Part:BBa_K4137006

CcdA expressing construct

This sequence codes for CcdA, the antitoxin which assures chi18h8’s production around the plant roots. The mleR regulatory site comes from p_mles, which binds with the RBS and CcdA, the antitoxin that will bind with CcdB when induced by malate acid. This will detoxify ccdB. The purification tag 6xHis, and the terminator B1006 (BBa_B1006) are flanked on the end of the open reading frame.

Fig.1 Construct design for ccdA antitoxin secretion.

Construct Designs

We attached a 6x His-Tag downstream of the ccdA sequence to determine the basal production of the antitoxin. A p_mleS promoter, which induces protein production when malate is present, as well as a 5'UTR RBS (AAGGAG), are attached upstream to the side of the open reading frame (ccdA-6x his tag). The terminator BBa_B1006 is attached downstream of the sequence.

Characterization

We performed protein expression in 50 ml of expression medium with BL21(DE3) E. coli and closely followed Berini et al's protocols. Cell harvest for ccdA was performed at 3h. We heated the mixture at 95˚C for 10 minutes, centrifuging at 4˚C, 16000g to extract the supernatant. An additional 95˚C heating and 100 µl PBS were added to eliminate clumping of pellets.

Sequence and Features

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]