Rabbit Anti-mCherry (a.a. 1-235) Polyclonal antibody

Reconciling transcription and DNA replication in the growing hyphae of the filamentous bacterium Streptomyces presents several physical constraints on growth due to their apically extending and branching, multigenomic cells and chromosome replication being independent of cell division. Using a GFP translational fusion to the beta'-subunit of RNA polymerase (rpoC-egfp), in its native chromosomal location, we observed growing Streptomyces hyphae using time-lapse microscopy throughout the life-cycle and under different growth conditions. The RpoC-eGFP fusion co-localized with DNA around 1.8 mu m behind the extending tip, whereas replisomes localize around 4-5 mu m behind the tip, indicating that at the growing tip, transcription and chromosome replication are to some degree spatially separated. Dual-labelled RpoC-egfp/DnaN-mCherry strains also indicate that there is limited co-localization of transcription and chromosome replication at the extending hyphal tip. This likely facilitates the use of the same DNA molecule for active transcription and chromosome replication in growing cells, independent of cell division. This represents a novel, but hitherto unknown mechanism for reconciling two fundamental processes that utilize the same macromolecular template that allows for rapid growth without compromising chromosome replication in filamentous bacteria and may have implications for evolution of filamentous growth in micro-organisms, where uncoupling of DNA replication from cell division is required.

Whole-cell biosensors (WCBs) have been designed to detect As(III), but most suffer from poor sensitivity and specificity. In this paper, we developed an arsenic WCB with a positive feedback amplifier in Escherichia coli DH5 alpha. The output signal from the reporter mCherry was significantly enhanced by the positive feedback amplifier. The sensitivity of the WCB with positive feedback is about 1 order of magnitude higher than that without positive feedback when evaluated using a half-saturation As(III) concentration. The minimum detection limit for As(III) was reduced by 1 order of magnitude to 0.1 AM, lower than the World Health Organization standard for the arsenic level in drinking water, 0.01 mg/liter or 0.13 mu M. Due to the amplification of the output signal, the WCB was able to give detectable signals within a shorter period, and a fast response is essential for in situ operations. Moreover, the WCB with the positive feedback amplifier showed exceptionally high specificity toward As(III) when compared with other metal ions. Collectively, the designed positive feedback amplifier WCB meets the requirements for As(III) detection with high sensitivity and specificity. This work also demonstrates the importance of genetic circuit engineering in designing WCBs, and the use of genetic positive feedback amplifiers is a good strategy to improve the performance of WCBs. IMPORTANCE Arsenic poisoning is a severe public health issue. Rapid and simple methods for the sensitive and specific monitoring of arsenic concentration in drinking water are needed. In this study, we designed an arsenic WCB with a positive feedback amplifier. It is highly sensitive and able to detect arsenic below the WHO limit level. In addition, it also significantly improves the specificity of the biosensor toward arsenic, giving a signal that is about 10 to 20 times stronger in response to As(III) than to other metals. This work not only provides simple but effective arsenic biosensors but also demonstrates the importance of genetic engineering, particularly the use of positive feedback amplifiers, in designing WCBs.