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Creating a Riboswitch‐Based Whole‐Cell Biosensor for Bisphenol A
Author(s) -
Zorawski Marek,
Shaffer Jeremy,
Velasquez Erick,
Liu Jane M.
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.805.3
Subject(s) - riboswitch , aptamer , plasmid , reporter gene , bisphenol a , computational biology , biosensor , biology , escherichia coli , green fluorescent protein , chemistry , rna , tetracycline , gene , gene expression , microbiology and biotechnology , genetics , biochemistry , non coding rna , antibiotics , organic chemistry , epoxy
Bisphenol A (BPA) is a known endocrine disruptor and potential carcinogen found in polycarbonate plastic products, food packaging, and drinking water supplies. A quick and affordable detector, such as a whole‐cell biosensor that measures the presence of BPA, would greatly benefit safety and health. We set out to develop such a method by using riboswitch‐based biosensors. Riboswitches are regions of mRNAs that control downstream gene expression and can be used as genetic sensors to create whole‐cell biosensors. Although there are no naturally occurring BPA‐riboswitches, we reasoned that we could use the power of genetic selections to generate one. Starting from a known riboswitch that regulates expression of the downstream selection marker tetA linked to the reporter gene gfp , we used PCR or Gibson Assembly to replace the aptamer domain of the switch with 40 random bases, generating plasmid libraries. We then transformed Escherichia coli with these plasmids such that each individual bacterium harbored a unique member of the library. Libraries made by Gibson Assembly resulted in 10 8 unique members, while those made by PCR were generally on the order of 10 6 members. With libraries in hand, the dualistic nature of TetA allowed us to apply dual genetic selection to identify riboswitches that express tetA‐gfp only in response to BPA. TetA is a transporter protein that pumps the antibiotic tetracycline out of the cell, rendering cells tetracycline resistant. TetA also allows toxic metals, such as Ni 2+ , to enter the cell, inhibiting growth. The E. coli library was grown in the presence of BPA and tetracycline. Surviving cells were switched to medium with Ni 2+ and no BPA. Only those cells that express tetA‐gfp exclusively in the presence of BPA were expected to survive both positive and negative selection. We succeeded in isolating unique riboswitches that converged to single sequences from starting libraries by bringing each library through three rounds of selection with varying tetracycline and Ni 2+ conditions. Ultimately, our hits proved largely unsuccessful in producing a significant increase in fluorescence when incubated with BPA for either 6 or 16 hours compared to no‐ligand controls. In order to improve the ON/OFF ratio of our riboswitches, we integrated fluorescence‐activated cell sorting into our riboswitch‐development platform, allowing us to both select for survival through dual genetic selection and screen for maximum or minimum fluorescence depending on presence or absence of the ligand of interest. Further studies will include optimizing the obtained riboswitches by mutagenesis and directed evolution. Support or Funding Information Research funding is provided by the NSF.