Difference between revisions of "Part:BBa K3634013"

Latest revision as of 12:30, 7 August 2020

ccdAB promoter + operator

The ccdAB toxin-antitoxin (TA) operon is a type II TA module found on the E.coli F plasmid. The module consists of the toxic ccdB (T) protein which poisons the enzyme DNA gyrase required for negative supercoiling of DNA (Bernard and Couturier, 1992). The activity of the unstable ccdA antitoxin (A) separates the ccdB-gyrase complex if present (Vandervelde et al., 2017).

Expression of ccdA and ccdB within the type II TA module is self-regulated by low specificity and affinity of ccdA for individual binding sites of the regulatory region upstream of both ccdA and ccdB genes. This 113bp ccdAB promoter/operator sequence extends into the first ccdA gene and is suspected to have a total of 8 ccdA operator binding sites (Tam & Kline, 1989). The antitoxin binds the operator DNA sites, with some sites overlapping the promoter, functioning as a repressor of ccdAB transcription. The toxin then functions as a co-repressor or de-repressor depending on the molar T:A ratio (Vandervelde et al., 2017).

At low T:A ratio, the operator is repressed however as the ratio is increased, repression is relieved by preferential formation of a V-shaped non-repressing heterohexamer (ccdB-ccdA-ccdB). Specifically, at the moment the molar ratio of T:A > 1, repression is rapidly lost (Vandervelde et al., 2017). Therefore the regulatory region, controlled by intracellular ccdAB concentrations, can be used for precise control of a desired output gene as shown in the native system.

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]

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 The ccdAB toxin-antitoxin (TA) operon is a type II TA module found on the E.coli F plasmid. The module consists of the toxic ccdB (T) protein which poisons the enzyme DNA gyrase required for negative supercoiling of DNA (Bernard and Couturier, 1992). The activity of the unstable ccdA antitoxin (A) separates the ccdB-gyrase complex if present (Vandervelde et al., 2017).
-Expression of ccdA and ccdB within the type II TA module is self-regulated by low specificity and affinity of ccdA for individual binding sites of the regulatory region upstream of both ccdA and ccdB genes. This 113bp ccdAB promoter/operator sequence extends into the first ccdA gene and is suspected to have a total of 8 ccdA operator binding sites. The antitoxin binds the operator DNA sites, with some sites overlapping the promoter, functioning as a repressor of ccdAB transcription. The toxin then functions as a co-repressor or de-repressor depending on the molar T:A ratio (Vandervelde et al., 2017).
+Expression of ccdA and ccdB within the type II TA module is self-regulated by low specificity and affinity of ccdA for individual binding sites of the regulatory region upstream of both ccdA and ccdB genes. This 113bp ccdAB promoter/operator sequence extends into the first ccdA gene and is suspected to have a total of 8 ccdA operator binding sites (Tam & Kline, 1989). The antitoxin binds the operator DNA sites, with some sites overlapping the promoter, functioning as a repressor of ccdAB transcription. The toxin then functions as a co-repressor or de-repressor depending on the molar T:A ratio (Vandervelde et al., 2017).
 At low T:A ratio, the operator is repressed however as the ratio is increased, repression is relieved by preferential formation of a V-shaped non-repressing heterohexamer (ccdB-ccdA-ccdB). Specifically, at the moment the molar ratio of T:A > 1, repression is rapidly lost (Vandervelde et al., 2017). Therefore the regulatory region, controlled by intracellular ccdAB concentrations, can be used for precise control of a desired output gene as shown in the native system.