Part:BBa_K174003

Heritable, Tunable, Stochastic Switch

This device is a tunable, heritable, stochastic switch for Bacillus subtilis. Essentially it encodes a heads or tails device that can be biased using two inducible promoters and by controlling the rate of degradation of the protein responsible for the switching, Hin invertase.

It can be tuned to control cell differention and fate in a stochastic manner.

The part employs Hin invertase to invert a middle segment flanked by hix sites.

Theinvertible region included an outward facing sigA promoter and the hin CDS [1,2,3]. The expression of Hin can be controlled by IPTG and Xylose using the pSpac or pXyl promoters that flank the invertable segment, and either of which can be used to drive the transcription of hin depending on the orientation of the middle segment [5,6].

As an indication of the segment orientation, either rfp on the left side of the switch or additional genes on the right side of the switch are expressed by the sigA promoter on the segment.

As a third level control, the modified version of ssrA tag has been translationally fused to the C-terminus of the Hin protein, so that it can be selectively degraded (See Part:BBa_K174002 for arabinose controlled SspB adaptor protein for the degradation).

When this part is used in combination Part:BBa_K174002 expression of hin, hence the rate of switching of the orientiation of the invertible region is controlled by the concentration of IPTG, Xylose, Arabinose.

Downstream genes on the left and right sides of the switch can be used to trigger cell fate decisions.

In our iGEM 2009 project, our switch was cloned into pGFP-rrnB [4] integration vector at the NheI site introducing gfp on the right side. However it was sent to the registry in pSB1AT3 since the pGFP-rrnB is not a Biobrick compatible vector.

For more information about this part go to Newcastle iGEM2009 [http://2009.igem.org/Team:Newcastle/Stochasticity Stochasticity] and [http://2009.igem.org/Team:Newcastle/Modelling Modelling] pages.

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
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1791
  Illegal AgeI site found at 105
  Illegal AgeI site found at 217
  
• 1000
  COMPATIBLE WITH RFC[1000]

References

1. Ham, T.S., et al., Design and Construction of a Double Inversion Recombination Switch for Heritable Sequential Genetic Memory. PLoS ONE, 2008. 3(7): p. e2815.
2. Haynes, K., M. Broderick, et al. (2008). "Engineering bacteria to solve the Burnt Pancake Problem." Journal of Biological Engineering 2(1): 8.
3. Kutsukake, K., et al., Two DNA Invertases Contribute to Flagellar Phase Variation in Salmonella enterica Serovar Typhimurium Strain LT2. J. Bacteriol., 2006. 188(3): p. 950-957.
4. Veening, J.-W., H. Murray, and J. Errington, A mechanism for cell cycle regulation of sporulation initiation in Bacillus subtilis. Genes & Development, 2009. 23(16): p. 1959-1970.
5. Kim, L., A. Mogk, et al. (1996). "A xylose-inducible Bacillus subtilis integration vector and its application." Gene 181: 71-76.
6. Kreuzer, P., D. Gartner, et al. (1989). "Identification and sequence analysis of the Bacillus subtilis W23 xylR gene and xyl operator." J. Bacteriol. 171(7): 3840-3845.