Recent advances in cell-free synthetic biology have spurred the development of in vitro molecular diagnostics that serve as effective alternatives to whole-cell biosensors. However, cell-free sensors for detecting manmade organic water contaminants such as pesticides are sparse, partially because few characterized natural biological sensors can directly detect such pollutants. Here, we present a platform for the cell-free detection of one critical water contaminant, atrazine, by combining a previously characterized cyanuric acid biosensor with a reconstituted atrazine-to-cyanuric acid metabolic pathway composed of several protein-enriched bacterial extracts mixed in a one pot reaction. Our cell-free sensor detects atrazine within an hour of incubation at an activation ratio superior to previously reported whole-cell atrazine sensors. We also show that the response characteristics of the atrazine sensor can be tuned by manipulating the component ratios of the cell-free reaction mixture. Our approach of utilizing multiple metabolic steps, encoded in protein-enriched cell-free extracts, to convert a target of interest into a molecule that can be sensed by a transcription factor is modularly designed, which should enable this work to serve as an effective proof-of-concept for rapid fielddeployable detection of complex organic water contaminants.
14Recent advances in cell-free synthetic biology have spurred the development of in vitro 15 molecular diagnostics that serve as effective alternatives to whole-cell biosensors. However, 16 cell-free sensors for detecting manmade organic water contaminants such as pesticides are 17 sparse, partially because few characterized natural biological sensors can directly detect such 18 pollutants. Here, we present a platform for the cell-free detection of one critical water 19 contaminant, atrazine, by combining a previously characterized cyanuric acid biosensor with a 20 reconstituted atrazine-to-cyanuric acid metabolic pathway composed of several protein-enriched 21 bacterial extracts mixed in a one pot reaction. Our cell-free sensor detects atrazine within an 22 hour of incubation at an activation ratio superior to previously reported whole-cell atrazine 23 sensors. We also show that the response characteristics of the atrazine sensor can be tuned by 24 manipulating the component ratios of the cell-free reaction mixture. Our approach of utilizing 25 multiple metabolic steps, encoded in protein-enriched cell-free extracts, to convert a target of 26 interest into a molecule that can be sensed by a transcription factor is modularly designed, 27 which should enable this work to serve as an effective proof-of-concept for rapid field-28 deployable detection of complex organic water contaminants. 29 30 KEYWORDS 31 cell-free, TX-TL, metabolism, transcription factor, biosensor, atrazine, cyanuric acid, synthetic 32 biology 33 34 INTRODUCTION 35Cell-free gene expression (CFE) has recently emerged as a powerful strategy for rapid, 36 field-deployable diagnostics for nucleic acids 1-5 and chemical contaminants. 6-9 One reason for 37 this success is that CFE reactions minimize many of the constraints of whole-cell sensors, 38including mass transfer barriers, cytotoxicity, genetic instability, plasmid loss, and the need for 39 biocontainment. 8,10 In addition, CFE reactions can be stabilized through freeze-drying and then 40 are activated upon rehydration, enabling the biosensors to be used outside the laboratory at the 41 point of sampling in the field. 1 However, previously reported cell-free biosensors have so far 42 been limited to detecting either nucleic acids 2,3 or chemical contaminants that can be directly 43 sensed with well-characterized allosteric protein transcription factors or riboswitches. 8,9,[11][12][13] 44 2 Here, we expand the ability of cell-free biosensors to detect complex organic molecules 45 by developing a combined metabolism and biosensing strategy. Our strategy is motivated by the 46 observation that the space of known natural transcription factors may be insufficient to directly 47 detect organic molecules of analytical interest, especially those that are man-made and 48 relatively new to natural environments. On the other hand, a wealth of metabolic biochemistry 49 often exists that could convert a target molecule of interest into a compound that can be directly 50 sensed by a transcription factor. T...
A mechanical obstruction is not a physiological entity, and when it occurs within the 30-day postoperative period, it is called an early postoperative small bowel obstruction. Kinking of small bowel segments at the ileostomy outlet secondary to a distended bladder is an unusual source of early postoperative small bowel obstruction. A 36-year-old female underwent a redo J-Pouch surgery and creation of loop ileostomy after pouch failure related to recurrent small bowel obstruction and perianal fistulae. Her foley catheter was removed on postoperative day 3 and she passed a trial of void test. On postoperative day 6, the abdomen became progressively more distended. Computerized tomography (CT) imaging with IV contrast showed small bowel distension extending to the midline anterior to the urinary bladder where it demonstrated a narrowed lumen. These findings were thought to be the cause of small bowel obstruction at this level before the ileostomy. Immediately after CT, a foley catheter was applied with which 2 L of urine was removed, and consequently, gas and stool were observed in the ostomy soon thereafter. Although rare, urinary retention may cause intestinal obstruction, especially in the presence of a loop ileostomy in close proximity.
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