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1) Product Images from "Designer diatom episomes delivered by bacterial conjugation"
Article Title: Designer diatom episomes delivered by bacterial conjugation
Journal: Nature Communications
Figure Legend Snippet: Demonstration that P. tricornutum episomes replicate as stable, circular, low-copy plasmids ( a – e ), and expression and localization of proteins encoded on the P. tricornutum episome p0521s (F-I). ( a ) Cultures of P. tricornutum containing p0521s (clone 9, Supplementary Fig. 3 ), were subcultured in seawater medium for 28 days with or without antibiotic selection and plated ( Supplementary Fig. 5 ). DNA from five antibiotic-resistant colonies from each culture ( Supplementary Fig. 5D ) was recovered in E. coli and isolated plasmids were separated by agarose gel electrophoresis. Shown are rescued plasmids derived from separate P. tricornutum colonies that were initially subcultured for 28 days without (lanes 1–5) or with (lanes 6–10) antibiotic selection. ‘M' designates supercoiled marker 41 and ‘C' designates the original plasmid (isolated from clone 9) introduced into P. tricornutum . Arrow denotes supercoiled plasmid band. ( b ) Stability of p0521-Se containing a 49-kb S. elongatus fragment. Ten independently transformed P. tricornutum lines containing p0521-Se were subcultured in liquid media with selection for 60 days, followed by episome rescue and separation of plasmids by agarose gel electrophoresis. Arrow denotes supercoiled plasmid band. ( c ) Plasmids extracted from P. tricornutum were untreated, treated with exonuclease, ClaI endonuclease or a combination of exonuclease and ClaI. Treated plasmids were transformed into E. coli and the number of transformed colonies was plotted (error bars indicate one s.d. of the mean from three biological replicates). ( d ) Agarose gel electrophoresis of plasmids extracted from P. tricornutum and treated with nucleases. Lanes from left to right: (1) 1 kb + ladder (NEB), (2) p0521s control (from E. coli ), (3) p0521s exonuclease-treated (extracted from P. tricornutum ), (4) p0521s untreated (extracted from P. tricornutum ), (5) 1 kb + ladder (NEB), ( e ) Copy number of p0521s in P. tricornutum determined by qPCR. Cm ( Cat gene) and His ( HIS3 gene) are loci found on the episome backbone; Ure (urease, protein ID 29702) and NR (nitrate reductase, protein ID 54983) are loci encoded on P. tricornutum nuclear chromosomes 18 and 20, respectively; Rbc (RuBisCO small subunit) and CytB (Cytochromoe B) are loci found on the P. tricornutum chloroplast and mitochondrial chromosomes, respectively. Error bars denote one s.d. of the mean from three biological replicates. ( f ) Wild-type P. tricornutum , fluorescence measured with GFP settings. ( g ) P. tricornutum expressing CFP translationally fused to beta-carbonic anhydrase (Protein ID 51305) localized to the chloroplast pyrenoid encoded on plasmid p0521s. ( h ) P. tricornutum expressing YFP translationally fused to mitochondrial urea transporter (Protein ID 39772) encoded on plasmid p0521s. ( i ) P. tricornutum expressing GFP localized to the cytoplasm encoded on plasmid p0521s. Scale bar for f – i indicates 5 μm.
