Part:BBa_K4212012

CotG_native

Native promoter of CotG, sequence of 200 bases upstream of the CotG CDS. Utilised in transcriptional units with CotG fusion proteins.

Usage and Biology

Given the tightly controlled process of sporulation, we also wanted to test the difference in burden, expression level and functionality using an anchor protein specific promoter vs. a constitutive one. One concern was that using a constitutive promoter might result in excessive metabolic load, affecting the standard functioning of the cell including sporulation. Furthermore due to the timeliness of CotG expression, another worry was that the fusion protein might integrate into the wrong layer of the spore coat. Thus, we designed a new part for the STK toolkit, a CotG native promoter + rbs. We accomplished this by taking 200bp upstream from the CotG CDS in the B. subtilis genome and adding the appropriate recognition sites and overhangs on either end. Assembly using strategies 1 and 3 were attempted, given the poor results from strategy.

Figure 1. The schematic design of CotG promoter

References

[1]Sacco, M., Ricca, E., Losick, R. & Cutting, S. (1995) An additional GerE-controlled gene encoding an abundant spore coat protein from Bacillus subtilis. Journal of Bacteriology. 177 (2), 372–377. doi:10.1128/jb.177.2.372-377.1995.

[2] Henriques, A.O., Melsen, L.R. & Moran, C.P. (1998) Involvement of Superoxide Dismutase in Spore Coat Assembly in Bacillus subtilis. Journal of Bacteriology. 180 (9), 2285–2291. doi:10.1128/JB.180.9.2285-2291.1998.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 101
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]