Difference between revisions of "Part:BBa K4137007"

Revision as of 05:02, 10 October 2022

pLac+RBS+CcdB-Myc+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 Myc epitope Tag downstream of the ccdB sequence for purification purposes. A pLac promoter is attached upstream to the side of the open reading frame (ccdB-Myc coding complex) and RBS (AAGGAG). 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. 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 200 µl, and resuspended the cells.

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

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
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 271

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 ===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. 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.
+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. 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 200 µl, and resuspended the cells.
 [[File: ccdb-sds.png|800px|thumb|center|Fig.2 SDS-PAGE analysis of 20 µl ccdB protein extract.]]