Microbial microfluidic biosensor

Abstract

Provided are a microfluidic biosensors that are suitable for continuously monitoring toxin levels in water supplies.

1 .- 89 . (canceled) 
     
     
         90 . A plurality of recombinant microbial cell strains, each strain comprising a different expression cassette comprising a biosensor or promoter operably linked to a polynucleotide encoding a detectable agent, wherein transcription of the biosensor or promoter is modulated by the presence of a heavy metal analyte. 
     
     
         91 . The plurality of recombinant microbial cell strains of  claim 90 , wherein the heavy metal analyte is selected from the group consisting of arsenic, cadmium, chromium VI, cobalt, copper, lead, mercury, and zinc. 
     
     
         92 . The plurality of recombinant microbial cell strains of  claim 90 , wherein the biosensor or promoter is selected from the group consisting of ParsR (arsenic), PcadC (cadmium), PcadR (cadmium), PzntA (cadmium), PchrB (chromium VI), PchrS (chromium VI), PrecN (chromium VI), PsulA (chromium VI), PumuD (chromium VI), PdadA (cobalt), Phmp (cobalt), PilvB (cobalt), PlipA (cobalt), PmmuP (cobalt), PnmtR (cobalt), PsoxR (cobalt), PtehA (cobalt), PygbA (cobalt), PyjbJ (cobalt), PyqfA (cobalt), PcopA (copper), PcusC (copper), PcusR (copper), PpbrR (lead), PmntH (lead), PshiA (lead), PybiI (lead), PyjjZ (lead), PmerR (mercury), PmntH (zinc), PshiA (zinc), PyjjZ (zinc), PzntA (zinc), and PzraP (zinc). 
     
     
         93 . The plurality of recombinant microbial cell strains of  claim 90 , wherein the detectable agent is a nucleic acid, detectable protein, antibody-linked reporter protein, enzymatic assay product, or electrochemical reaction product. 
     
     
         94 . The plurality of recombinant microbial cell strains of  claim 90 , wherein the detectable agent is a fluorescent protein or a luminescent protein. 
     
     
         95 . The plurality of recombinant microbial cell strains of  claim 90 , wherein the microbial cell is a bacterium, cyanobacterium, a fungus, a microalga, or an alga. 
     
     
         96 . The plurality of recombinant microbial cell strains of  claim 95 , wherein the bacterium is selected from the group consisting of  Escherichia coli, Bacillus subtilis, Salmonella  sp.,  Aliivibrio fischeri, Pseudomonas fluorescens, Bacillus  sp.,  Cupriavidus metallidurans, Deinococcus radiodurans , and  Staphylococcus aureus.    
     
     
         97 . The plurality of recombinant microbial cell strains of  claim 95 , wherein the fungus is selected from the group consisting of  Saccharomyces cerevisiae  and  Trichosporon cutaneum.    
     
     
         98 . The plurality of recombinant microbial cell strains of  claim 95 , wherein the cyanobacterium is  Synechocystis  sp. 
     
     
         99 . A method of detecting the presence of a heavy metal analyte of interest, comprising:
 (a) culturing, in a defined array, the plurality of recombinant microbial cell strains of  claim 90 , each strain of the plurality of recombinant microbial cell strains, comprising a different expression cassette comprising a biosensor or promoter operably linked to a polynucleotide encoding a detectable agent, wherein transcription of the biosensor or promoter is modulated by the presence of a heavy metal analyte of interest;   (b) delivering an aqueous sample suspected of comprising the heavy metal analyte of interest to each strain of the plurality of recombinant microbial cell strains in the defined array under environmental conditions permitting modulated expression of the detectable agent in the presence of the heavy metal analyte of interest;   (c) measuring in the defined array, an amount of the detectable agent, corresponding to a quantifiable level of the heavy metal analyte of interest; and   (d) quantifying the quantifiable level of the heavy metal analyte of interest in the aqueous sample corresponding to the amount of the detectable agent measured in step (c).   
     
     
         100 . The method of  claim 99 , wherein the heavy metal analyte is selected from the group consisting of arsenic, cadmium, chromium VI, cobalt, copper, lead, mercury, and zinc, or is more than one of these members. 
     
     
         101 . The method of  claim 99 , wherein the biosensor or promoter is selected from the group consisting of ParsR (arsenic), PcadC (cadmium), PcadR (cadmium), PzntA (cadmium), PchrB (chromium VI), PchrS (chromium VI), PrecN (chromium VI), PsulA (chromium VI), PumuD (chromium VI), PdadA (cobalt), Phmp (cobalt), PilvB (cobalt), PlipA (cobalt), PmmuP (cobalt), PnmtR (cobalt), PsoxR (cobalt), PtehA (cobalt), PygbA (cobalt), PyjbJ (cobalt), PyqfA (cobalt), PcopA (copper), PcusC (copper), PcusR (copper), PpbrR (lead), PmntH (lead), PshiA (lead), PybiI (lead), PyjjZ (lead), PmerR (mercury), PmntH (zinc), PshiA (zinc), PyjjZ (zinc), PzntA (zinc) and PzraP (zinc). 
     
     
         102 . The method of  claim 99 , wherein the detectable agent is a nucleic acid, detectable protein, antibody-linked reporter protein, enzymatic assay product, or electrochemical reaction product. 
     
     
         103 . The method of  claim 99 , wherein the detectable agent is a fluorescent protein or a luminescent protein. 
     
     
         104 . The method of  claim 99 , wherein the microbial cell is a bacterium, cyanobacterium, a fungus, a microalga, or an alga. 
     
     
         105 . The method of  claim 104 , wherein the bacterium is selected from the group consisting of  Escherichia coli, Bacillus subtilis, Salmonella  sp.,  Aliivibrio fischeri, Pseudomonas fluorescens, Bacillus  sp.,  Cupriavidus metallidurans, Deinococcus radiodurans , and  Staphylococcus aureus.    
     
     
         106 . The method of  claim 104 , wherein the fungus is selected from the group consisting of  Saccharomyces cerevisiae  and  Trichosporon cutaneum.    
     
     
         107 . The method of  claim 104 , wherein the cyanobacterium is  Synechocystis  sp. 
     
     
         108 . The method of  claim 99 , wherein step (c) comprises measuring a transcription level and/or an activation level of the detectable agent, wherein the transcription level and/or the activation level is expressed by a strain of the plurality of recombinant microbial cell strains, in the defined array. 
     
     
         109 . The method of  claim 108 , wherein the transcription level and/or the activation level of the detectable agent corresponds to the quantifiable level of the heavy metal analyte of interest. 
     
     
         110 . The method of  claim 99 , wherein step (c) comprises measuring a fluorescence and/or a luminescence of the detectable agent.