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 stochatic manner.

As an indication of the decision, either Rfp on the left side of the switch or the downstream genes on the right side of the switch is expressed.

An invertible region including a sigA promoter and the hin CDS is inverted by Hin [1,2,3]. The expression of Hin can be controlled by IPTG and Xylose using the pSpac or pXyl promoters that flank the hin gene, depending on the orientation of the middle segment [5,6].

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 affected 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 on NheI site and ended up with 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.