IGEM:Paris Bettencourt 2012/Notebooks/Delay group - Revision history

Ernest Mordret at 13:57, 19 July 2012

2012-07-19T13:57:21Z

←Older revision		Revision as of 13:57, 19 July 2012
Line 25:		Line 25:
	= References =		= References =
	1. Topp, S. & Gallivan, J.P. Riboswitches in unexpected places - A synthetic riboswitch in a protein coding region. RNA journal 2498-2503 (2008) [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2590966/\| Paper]		1. Topp, S. & Gallivan, J.P. Riboswitches in unexpected places - A synthetic riboswitch in a protein coding region. RNA journal 2498-2503 (2008) [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2590966/\| Paper]
		+
		+	=sRNA control=
		+	To achieve a strong repression of the colicin expression (and avoid the problems linked to its toxicity for the cell), we want to couple a repression at the level of the promoter (yiaGp is transcribed only in stationnary phase) to a post-trancriptional control using sRNAs. To do so, we will adapt a previously decribed system [1]. We hope to build and characterize the following design.
		+	[[Image:Capture_d’écran_2012-07-19_à_15.41.23.png\|center\|600px]]
		+
		+	We will use the leader sequence of OmpF (a porine). The authors of the article found a post-translational repression of up to 145-fold.
		+	= References =
		+	1. Sharma, V., Yamamura, A., & Yokobayashi, Y. (2012). Engineering Artificial Small RNAs for Conditional Gene Silencing in Escherichia coli, 6-13.
		+	[http://pubs.acs.org/doi/abs/10.1021/sb200001q\| Paper]

Aishah Prastowo: /* Alginate beads */

2012-07-18T09:45:11Z

Alginate beads

←Older revision		Revision as of 09:45, 18 July 2012
Line 17:		Line 17:

	=Alginate beads=		=Alginate beads=
-	Here, we would place alginate beads, with controlled alginate matrix size into the capsule. The alginate beads contains theophylline, that would act as an activator of a [http://en.wikipedia.org/wiki/Riboswitch\| riboswitch] that would repress gene expression [1]. On of our options is to deliver theophylline through bits of complex sugars that would be the carbon source of our bacteria. The riboswitch would be fused into the 5' end of the gene we want to repress (see figure 1). That gene would be one of the [http://en.wikipedia.org/wiki/Colicin \| colicin] gene, that can diffuse and kill other ''Enterobacteriaceae''. The riboswitch will be translated, and thus we need to ensure that it will not affect the function of the protein. The colicine gene is under Rpos promoter which is activated when bacteria is on stationary phase (e.g. YiaGp). So, bacteria will die on stationary phase and when theophylline is under a certain threshold, thanks to its diffusion through beads and capsule.	+	Here, we would place alginate beads, with controlled alginate matrix size into the capsule. The alginate beads contains theophylline, that would act as an activator of a [http://en.wikipedia.org/wiki/Riboswitch riboswitch] that would repress gene expression [1]. On of our options is to deliver theophylline through bits of complex sugars that would be the carbon source of our bacteria. The riboswitch would be fused into the 5' end of the gene we want to repress (see figure 1). That gene would be one of the [http://en.wikipedia.org/wiki/Colicin colicin] gene, that can diffuse and kill other ''Enterobacteriaceae''. The riboswitch will be translated, and thus we need to ensure that it will not affect the function of the protein. The colicine gene is under Rpos promoter which is activated when bacteria is on stationary phase (e.g. YiaGp). So, bacteria will die on stationary phase and when theophylline is under a certain threshold, thanks to its diffusion through beads and capsule.
	However, we need to ensure that the expression of this toxin protein is very repressed and that it will not affect the fitness of the bacteria as long as it is in stationary phase with theophylline.		However, we need to ensure that the expression of this toxin protein is very repressed and that it will not affect the fitness of the bacteria as long as it is in stationary phase with theophylline.
	If possible, we would like to try to replace the nuclease domain of the colicin gene with a restriction enzyme domain to target exclusively the synthetic genes carried by the plasmid.		If possible, we would like to try to replace the nuclease domain of the colicin gene with a restriction enzyme domain to target exclusively the synthetic genes carried by the plasmid.

