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Characterization of IPTG inducable KinA sporulation trigger device

We successfully characterized our IPTG inducible kinA sporulation tuning device. Although Spo0A is the master regulator of sporulation in B. subtilis, only an increase in phosphorylated Spo0A can trigger the sporulation(1). We achieved this scenario by artificially inducing kinA by IPTG to participate in a multicomponent phosphorelay. We used microscopy to verify our results.

For more information about the design of this device go to our sporulation tuning page.

Our synthesised device was cloned into the integration vector pGFP-rrnb constructed by Dr. JW Veening. By placing our device just before the gfp CDS in the integration vector, we hoped to see GFP when we induce the device with IPTG. However for IPTG to work, we needed LacI expressed in the cells.

When we first did our transformation for B. subtilis we forgot this link and we got similar results with cells induced with IPTG and not induced with IPTG. Obviously LacI was not present in the cells and adding IPTG did not make any difference.

We then used a Bacillus subtilis mutant, BFS687, which has pMutin4 integrated into the chromosomal DNA (2). We selected this mutant especially since it does not have any phenotype under a wide range of conditions as described in the [http://locus.jouy.inra.fr/cgi-bin/genmic/madbase/progs/madbase.operl Micado] database. Since pMutin4 has LacI gene, we were able to get LacI expressed in the cells. We successfully transformed the mutant with pGFP-rrnb carrying out kinA based sporulation rate tuning brick . amyE homologous regions in the integration vector mediated a double crossover into the chromosome. As a result of this crossover, cells lose the amyE gene and cannot break down starch. When transformed colonies are plated into starch plates, the transformed colonies that have successfully inetgrated the plasmid can be seen without any halos around them when we expose the plates to iodine.

We then selected two transformed colonies from the starch plate and used them to induce sporulation by adding IPTG.

To select the transformed colonies we prepared our plates with LB + Erythromycin + Chloramphenicol. Chloramphenicol was used to select pgf-rrnb based transformations and Erythromycin was used to select the colonies with pMutin4 intgrated. Hence, by adding these two antibiotics we made sure that we were using BFS867 mutants transformed with pgfp-rrnb integration vector containing our biobrick as an insert.

Concentrations used to characterize our device:

IPTG : 1mM
Erythromycin :0.3ug/ml
Chloramphenicol: 5ug/ml

Experiment Overview

Each 10ml of overnight cultures was split into two flasks with 60ml of LB+Em+CHL. Every half an hour we meaured the optical density of the cells to get the growth curve of the cells. We also stored samples at each time point. We added IPTG after an hour we started to our experiments. We also used wild type B. subtilis and wild type cells transformed with pgfp-rrnb as the negative and positive controls respectively.

After time point 5, before the cells reached stationary phase, we used the samples from time point 1 and time point 5 and prepared them for microscopy by resuspending the cells in SMM medium.

Growth Curves

	Shows Wild type B. subtilis, WT. B. Subtilis transformed with pgfp-rrnb, and BFS867 mutant transformed with pgfp-rrnb. (+ sign represents IPTG added)
	Growth curve of the mutant with and without IPTG

Microscopy results

At time point 1, colonies did not have much Gfp and spores. When it is looked to the sample taken at time point 5, spores inside the mother cells can be noticed. As it can be seen there are many spores. Original copy of KinA also works and make the cells sporulate. However as it can be seen, there are lots of cells with Gfp about to sporulate. Hence we can conclude that the sporulation was mainly triggered by our device.

Bright Field image of BFS 867 at time point 5, No IPTG	GFPs shown for BFS 867 at time point 5, No IPTG
Bright Field image of BFS 867 at time point 5, WithIPTG	GFPs shown for BFS 867 at time point 5, With IPTG

1. 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.
2. Retrieved 20/10/2009, from http://bacillus.genome.jp/bsorf_mutant_list/Page12.htm