Journal: Microbial Biotechnology

Article Title: Unlocking the strength of inducible promoters in Gram‐negative bacteria

doi: 10.1111/1751-7915.14219

Figure Lengend Snippet: Comparison of sfGFP and mCardinal emissions in Gram‐negative bacteria. (A) Absolute values of fluorescence measured in arbitrary units with far red wavelengths (excitation 604, emission 659, left panel), and green wavelengths (excitation 485, emission 510) E. coli DH10B (red), P. putida (green) and V. natriegens (blue). Note that these strains are wild type and do not contain any fluorescent protein genes. N = 3. Error bars ± SD. (B–D) Fluorescence signal‐to‐background ratio of recombinant strains expressing sfGFP (green lines) and mCardinal (red lines) from the constitutive tacI promoter (these represent ‘signal’) versus wild‐type strains (black lines) of (B) E. coli (C) P. putida and (D) V. natriegens (these wild‐type strains represent ‘background’). Signal‐to‐background measurement ratios are then plotted over time (left panels) The fluorescence values (in arbitrary units, AU) measured to create the signal‐to‐background ratios are shown in the middle (sfGFP) and right (mCardinal) panels. N = 3. Error bars ± SD

Article Snippet: E. coli DH10B, P. putida JE90 derivative of KT2440 with BxB1int‐attB site (Elmore et al., ) and V. natriegens Vmax X2 (Codex DNA, Inc.) were used to evaluate the synthetic lac and tet promoters.

Techniques: Comparison, Bacteria, Fluorescence, Recombinant, Expressing

Journal: Microbial Biotechnology

Article Title: Unlocking the strength of inducible promoters in Gram‐negative bacteria

doi: 10.1111/1751-7915.14219

Figure Lengend Snippet: Time courses of mCardinal production in V. natriegens . Synthetic lac (A) and tet (B) promoters in their constitutive, repressed and induced states. (C) Direct comparison of each promoter under evaluation against the constitutive promoter tacI in V. natriegens . V. natriegens carrying tacI‐mCardinal was normalized to 100% mCardinal production and the wild‐type V. natriegens strain normalized to 0% mCardinal production. Green was used to indicate the highest expressing conditions among the samples, red for lowest expression, and yellow for intermediate expression (the hues approximate the range of expression). For all samples, the fluorescence mean of mCardinal signal (excitation 605, emission 659) was normalized by the cell density (OD 600 ). N = 4. Error bars ± SD

Article Snippet: E. coli DH10B, P. putida JE90 derivative of KT2440 with BxB1int‐attB site (Elmore et al., ) and V. natriegens Vmax X2 (Codex DNA, Inc.) were used to evaluate the synthetic lac and tet promoters.

Techniques: Comparison, Expressing, Fluorescence

Journal: Scientific Reports

Article Title: CRISPR/Cas9 nickase-mediated disruption of hepatitis B virus open reading frame S and X

doi: 10.1038/srep13734

Figure Lengend Snippet: ( A ) Schematic representation of the hepatitis B virus genome. Relaxed circular DNA (rcDNA) of the HB virion (thin continuous line), which is converted to cccDNA (thin continuous and dotted line) following hepatocyte infection, is indicated in the centre of the map. The four viral transcripts of the core (C), polymerase (P), and surface (S) and X proteins are indicated around the outside. Regions targeted by Cas9n via guide RNA (gRNA) specific to S and X sequences are indicated by arrows and scissors (scissors were drawn by Niklas Beschorner). ( B ) DNA sequence and sequence conservation of the regions targeted by Cas9n within the S and X gene of HBV. The sequence shown is based on genotype A consensus. Target sequences in ORF S and X are depicted (S1 and S2, or X1 and X2), each encompassing proto-spacer adjacent motifs (PAM, bold and boxed), 2 × 20 nucleotides complementary to gRNA (boxed) and offset distance between the two sequences complementary to gRNA (underlined).

Article Snippet: Cultures were grown to ~ 70–80% confluence and subsequently transfected with a total of 1.5 μg plasmid DNA (i.e. RFP-GFP reporter constructs together with the expression plasmids Cas9n/sgRNA-S1 and Cas9n/sgRNA-S2, or Cas9n/sgRNA-X1 and Cas9n/sgRNA-X2, respectively) using Lipofectamin 2000 (Invitrogen) according to the manufacturer’s protocol.

Techniques: Virus, Infection, Sequencing

Journal: Scientific Reports

Article Title: CRISPR/Cas9 nickase-mediated disruption of hepatitis B virus open reading frame S and X

doi: 10.1038/srep13734

Figure Lengend Snippet: ( A ) T7EI assays were performed using PCR primers (indicated by arrows) flanking the HBV S or X sequence in the respective reporter plasmid. ( B ) Detection of Cas9n-specific activity was visualized by gel electrophoresis. HEK293 cells were transfected as before and total genomic DNA was isolated at 72 h post transfection for subsequent T7EI cleavage. Arrows depict the sizes of wild-type and Cas9n-mutagenized DNA fragments. ( C ) T7EI assay using genomic DNA from GFP + HEK293 cell cultures at 24 h post transfection. ( D ) Sequence analysis of corresponding DNA samples. Alignment to the wild-type ORF S reporter sequence is shown. gRNA sequences (boxed), PAM (boxed and bold) and Cas9n-mediated deletions are indicated.

Article Snippet: Cultures were grown to ~ 70–80% confluence and subsequently transfected with a total of 1.5 μg plasmid DNA (i.e. RFP-GFP reporter constructs together with the expression plasmids Cas9n/sgRNA-S1 and Cas9n/sgRNA-S2, or Cas9n/sgRNA-X1 and Cas9n/sgRNA-X2, respectively) using Lipofectamin 2000 (Invitrogen) according to the manufacturer’s protocol.

Techniques: Sequencing, Plasmid Preparation, Activity Assay, Nucleic Acid Electrophoresis, Transfection, Isolation, T7EI Assay