Improving Cell‐Free Protein Synthesis through Genome Engineering of Escherichia coli Lacking Release Factor 1

Site‐specific incorporation of non‐standard amino acids (NSAAs) into proteins opens the way to novel biological insights and applications in biotechnology. Here, we describe the development of a high yielding cell‐free protein synthesis (CFPS) platform for NSAA incorporation from crude extracts of genomically recoded Escherichia coli lacking release factor 1. We used genome engineering to construct synthetic organisms that, upon cell lysis, lead to improved extract performance. We targeted five potential negative effectors to be disabled: the nuclease genes rna, rnb, csdA, mazF, and endA. Using our most productive extract from strain MCJ.559 (csdA^? endA^?), we synthesized 550±40 μg mL^?1 of modified superfolder green fluorescent protein containing p‐acetyl‐L ‐phenylalanine. This yield was increased to ～1300 μg mL^?1 when using a semicontinuous method. Our work has implications for using whole genome editing for CFPS strain development, expanding the chemistry of biological systems, and cell‐free synthetic biology.     

Serotonergic and cholinergic involvement in habituation of activity and spontaneous alternation of rats in a maze.

Studied the effects of drug administration to 220 male Sprague-Dawley rats on spontaneous alternation (SA) in a -maze which Ss were permitted to freely explore for 8-min sessions. Results show that SA was not affected by administration of methysergide or the serotonin (5-HT) depletors DL-chloroamphetamine (PCA) and DL-p-chlorophenylalanine (PCPA). However, either LSD, or d-amphetamine in combination with methysergide, PCA, or PCPA interfered with SA. Scopolamine also disrupted SA, but pretreatment with 5-hydroxytryptophan or amphetamine blocked this action. Amphetamine reversal of the scopolamine-induced disruption of SA did not occur in Ss depleted of 5-HT or pretreated with methysergide. Amphetamine disrupted habituation of exploratory activity alone or after PCPA or PCA. PCPA or PCA alone did not affect habituation. Scopolamine interfered with habituation of activity. Methysergide caused an increase in the initial activity level, while LSD produced a dose-dependent decrease. (34 ref.) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Seroprevalence of human granulocytic ehrlichiosis among permanent residents of northwestern Wisconsin

Four-hundred seventy-five permanent residents of Wisconsin were tested for antibodies to the agent of human granulocytic ehrlichiosis (HGE) by indirect immunofluorescent antibody (IFA) testing with Ehrlichia equi as antigen marker. Each resident completed a standard survey questionnaire about outdoor activities, animal and tick exposure, and any febrile illness during the preceding 12 months. Seventy-one serum samples (14.9%) contained E. equi antibodies. The mean IFA titer for seropositive residents was 250 (range, 80-10,240). Seropositive residents were older than seronegative ones (62 vs. 56 years; P = .019). None of the seropositive residents had a history suggestive of ehrlichiosis. There was no association between the IFA test outcome and specific demographic variables or history of tick bites. HGE appears to be a common subclinical or mild infection among residents in northwestern Wisconsin.