Techniques Used: Expressing, Selection, Isolation, Agarose Gel Electrophoresis, Derivative Assay, Marker, Plasmid Preparation, Transformation Assay, Real-time Polymerase Chain Reaction, Fluorescence
2) Product Images from "Transduction of human embryonic stem cells by ecotropic retroviral vectors"
Article Title: Transduction of human embryonic stem cells by ecotropic retroviral vectors
Journal: Nucleic Acids Research
Figure Legend Snippet: Transduction of hES cells with murine retroviral vectors. ( A ) Transfection efficiencies of undifferentiated hES cells using optimized protocols for lipofection (L), electroporation (E) and nucleofection (N) techniques. ( B ) mCAT1-HA expression (green) in nucleofected hES cells cultured on matrigel. Twenty-four hours after plating, the cells show a flattened morphology typical for colonies propagated on matrigel. Nuclear expression of Oct-4 (red) reflects their undifferentiated state. ( C ) Schematic illustration of the two-step protocol used for transduction of hES cells with ecotropic retroviral vectors. First cells are nucleofected with a construct encoding the murine retrovirus receptor mCAT1. Twenty-four hours later they are transduced with a murine retroviral vector. Transduced cultures are either analyzed for transgene expression after 48 h or subjected to selection of permanently transduced clones. The integrated provirus expresses the EGFP transgene from the viral LTR linked to a neomycin resistance gene (neoR) by an internal ribosome entry site (IRES). ( D ) Forty-eight hours after infection, 16.4 ± 5.9% of the total cell population showed EGFP-expression. Normalized to the proportion of mCAT1-expressing cells determined in (A), this corresponds to a calculated transduction efficiency of ∼30% of the mCAT1-expressing cells (mean values from n = 5 independent experiments). ( E – G ) Transduced EGFP-positive cells continue to express the pluripotency-associated markers Oct-4 (E), Tra-1-60 (F) and Tra-1-81 (G) (all red; counterstain DAPI). ( H ) Five passages after transduction, four clones were subjected to Southern analysis. A single integration of the EGFP transgene could be detected in clones PK2, PK4 and PK7. Clone PK5 displays two bands, which could be the result of a double integration, a mixed clone population or a mutation of the vector provirus. Restriction analysis was performed with EcoRI (for genomic DNA) and ClaI (unique site within the retroviral vector). Scale bars: B,E: 100 μm; F: 30 μm; G: 50 μm.
Techniques Used: Transduction, Transfection, Electroporation, Expressing, Cell Culture, Construct, Plasmid Preparation, Selection, Clone Assay, Infection, Mutagenesis
3) Product Images from "Biochemical reconstitution of abasic DNA lesion replication in Xenopus extracts"
Article Title: Biochemical reconstitution of abasic DNA lesion replication in Xenopus extracts
Journal: Nucleic Acids Research
Figure Legend Snippet: Determination of the nucleotides opposite the AP site in the final replication products (after 75 min of incubation in NPE). ( A ) Restriction digestion of the gel-purified supercoiled final replication products (detected by 32 P) and the control pET28a DNA (detected by SYBR Gold). R: relaxed; L: linear; S: supercoiled. ( B ) Transformation efficiency of pET28a (kan R ; expressed in percentages of the number of transformants of the uncut DNA) and AP DNA (amp R ; expressed in absolute colony numbers). ( C ) The nucleotides found at the position opposite the AP lesion in the plasmids isolated from the transformants of the DpnI and ClaI-digested Δ:C and Δ:G replication products. ( D ) The average ratios and absolute deviations of each nucleotide inserted at the position opposite the AP lesion for the Δ:C and Δ:G replication products. The data were from two independent experiments for each substrate. The right-most column listed the expected ratios if the AP lesion was replicated by random insertion of the 4 nt.
Techniques Used: Incubation, Purification, Transformation Assay, Isolation
Figure Legend Snippet: Analysis of the replication products that still carried the AP lesion (after 75 min of incubation in NPE). ( A ) Six potential types of DNA and their sensitivity to various enzymes. The lesion-carrying DNA would be nicked on the AP strand but intact on the complementary strand. BER: base excision repair; H: non-G; D: non-C. ( B ) Sequence analysis of the cloned PCR products amplified from Δ:G replication products that had been digested with DpnI, ClaI, APE and KpnI. ( C ) Sequence analysis of the cloned PCR products amplified from Δ:T replication products that had been digested with DpnI, ClaI, APE and KpnI.
Techniques Used: Incubation, Sequencing, Clone Assay, Polymerase Chain Reaction, Amplification