Part:BBa_K1085014

pSB1AC3-HySp: Integrational backbone into B. subtilis amyE locus with IPTG inducible promoter

To transform B. subtilis with our developed biobricks we have improved the amyE intergrational backbone from Munich's iGEM team 2012 (BBa_K823023) that had no inducible promoter. We've added the HyperSpank IPTG inducible promoter from Rudner's Lab 2004. This promoter is placed right in front of the prefix, so any biobrick can be inserted in this backbone (BBa_K1085014) and can be induced with IPTG.

In figure 1 a close-up of the promoter is shown.

The HyperSpank promoter has a single nucleotide change (G->T) at the -1 position. This increases the expression levels but also causes leaky expression when IPTG is absent [1]. To improve repression, a second lacO operator site has been inserted 71 bp upstream of the first. (David Rudner, Harvard Medical School)

Figure 1: The HyperSpank promoter with in orange the lacO operators, in red the -35, -10, +1 signals and in green the prefix.

Figure 2: BBa_K1085014 with: The amp gene for ampicillin resistance in E. coli; The cat gene for chloramphenicol resistance in B. subtilis; The HyperSpank promoter with its repressor lacI; The amyE up- and downstream fragments for intergration in the amyE locus.

Promoter activity

BBa_K1085014 was constructed with two variants of GFP (one form our group and one from the biobrick system, BBa_E0840). BBa_K1085014-GFPmg and BBa_K1085014-BBa_E0840 were transformed to B. subtilis. Overnight cultivated strains were diluted 1:100 in fresh medium and grown till OD~0.45 and induced with 1mM IPTG. A 1,2 and 3 hour sample was analysed under the microscope, results are shown in figure 3.

Figure 3 A:	B:	C:

Figure 3: In (a) An overlay picture is shown from the phase-contrast and GFP-channel picture. In (b,c) the intensity in of ~40 cells in the GFP-channel, were analyzed from two pictures. The average intensity (AU) from the cells are plotted above with the standard deviation. In (b) a GFP from our group is shown and in (c)a biobricked GFP (BBa_E0840) is shown in a plot. wt=wildtype, T0= before induction, T1,2,3=#h of induction with IPTG.

Sequence and Features

Assembly Compatibility:

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

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 7604
  Illegal NheI site found at 7428
  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  Illegal NotI site found at 9
  Illegal NotI site found at 7610
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 7604
  Illegal BamHI site found at 6066
  Illegal XhoI site found at 3574
  Illegal XhoI site found at 7488
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found at 7604
  Illegal suffix found at 2
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found at 7604
  Plasmid lacks a suffix.
  Illegal XbaI site found at 7619
  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  Illegal NgoMIV site found at 1128
  Illegal NgoMIV site found at 3538
  Illegal NgoMIV site found at 4677
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal BsaI.rc site found at 4501