Aishah Prastowo: /* Alginate beads */

2012-07-18T09:44:46Z

Alginate beads

←Older revision		Revision as of 09:44, 18 July 2012
Line 17:		Line 17:

	=Alginate beads=		=Alginate beads=
-	Here, we would place alginate beads, with controlled alginate matrix size into the capsule. The alginate beads contains theophylline, that would act as an activator of a [http://en.wikipedia.org/wiki/Riboswitch\| riboswitch] that would repress gene expression [1]. On of our options is to deliver theophylline through bits of complex sugars that would be the carbon source of our bacteria. The riboswitch would be fused into the 5' end of the gene we want to repress (see figure 1). That gene would be one of the [http://en.wikipedia.org/wiki/Colicin\| colicin] gene, that can diffuse and kill other ''Enterobacteriaceae''. The riboswitch will be translated, and thus we need to ensure that it will not affect the function of the protein. The colicine gene is under Rpos promoter which is activated when bacteria is on stationary phase (e.g. YiaGp). So, bacteria will die on stationary phase and when theophylline is under a certain threshold, thanks to its diffusion through beads and capsule.	+	Here, we would place alginate beads, with controlled alginate matrix size into the capsule. The alginate beads contains theophylline, that would act as an activator of a [http://en.wikipedia.org/wiki/Riboswitch\| riboswitch] that would repress gene expression [1]. On of our options is to deliver theophylline through bits of complex sugars that would be the carbon source of our bacteria. The riboswitch would be fused into the 5' end of the gene we want to repress (see figure 1). That gene would be one of the [http://en.wikipedia.org/wiki/Colicin \| colicin] gene, that can diffuse and kill other ''Enterobacteriaceae''. The riboswitch will be translated, and thus we need to ensure that it will not affect the function of the protein. The colicine gene is under Rpos promoter which is activated when bacteria is on stationary phase (e.g. YiaGp). So, bacteria will die on stationary phase and when theophylline is under a certain threshold, thanks to its diffusion through beads and capsule.
	However, we need to ensure that the expression of this toxin protein is very repressed and that it will not affect the fitness of the bacteria as long as it is in stationary phase with theophylline.		However, we need to ensure that the expression of this toxin protein is very repressed and that it will not affect the fitness of the bacteria as long as it is in stationary phase with theophylline.
	If possible, we would like to try to replace the nuclease domain of the colicin gene with a restriction enzyme domain to target exclusively the synthetic genes carried by the plasmid.		If possible, we would like to try to replace the nuclease domain of the colicin gene with a restriction enzyme domain to target exclusively the synthetic genes carried by the plasmid.

Ernest Mordret at 14:08, 14 July 2012

2012-07-14T14:08:57Z

←Older revision		Revision as of 14:08, 14 July 2012
Line 8:		Line 8:
	! [[IGEM:Paris_Bettencourt_2012/Previous_Biosafety_iGEM_Projects \| Previous Biosafety iGEM projects]]		! [[IGEM:Paris_Bettencourt_2012/Previous_Biosafety_iGEM_Projects \| Previous Biosafety iGEM projects]]
	\|}		\|}
-		+	[[IGEM:Paris_Bettencourt_2012/Notebooks/Delay_group/Lab_notes \| Lab notes]]
	<center>'''Delay group'''</center>		<center>'''Delay group'''</center>
	----		----

Dylan Iverson: IGEM:Paris Bettencourt 2012/Notebook/Delay group moved to IGEM:Paris Bettencourt 2012/Notebooks/Delay group over redirect

2012-07-03T17:13:27Z

IGEM:Paris Bettencourt 2012/Notebook/Delay group moved to IGEM:Paris Bettencourt 2012/Notebooks/Delay group over redirect

←Older revision

Revision as of 17:13, 3 July 2012

Dylan Iverson: IGEM:Paris Bettencourt 2012/Notebooks/Delay group moved to IGEM:Paris Bettencourt 2012/Notebook/Delay group over redirect

2012-07-03T17:13:03Z

IGEM:Paris Bettencourt 2012/Notebooks/Delay group moved to IGEM:Paris Bettencourt 2012/Notebook/Delay group over redirect

←Older revision

Revision as of 17:13, 3 July 2012

Dylan Iverson: IGEM:Paris Bettencourt 2012/Notebook/Delay group moved to IGEM:Paris Bettencourt 2012/Notebooks/Delay group

2012-07-03T17:12:28Z

IGEM:Paris Bettencourt 2012/Notebook/Delay group moved to IGEM:Paris Bettencourt 2012/Notebooks/Delay group

