Team:Newcastle/Characterisation

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(Difference between revisions)
(→Characterization of IPTG inducable KinA sporulation trigger device)
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=Characterization of IPTG inducable KinA sporulation trigger device=
=Characterization of IPTG inducable KinA sporulation trigger device=
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We successfully characterized IPTG inducible KinA sporulation trigger device. Although Spo0A is the master regulator of sporulation in ''B. subtilis'', only a gradual 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.  
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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  [https://2009.igem.org/Team:Newcastle/SporulationTuning sporulation tuning] page.
For more information about the design of this device go to our  [https://2009.igem.org/Team:Newcastle/SporulationTuning sporulation tuning] page.
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Our synthesised device was cloned into pgfp-rrnb integration vector. 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.  
+
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.
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. Since pmutin4 has LacI gene, we were able to get LacI expressed in the cells. We successfully transformed the mutant with pgfp-rrnb. AmyE regions in the integration vector provided a double crossover into the chromosome. As a result of this crossover, cells loose amyE gene and cannot break starch. When transformed colonies are plated into starch plates, the transformed colonies can be seen without any halos around them.   
+
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.
We then selected two transformed colonies from the starch plate and used them to induce sporulation by adding IPTG.
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To select the transformed colonies we prepared our plates with LB + Erythromycin + Chloramphenicol. Chloramphenicol was used to select pgf-rrnb transformations and Erythromycin  was used to select the colonies with pmutin4. Hence by adding these two antibiotics we made sure that we were using BFS867 mutants transformed with pgfp-rrnb integration vector hence with our biobrick.
+
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:
Concentrations used to characterize our device:
 +
  IPTG : 1mM
  IPTG : 1mM
  Erythromycin :0.3ug/ml
  Erythromycin :0.3ug/ml
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===Experiment Overview===
===Experiment Overview===
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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 controls.
+
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 they reach to stationary phase, we got the samples from time point 1 and time point 5 and prepared them for microscopy by resuspending the cells in SMM medium.
+
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===
===Growth Curves===

Revision as of 20:29, 21 October 2009

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

Team Newcastle iGEM 2009 GrowthCurve 1.png Shows Wild type B. subtilis, WT. B. Subtilis transformed with pgfp-rrnb, and BFS867 mutant transformed with pgfp-rrnb. (+ sign represents IPTG added)
Team Newcastle iGEM 2009 GrowthCurve 2.png
Team Newcastle iGEM 2009 GrowthCurve 3.png 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



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