Part:BBa_K4137002

Malate-binding Transcriptional Activator + 6xHis

BBa_K4137002, nicknamed mleR for the purposes of our project, is a malate-binding transcriptional activator belonging to l. lactis that has a 6xHis tag attached to permit protein purification. The mleR transcriptional activator portion of the part is responsible for governing malolactic fermentation in l. lactis, and responds to malate to induce malolactic enzyme production.

Fig.1 mleR with C terminal 6x his tag.

Construct Design

During the gBlocks fragment synthesis stage of our construct design, we were forced to split mleR along the restriction sites NdeI due to errors in IDT gblock fragment synthesis, in which the first half would have cut sites at EcoRI and NdeI and the second half would have cut sites at NdeI and SpeI. We first ligated the first half of mleR onto our pSB1C3 backbone before attaching the second half. The full mleR construct would have EcoRI and SpeI cut sites.

Fig.2 mleR first half with E-N cut sites and second half with N-S cut sites.

Since we aimed to develop determine how different combinations of our toxin-antitoxin system components (ccdA, ccdB, mleR) affect one another's activity to verify our kill switch theory, we designed our constructs with biobrick restriction sites that bracket our genetic components--to insert one of our components into our backbone pSB1C3, we would cut at EcoRI and SpeI for both the insert and the vector. We also flanked each component with a purification tag to observe their expression levels.

Fig.3 mleR with E-S cut sites.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 18
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]