Part:BBa_K4137008

mleR expressing construct

This construct produces and purifies mleR, the malate-induced regulator that binds to p_mleS promoter for the production of ccdA antitoxin.

Fig.1 Malate-binding transcriptional activator + 6x His-Tag complete construct.

Construct Designs

We attached a 6x His-Tag downstream of the mleR sequence for purification purposes. A T7 and RBS (AAGGAG) are attached upstream to the side of the open reading frame. The terminator BBa_B1006 is attached downstream of the sequence.

Characterization

Due to the lack of equipment for performing sonication and chemical lysis during protein extraction, we modified a thermolysis method based on tips from Lab 106 personnel and theory to resolve this issue. We performed protein expression in 50 ml of expression medium with BL21(DE3) E. coli and followed Berini et al.’s protocol. Cell harvest for mleR was performed at time intervals of 0h, 0.5h, 1h, 2h, 3h, and 4h. We added phosphate Buffered Saline (PBS) and 4X Sample buffer to pelletized cells in a volume ratio of 3:1, adjusted the total fluid volume to 100 µl, and resuspended the cells.

Fig.2 SDS-PAGE analysis of 20 µl mleR protein extract..

Sequence and Features

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 49
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]