Difference between revisions of "Part:BBa K4137014"

Revision as of 08:06, 10 October 2022

CcdA regulatory and expression device

The mleR regulatory site which comes from p_mles binds with the RBS and CcdA, the antitoxin that will bind with CcdB when induced by malate acid and detoxifies it, the purification tag 6xHis, and the terminator B1006 (BBa_B1006). This sequence codes for CcdA, the antitoxin which assures chi18h8’s production around the plant roots. The second half contains a T7 promoter, an RBS, a mleR coding complex, a mleR purification tag—6xHis, and a terminator B1006. This half produces and purifies mleR, the regulator of CcdA, which produces when malate acid is present. The construct as a whole code for Ccda is regulated by mleR when malate acid is presented.

In theory, mleR regulatory site requires mleR to achieve maximal expression; mleR cannot be activated without malate, and thus transcription via the mleR regulon will not induced, causing low CcdA production.

Fig.1 Design for ccdA-mleR malate-inducing antitoxin construct.

Construct Design

We attached a Myc epitope tag downstream of the ccdA sequence for purification purposes. A p_mleS promoter, which induces protein production when malate is present, as well as RBS, are attached upstream to the side of our first open reading frame (ccdA-Myc). A T7 and RBS (AAGGAG) are attached upstream to the side of our open reading frame (mleR-6x his). Terminators BBa_B1006 are attached downstream of each of the open reading frames 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 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 200 µl, and resuspended the cells. We heated the mixture at 95˚C for 20 minutes, centrifuging at 4˚C, 16000g to extract the supernatant

Fig.2 SDS-PAGE analysis of 20 µl sample ccdA-mleR extraction.

Sequence and Features

Sequence and Features