Journal: Nature Communications

Article Title: Evolution of a General RNA-Cleaving FANA Enzyme

doi: 10.1038/s41467-018-07611-1

Figure Lengend Snippet: FANA transcription and reverse transcription in vitro. a Constitutional structures for 2’-deoxyribonucleic acid (DNA) and 2’-fluoroarabino nucleic acid (FANA). b FANA transcription activity for wild-type archaeal DNA polymerases (exo−) from 9°N, DV, Kod, and Tgo (left panel). Samples were analyzed after 15 and 30 min at 55 °C. FANA reverse transcriptase activity of Bst DNA polymerase LF, 2.0, 3.0, and LF* (right panel). LF* denotes wild-type Bst DNA polymerase, large fragment, expressed and purified from E. coli . Samples were analyzed after 30 min at 50 °C. All samples were resolved on denaturing PAGE and visualized using a LI-COR Odyssey CLx. c Fidelity profile observed for FANA replication using Tgo and Bst LF* polymerases. The mutation profile reveals a mutation rate of 8 × 10 -4 and an overall fidelity of ~99.9%. d Catalytic rates observed for FANA synthesis with Tgo (left panel) and reverse transcription with Bst LF* (right panel)

Article Snippet: After 12 rounds of selective amplification, cDNA amplicons were cloned into E. coli DH5α cells for Sanger sequencing by Retrogen (San Diego, CA).

Techniques: In Vitro, Activity Assay, Purification, Mutagenesis

Journal: Nucleic Acids Research

Article Title: The CRISPR ancillary effector Can2 is a dual-specificity nuclease potentiating type III CRISPR defence

doi: 10.1093/nar/gkab073

Figure Lengend Snippet: Can2 protects against MGE in vivo . ( A ) Plasmid challenge assay of mycobacterial type III-A system in E. coli host. E. coli cells harbouring the mycobacterial type III-A interference complex Csm1-5 and TetR targeting spacer were transformed with plasmid containing TsuCan2 effector and tetracycline-resistance gene (Wild-type). Other strains are indicated as ‘No Can2’ where the TsuCan2 is absent, ‘Cyclase variant’ where the interference complex is unable to produce cOA and ‘No targeting crRNA’ where the TetR targeting spacer is replaced with a spacer targeting pUC19 MCS. A 10-fold dilution series of the transformation mixture was applied onto tetracycline selective plates to determine the number of viable transformants. ( B ) Growth curves of E. coli cells harbouring the interference complex and phage P1 lpa gene-targeting spacer supplemented with TsuCan2 effector (indicated as Wild-type). Cells were grown in LB broth in a 96-well plate with shaking at 37°C and infected with phage P1 at a MOI of ∼1. OD 595 of the culture was measured every 15 min to plot against time over the 16 h incubation. Other strains are indicated as ‘Cyclase variant’ (green) where the interference complex is unable to produce cOA molecules; ‘No Lpa target’ (black) where lpa gene-targeting spacer is replaced with a spacer targeting pUC19 (MCS); ‘Can2 E302A/K304A’ (grey) which is a TsuCan2 nuclease variant. ‘Control’ (purple) represents wild-type cells (orange) incubated without phage infection under the same conditions. Data points represent the mean of eight experimental replicates (four biological replicates with two technical replicates each) with the standard deviation shown. ( C ) The OD 595 values of all strains after 4 and 10 h growth are shown, coloured as in panel B. Statistical analysis was carried out with RStudio using the unpaired Welch two sample test to calculate P -values. NS (not significant) indicates P -values > 0.05 and *** indicates P -values < 1E–05.

Article Snippet: Competent DH5α ( E. coli ) cells were transformed with the construct and sequence integrity was confirmed by sequencing (GATC Biotech, Eurofins Genomics, DE).

Techniques: In Vivo, Plasmid Preparation, Transformation Assay, Variant Assay, Infection, Incubation, Control, Standard Deviation