Part:BBa_K1088026

B. subtilis dxs-GFP protein fusion (lac promoter with lac inhibitor: IPTG inducible)

This part consist of the dxs gene derived from B. subtilis fused to GFP at the translational level with a 10 AA linker between the proteins. The reporter fusion is under the control of the lac promoter and has a strong RBS.

To repress expression from the lac promoter, the lacI gene under a constitutive promoter, with a strong RBS and a efficient terminator is placed upstream to the reporter fusion. The repression can then be relieved with addition of IPTG, which binds and inhibits the function of LacI.

The levels of expression were meassured in E. coli K-12 MG1655 carrying the part using fluorescence activated cell sorting (FACS). The experiment proved that the reporter fusion wasn't expressed when the strain was grown without IPTG in the media, and that the reporter fusion was expressed after addition with IPTG.

FACS results and growth curves of +/-lacI:LVA carrying strains. 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 percent of +lacI:LVA 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_K1088026) (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. This brick was build to test induction time and IPTG concentration for induction of a similar device (BBa_K1088027) which lacks the linker and GFP. Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Unknown
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 2460
  Illegal EcoRI site found at 3117
  Illegal NheI site found at 7
  Illegal NheI site found at 30
  Illegal PstI site found at 2518
  Illegal PstI site found at 2964
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Unknown
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 2460
  Illegal EcoRI site found at 3117
  Illegal PstI site found at 2518
  Illegal PstI site found at 2964
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 2460
  Illegal EcoRI site found at 3117
  Illegal PstI site found at 2518
  Illegal PstI site found at 2964
  Illegal NgoMIV site found at 2417
  Illegal AgeI site found at 2310
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 2259
  Illegal BsaI.rc site found at 3973
  Illegal SapI.rc site found at 2958