←Older revision

Revision as of 17:12, 3 July 2012

Jean Cury at 17:09, 3 July 2012

2012-07-03T17:09:40Z

←Older revision		Revision as of 17:09, 3 July 2012
Line 1:		Line 1:
-	{\| border = "1" width="100%"	+	{\| border = "1" width="100%"
-	! [[IGEM:Paris_Bettencourt_2012/~~Notebook/~~\| ~~Notebook~~]]	+	! [[IGEM:Paris_Bettencourt_2012/Notebooks \| Notebooks]]
-	! [[IGEM:Paris_Bettencourt_2012/Design/\| Design]]	+	! [[IGEM:Paris_Bettencourt_2012/Design \| Design]]
	! [[IGEM:Paris_Bettencourt_2012/Roadmap \| Roadmap]]		! [[IGEM:Paris_Bettencourt_2012/Roadmap \| Roadmap]]
	! [[Meetings and to-dos]]		! [[Meetings and to-dos]]
Line 8:		Line 8:
	! [[IGEM:Paris_Bettencourt_2012/Previous_Biosafety_iGEM_Projects \| Previous Biosafety iGEM projects]]		! [[IGEM:Paris_Bettencourt_2012/Previous_Biosafety_iGEM_Projects \| Previous Biosafety iGEM projects]]
	\|}		\|}
-

	<center>'''Delay group'''</center>		<center>'''Delay group'''</center>

Dylan Iverson: IGEM:Paris Bettencourt 2012/Notebook/Delay moved to IGEM:Paris Bettencourt 2012/Notebook/Delay group: harmonisation

2012-07-03T15:59:19Z

IGEM:Paris Bettencourt 2012/Notebook/Delay moved to IGEM:Paris Bettencourt 2012/Notebook/Delay group: harmonisation

←Older revision

Revision as of 15:59, 3 July 2012

Ernest Mordret at 16:11, 29 June 2012

2012-06-29T16:11:20Z

←Older revision		Revision as of 16:11, 29 June 2012
Line 18:		Line 18:

	=Alginate beads=		=Alginate beads=
-	Here, we would place alginate beads, with controlled alginate matrix size into the capsule. The alginate beads contains theophylline, that would act as an activator of a [http://en.wikipedia.org/wiki/Riboswitch\| riboswitch] that would repress gene expression [1]. ~~This~~ riboswitch would be fused into the 5'~~-UTR~~ of the gene we want to repress (see figure 1). That gene would be one of the [http://en.wikipedia.org/wiki/Colicin\| colicin] gene, that can diffuse and kill other ''Enterobacteriaceae''. The colicine gene is under Rpos promoter which is activated when bacteria is on stationary phase. So, bacteria will die on stationary phase and when theophylline is under a certain threshold, thanks to its diffusion through beads and capsule.	+	Here, we would place alginate beads, with controlled alginate matrix size into the capsule. The alginate beads contains theophylline, that would act as an activator of a [http://en.wikipedia.org/wiki/Riboswitch\| riboswitch] that would repress gene expression [1]. On of our options is to deliver theophylline through bits of complex sugars that would be the carbon source of our bacteria. The riboswitch would be fused into the 5' end of the gene we want to repress (see figure 1). That gene would be one of the [http://en.wikipedia.org/wiki/Colicin\| colicin] gene, that can diffuse and kill other ''Enterobacteriaceae''. The riboswitch will be translated, and thus we need to ensure that it will not affect the function of the protein. The colicine gene is under Rpos promoter which is activated when bacteria is on stationary phase (e.g. YiaGp). So, bacteria will die on stationary phase and when theophylline is under a certain threshold, thanks to its diffusion through beads and capsule.
-		+	However, we need to ensure that the expression of this toxin protein is very repressed and that it will not affect the fitness of the bacteria as long as it is in stationary phase with theophylline.
		+	If possible, we would like to try to replace the nuclease domain of the colicin gene with a restriction enzyme domain to target exclusively the synthetic genes carried by the plasmid.
	[[Image:Theophuline_riboswitch.png\|center\|500px]]		[[Image:Theophuline_riboswitch.png\|center\|500px]]
	<center>Figure 1 : Theophylline riboswitch, adapted from [1]</center>		<center>Figure 1 : Theophylline riboswitch, adapted from [1]</center>