3-kb fragment of cj1136 interrupted by Kan r cassette This study pAJ20 2.3-kb DNA fragment flanking the intergenic region between cj0652 and cj0653c with additional BamHI restriction site in the intergenic region, cloned into pGEM-T Easy vector This study pAJ22 Chloramphenicol resistance cassette cloned into pAJ20 This study pAJ22-1136 Wild-type copy of cj1136 with its own promoter cloned into pAJ22 vector This study pMA24 Source of kanamycin resistance cassette 1 pUOA18 Source of chloramphenicol resistance cassette 51 Open in a separate window Bacterial strains and plasmids

Techniques: Plasmid Preparation, Mutagenesis, Generated, Cloning, Clone Assay

Journal: Nucleic Acids Research

Article Title: Short tandem repeat stutter model inferred from direct measurement of in vitro stutter noise

doi: 10.1093/nar/gky1318

Figure Lengend Snippet: The synthetic STR experiment summary. ( A ) Schematic description of the synthetic library. In each plasmid, a different synthetic STR construct was designed, synthesized and clone-sequenced for various STR types and length. The STR was designed within a context of an Illumina Truseq-HT dual index library to enable for nested PCR amplification at two time points (T 2 - amplification using outer primers only, T 3 -amplification using inner primers followed amplification by outer primers). The library is flanked by BsrDI restriction sites to enable direct sequencing of the STR library without amplification (T 1 ). Internal barcode (yellow triangle) is a short sequence, unique to each STR length to detect for cross-contamination. See text and methods for elaboration and for the designed constructs. ( B ) AC STRs repeat-number histograms, as were interpreted from sequencing results (T 1 , T 2 and T 3 ), compared to their expected length, T 0 (designed sequence). ( C ) Sequencing analysis results of each STR type, repeat-number and time point described as the percentage of the original (designed) signal from all the reads. Dashed line at the 5% marks the lower threshold of analysis: data points below the mark were deemed too noisy and were excluded from downstream analysis.

Article Snippet: STR plasmid design: Sequence verified cloned plasmids containing synthetic STRs of different types and sizes ( ) were ordered from either IDT or GenScript (pIDT-kan and modified puc57-Kan vectors, respectively).

Techniques: Plasmid Preparation, Construct, Synthesized, Nested PCR, Amplification, Sequencing

Journal: The Journal of Biological Chemistry

Article Title: A formamidopyrimidine derivative from the deoxyguanosine adduct produced by food contaminant acrylamide induces DNA replication block and mutagenesis

doi: 10.1016/j.jbc.2023.105002

Figure Lengend Snippet: Schematic diagram of the intracellular TLS assay using a shuttle vector. dG, dfG, and GA-FAPy-dfG were introduced at a specific position (denoted as X) on one side of the shuttle vector (modified strand), whereas the other strand (unmodified strand) had a three-base mismatch ( underlined ) for the GA-FAPy-dfG and SmiI recognition sequence ( blue box ). The vector was transfected into XP4PASV cells and allowed to replicate for 48 h. Progeny plasmids were then recovered and digested with DpnI to remove nonreplicated original DNA. Escherichia coli was transformed with progeny plasmids, and resultant clones were subjected to PCR to amplify the lesion site. The PCR products were treated with SmiI to digest unmodified strand progeny. Replication efficiency was calculated from the ratio of the modified strand progeny in the recovered plasmids and is shown relative to dG. Mutation spectra were analyzed using Sanger sequencing of the PCR products of the modified strand progeny. GA-FAPy-dfG, N 6 -(2-deoxy-2-fluoro- d -arabinofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-[ N -(2-carbamoyl-2-hydroxyethyl)formamido]pyrimidine; TLS, translesion DNA synthesis.

Article Snippet: NEB10β electrocompetent E. coli ( araD 139 Δ( ara-leu )7697 fhuA lacX74 galK (φ80 Δ( lacZ ) M15 ) mcrA galU recA1 endA1 nupG rpsL (Str r ) Δ [ mrr-hsdRMS-mcrBC ]; New England Biolabs) was transformed with the progeny plasmids and plated on 1× YT plates containing 100 μg/ml carbenicillin (Sigma–Aldrich) and 50 μg/ml blasticidin S (Kaken Pharmaceutical).

Techniques: Plasmid Preparation, Modification, Sequencing, Transfection, Transformation Assay, Clone Assay, Mutagenesis, DNA Synthesis