Part:BBa_K1088027

Dxs from B. subtilis with lac promoter and lacI (IPTG inducible)

The part consist of the dxs gene derived from B. subtilis 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 purpose of the brick is to increase the flow through the MEP pathway upon addition of IPTG.

A similar part (BBa_K1088026) with a GFP fused to Dxs, was used to prove that the part is IPTG inducible.

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 grows slightly faster than strains bearing plasmids. B) Per cent 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 increasingly per cent of +lacI:LVA became fluorescent and reaches a maximum of 70-75 per cent after 90 min. C) Mean GFP fluorescence of entire population. The -lacI:LVA cells became increasingly more fluorescent over time, both with and without induction. Though, 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 is reflected.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 2460
  Illegal EcoRI site found at 3117
  Illegal PstI site found at 2518
  Illegal PstI site found at 2964
  
• 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]

  Illegal EcoRI site found at 2460
  Illegal EcoRI site found at 3117
  
• 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 SapI.rc site found at 2958