Antibiotic-Efficient Genetic Cassette for the TEM-1 β-Lactamase That Improves Plasmid Performance.

Antibiotic resistance cassettes are indispensable tools in recombinant DNA technology, synthetic biology, and metabolic engineering. The genetic cassette encoding the TEM-1 β-lactamase (denoted Tn3.1) is one of the most commonly used and can be found in more than 120 commercially available bacterial expression plasmids ( e.g. , the pET , pUC , pGEM , pQE , pGEX , pBAD , and pSEVA series). A widely acknowledged problem with the cassette is that it produces excessively high titers of β-lactamase that rapidly degrade β-lactam antibiotics in the culture media, leading to loss of selective pressure, and eventually a large percentage of cells that do not have a plasmid. To address these shortcomings, we have engineered a next-generation version that expresses minimal levels of β-lactamase (denoted Tn3.1 MIN ). We have also engineered a version that is compatible with the Standard European Vector Architecture (SEVA) (denoted Ap (pSEVA#1 MIN --)). Expression plasmids containing either Tn3.1 MIN or Ap (pSEVA#1 MIN --) can be selected using a 5-fold lower concentration of β-lactam antibiotics and benefit from the increased half-life of the β-lactam antibiotics in the culture medium (3- to 10-fold). Moreover, more cells in the culture retain the plasmid. In summary, we present two antibiotic-efficient genetic cassettes encoding the TEM-1 β-lactamase that reduce antibiotic consumption (an integral part of antibiotic stewardship), reduce production costs, and improve plasmid performance in bacterial cell factories.