Part:BBa_K174011

IPTG inducable kinA Bacillus sporulation trigger

Spo0A is the master regulator of sporulation in B. subtilis. When it is phosphorylated, it activates the expression of more than 100 genes(1). It has been shown that, activation of spo0A is sufficient to trigger sporulation.

By artifically synthesising KinA during growth, spoulation can be triggered in a multicomponent phosphorelay in which KinA phosphorylates Spo0F, Spo0F~P in turn phosphorylates Spo0B, and finally Spo0B~P phosphorylates Spo0A. (2)

Expressing Spo0A itself alone in the cell would not initiate the sporulation. Spo0A~ P initiates sporulation, first by activating low-threshold genes, and then by gradually increasing and activating high-threshold genes. Hence the multicomponent phosphorelay’s elements, KinA, Spo0F and spo0B, gradually increases spo0A and its activity, and initiates the sporulation. (2).

By adding pspac promoter and RBS to the front of KinA, we made our device to express KinA when induced by IPTG. Hence this device will trigger sporulation when IPTG is added to the medium.

We designed the device with a pluggable promoter. The promoter is flanked by AccIII and BalI cut sites and can easily be replaced. Hence other inducers can also be used to trigger the sporulation with this device.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Characterisation

• To learn more about how we characterised this part, go to our iGEM 2009 [http://2009.igem.org/Team:Newcastle/Characterisation characterisation] page.

• To learn more design and modelling of our construct, visit our iGEM 2009 [http://2009.igem.org/Team:Newcastle/SporulationTuning sporulation tuning] sub-project page.

References

1. Veening, J.-W., W. K. Smits, et al. (2008). "Bistability, Epigenetics, and Bet-Hedging in Bacteria." Annual Review of Microbiology 62(1): 193-210.
2. Fujita, M. and R. Losick (2005). "Evidence that entry into sporulation in Bacillus subtilis is governed by a gradual increase in the level and activity of the master regulator Spo0A." Genes & Development 19(18): 2236-2244.