Part:BBa_K3634014

CcdAB-Controlled mf-Lon Protease

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.

Here, the output protein expressed will be mf-Lon protease (BBa_K2333011). mf-Lon protease is evolutionary related to the native Lon protease in E.coli and functions similarly in that recognition of a specific degradation tag allows for rapid endogenous breakdown of the protein to which it was attached. The two enzymes differ in their ability to recognise these specific tags; mf-Lon protease cannot recognise degradation tags associated to E.coli lon protease and vice versa. By the ability to alter the strength of signals associated with these degradation tags through sequence specific alterations (Collins et al., 2014), intracellular concentrations of proteins can be precisely modulated.

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
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1934
  Illegal AgeI site found at 2018
  Illegal AgeI site found at 2224
  Illegal AgeI site found at 2249
  
• 1000
  COMPATIBLE WITH RFC[1000]