Transformation efficiency: 1–3 x 107 cfu/µg pUC19 DNA
BL21(DE3) containing the Lemo System™
Tunable T7 Expression Strain for difficult targets: membrane proteins, toxic proteins and proteins prone to insoluble expression
Deficient in proteases Lon and OmpT
Resistant to phage T1 (fhuA2)
Lemo System™ maintained by chloramphenicol
Description: Chemically competent E. coli cells suitable for transformation and protein expression. Fine tuning of T7 expression can alleviate inclusion body formation or growth inhibitory effects from toxic proteins. In many cases, less expression equals more protein of interest produced in the desired form. This is particularly true for membrane protein expression (Wagner et al. 2008). Membrane protein expression and protein export in E. coli are both limited by the throughput capacity of the Sec translocase and in some cases the Tat translocase. T7 expression of proteins targeted to the Sec translocase often leads to accumulation of inclusion bodies or inhibition of cell division, if expression is not regulated.
Lemo21(DE3) offers the host features of BL21(DE3) while also allowing for tunable expression of difficult clones. Tunable expression is achieved by varying the level of lysozyme (lysY), the natural inhibitor of T7 RNA polymerase. The level of lysozyme is modulated by adding L-rhamnose to the expression culture at levels from zero to 2000 µM. When Lemo21(DE3) is grown without rhamnose, the strain performs the same as a pLysS containing strain. However, optional addition of rhamnose tunes the expression of the protein of interest. For difficult soluble proteins, tuning the expression level may also result in more soluble, properly folded protein.
Reagents Supplied:
6 x 0.05 ml/tube of chemically competent Lemo21(DE3) Competent E. coli cells (Store at -80°C)
12.5 ml of 0.5 M L-rhamnose solution (Store at room temperature)
0.025 ml of 50 pg/µl pUC19 Control DNA (Store at -20°C)
Transformation Protocol Variables: Thawing: Cells are best thawed on ice and DNA added as soon as the last bit of ice in the tube disappears. Cells can also be thawed by hand, but warming above 0°C will decrease the transformation efficiency.
Incubation of DNA with Cells on Ice: For maximum transformation efficiency, cells and DNA should be incubated together on ice for 30 minutes. Expect a 2-fold loss in transformation efficiency for every 10 minutes this step is shortened.
Heat Shock: Both the temperature and the timing of the heat shock step are important and specific to the transformation volume and vessel. Using the transformation tube provided, 10 seconds at 42°C is optimal.
Outgrowth: Outgrowth at 37°C for 1 hour is best for cell recovery and for expression of antibiotic resistance. Expect a 2-fold loss in transformation efficiency for every 15 minutes this step is shortened. SOC gives 2-fold higher transformation efficiency than LB medium; and incubation with shaking or rotating the tube gives 2-fold higher transformation efficiency than incubation without shaking.
Plating: Selection plates can be used warm or cold, wet or dry without significantly affecting the transformation efficiency. However, warm, dry plates are easier to spread and allow for the most rapid colony formation.
Western analysis of 6-His tagged Brugia malayi protein. A) B. malayi protein expressed at 20°C in BL21(DE3). B) Soluble fractions of B. malayi protein expressed at 30°C. Overnight expression of a membrane protein – PhoA fusion: Lemo System™ enables simple, rapid optimization of membrane protein expression.
原厂资料：
注意事项：
Usage notes:
CAUTION: This product contains DMSO, a hazardous material. Review the MSDS before handling.
STORAGE AND HANDLING: Competent cells should be stored at -80°C. Storage at -20°C will result in a significant decrease in transformation efficiency. Cells lose efficiency whenever they are warmed above -80°C, even if they do not thaw.
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