Part:BBa_K1088020

lacI:LVA device (constitutive promoter, RBS and terminator)

The trancsriptional repressor LacI with LVA tag for faster degradation (BBa_C0012) under the constitutively active promoter (BBa_J23106) with a RBS (BBa_B0030) and 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. However, we have build a similar device (BBa_K1088019) without the LVA-tag which proved to respond better and faster to induction.

To prove the function of this regulatory device another device carrying a GFP-protein fusion under the lactose promoter with and without this regulatory device was assayed (BBa_K1088009 and BBa_K1088008, respectively)

FACS (fluorescence assorted cell sorting) results and growth curves of +/-lacI:LVA carrying strains. LacI(N) stands for natural lacI, whereas lacI(LVA) contains a LVA tag promoting its fast degradation in the cell and should facilitate a more fine-tuned control. One triplicate of MG1655 (WT) and two triplicates of MG1655 strains carrying either BBa_K1088008 (-lacI:LVA) or BBa_K1088009 (+lacI:LVA) were grown from OD₆₀₀ 0.005 to approximately 0.2. At this OD the MG1655 triplicate and one triplicate of each strain carrying constructs were induced with 1 mM IPTG at time 0 min. FACS measurements were done at times: -30, 0, 30, 60, 90, 120, and 150 min.

A) Growth curve shows that WT cells grow slightly faster than strains carrying plasmids. B) Percentage of population above fluorescence threshold. None of the WT cells were fluorescent, almost all of the -lacI:LVA cells were constitutively fluorescent, and only cells overexpressing LacI:LVA weren’t fluorescent when not induced. Upon induction, increasing percentage of +lacI:LVA carrying cells became fluorescent and reached a maximum of 70-75 percent after 90 min. C) Mean GFP fluorescence of the entire population. The -lacI:LVA cells became increasingly more fluorescent over time, both with and without induction. The induced cells were slightly more fluorescent, which is probably because of the relief of repression from LacI naturally present in the cells. For the +lacI:LVA cells the results from B are reflected.

Explanatory text: FACS results and growth curves of lacI(N) and lacI:LVA carrying strains. Dublicates of MG1655 carrying either pSB1C3-Pcon-lacI(N)-term-Plac-dxs (B. subtilis)-GFP (BBa_K1088028) (lacI(N)) or pSB1C3-Pcon-lacI:LVA-term-Plac-dxs (B. subtilis)-GFP (BBa_K1088009) (lacI(lva)) were grown from OD₆₀₀=0.005 to approximately OD₆₀₀=0.2. At this OD, the one triplicate of each strain was induced with 0.05, 0.125, 0.25, or 0.5 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 the same pace B) Percentage of population above fluorescenct threshold. Upon induction of lacI(lva) with different concentrations of IPTG, the percentage of bacteria becoming fluorescent slowly increases. After approximately 180 min, 25-30 % of lacI(lva) induced bacteria in the range from 0.05-0.5 mM IPTG are above the fluorescenct threshold. After 180 min, there is a slightly higher percentage of bacteria above the fluorescenct threshold than for the lacI(lva) strain induced with 10-fold higher IPTG concentration. All lacI(N) induced with IPTG concentrations from 0.125-0.5mM become fluorescent within 180 min, and cultures with higher IPTG concentrations reach this point sooner. 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]