Backbone

An amyE locus integration backbone

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.

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