pET expression plasmids are widely used for producing recombinant proteins in Escherichia coli. Selection and maintenance of cells harboring a pET plasmid are possible using either a Tn3.1-type genetic fragment (which encodes a ß-lactamase and confers resistance to ß-lactam antibiotics) or a Tn903.1-type genetic fragment (which encodes an aminoglycoside-3’-phosphotransferase and confers resistance aminoglycoside antibiotics). Herein we have investigated how efficiently pET plasmids are maintained using these two fragments. The study reveals that pET plasmids are efficiently maintained with both Tn3.1 and Tn903.1 genetic fragments prior to the induction of recombinant protein production, and over short induction times (i.e., 2 h). However, over longer induction times (i.e., 20 h), the efficiency of plasmid maintenance depends on the host strain used, and the type of antibiotic selection cassette used. Based on our collective observations, we have 2 general tips for efficiently maintaining pET plasmids during recombinant production experiments.

• Tip #1: Use a strain with lowered levels of the T7 RNA polymerase, such as C41(DE3). pET plasmids will be efficiently maintained over long induction times with both the Tn3.1 and Tn903.1 genetic fragments, regardless of whether antibiotics are present during cultivation.

• Tip #2: If a strain with higher levels of T7 RNA polymerase strain is necessary, such as BL21(DE3)), keep induction times short or use a plasmid containing a Tn903.1-type fragment and select with kanamycin.

pET 表达质粒广泛用于在大肠杆菌中生产重组蛋白。使用 Tn3.1 型遗传片段（编码 β-内酰胺酶并赋予对 β-内酰胺抗生素抗性）或 Tn903.1 型遗传片段（编码氨基糖苷-3'-磷酸转移酶并赋予氨基糖苷类抗生素耐药性）。在此，我们研究了使用这两个片段维持 pET 质粒的效率。研究表明，在诱导重组蛋白产生之前，pET 质粒可在较短的诱导时间（即 2 小时）内有效地与 Tn3.1 和 Tn903.1 遗传片段一起维持。然而，在较长的诱导时间（即 20 小时）内，质粒维持的效率取决于所使用的宿主菌株和所使用的抗生素选择盒的类型。根据我们的集体观察，我们有 2 个在重组生产实验期间有效维护 pET 质粒的一般技巧。

• 提示 #1：使用 T7 RNA 聚合酶水平较低的菌株，例如 C41( DE3 )。无论培养过程中是否存在抗生素，pET 质粒都可以在较长的诱导时间内有效维持 Tn3.1 和 Tn903.1 基因片段。

• 提示#2：如果需要具有较高水平 T7 RNA 聚合酶菌株的菌株，例如 BL21( DE3 ))，请缩短诱导时间或使用包含 Tn903.1 型片段的质粒并用卡那霉素进行选择。