Part:BBa_K1088019

LacI device (constitutive promoter, RBS and terminator)

The trancsriptional repressor lacI (BBa_K1088018)under the constitutivly active promoter (BBa_J23106), with a RBS (BBa_B0030) and with a terminator (BBa_B1002).

This device can be used to overexpress lacI with the purpose of repressing transcription from high-copy plasmids containting the lac promoter (BBa_R0010). The repression can be relieved by allosteric binding of IPTG to lacI, thereby promoting transcription from the lac promoter.

FACS results and growth curves of lacI(N) and lacI:LVA carrying strains. Two triplicates of MG1655 strains carrying either pSB1C3-Pcon-lacI(N)-term-Plac-dxs(B. subtilits)-GFP (BBa_K1088026) (lacI(N)) or pSB1C3-Pcon-lacI:LVA-term-Plac-dxs(B. subtilits)-GFP (BBa_K1088009) (lacI:LVA) were grown from OD₆₀₀ 0.005 to approximately 0.2. At this OD the one triplicate of each strain were induced with 1 mM IPTG at time 0 min. FACS measurements were done at times: -30, 0, 30, 60, 90, 120, 150 and 180 min. A) lacI(lva) and lacI(N) strains grew at same pace both with and without induction. B) Per cent of population above fluorescence threshold. Both lacI(lva) and lacI(N) represses the expression, when not induced with IPTG. lacI(lva) reaches a maximum of merely 70-75 % after 150 min, which is both a poor response time and maximum cell per cent fluorescent compared to lacI(N). lacI(N) reaches a maximum just below 100 % at a time between 30 and 60 min. C) Mean GFP fluorescence of entire population. These results reflect what is seen in B, and clearly indicates that overexpression of natural LacI instead of LacI:LVA is the better, when a quick response and high level of expression is wanted.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]