Difference between revisions of "Part:BBa K174011"
(→Characterisation) |
(→Characterisation) |
||
| (One intermediate revision by the same user not shown) | |||
| Line 27: | Line 27: | ||
===Characterisation=== | ===Characterisation=== | ||
| − | This part was characterised by integrating it into the genome of ''B. subtilis'' 168 at the ''amyE'' locus and | + | This part was characterised by integrating it into the genome of ''B. subtilis'' 168 at the ''amyE'' locus and including a downstream ''gfp'' reporter. The response of correctly transformed strains to different levels of IPTG was examined using 1) Growth curves 2) Fluorescence Microscopy. The induction of the expression of ''kinA'', and the downstream transcriptional reporter, should result in increased sporulation rates and increased GFP based fluorescence, respectively. |
Strains with this brick integrated in the genome were grown in rich media for three hours. | Strains with this brick integrated in the genome were grown in rich media for three hours. | ||
Latest revision as of 14:01, 31 October 2009
IPTG inducable kinA Bacillus sporulation trigger
Spo0A is the master regulator of sporulation in B. subtilis. When Spo0A 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 upstream of kinA, we made our device able 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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Unknown
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Characterisation
This part was characterised by integrating it into the genome of B. subtilis 168 at the amyE locus and including a downstream gfp reporter. The response of correctly transformed strains to different levels of IPTG was examined using 1) Growth curves 2) Fluorescence Microscopy. The induction of the expression of kinA, and the downstream transcriptional reporter, should result in increased sporulation rates and increased GFP based fluorescence, respectively.
Strains with this brick integrated in the genome were grown in rich media for three hours.
In the absence of IPTG only minimal sporulation rates were observed and only background fluorescence, throughout the growth curve.
In the presence of 1mM IPTG after three hours most cells could be seen to be sporulating and high levels of GFP fluorescence were observed.
- 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 about the design and modelling of our construct, visit our iGEM 2009 [http://2009.igem.org/Team:Newcastle/SporulationTuning sporulation tuning] sub-project page.
References
- Veening, J.-W., W. K. Smits, et al. (2008). "Bistability, Epigenetics, and Bet-Hedging in Bacteria." Annual Review of Microbiology 62(1): 193-210.
- 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.