Difference between revisions of "Part:BBa K4137013"

Revision as of 07:46, 10 October 2022

ccdB toxin secretion pathway

This part consists of a pLac promoter, RBS, a ccdB-6xHis coding complex, and a terminator (BBa_B1006). This part produces a constant expression of ccdB toxin under the LB medium, inhibiting the GyrA subunit of DNA gyrase and prevents negative supercoiling of DNA. An accumulation of ccdB without sufficient suppression by antitoxin ccdA leads to risks in host viability.

Fig.1 Construct design for ccdB toxin secretion.

Construct Design

We attached a 6x His-Tag downstream of the ccdB sequence for purification purposes. A pLac promoter, which induces protein production when malate is present, as well as 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. CcdB cell harvest 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. 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