Riboregulators - Revision history

WikiSysop: /* From Concept to Wet Lab */

2007-12-11T15:30:33Z

‎From Concept to Wet Lab

Erzwack: /* Modularity */

2007-12-06T20:11:37Z

‎Modularity

Erzwack: /* From Concept to Wet Lab */

2007-12-06T20:10:28Z

‎From Concept to Wet Lab

Erzwack: /* From Concept to Wet Lab */

2007-12-06T20:09:55Z

‎From Concept to Wet Lab

Erzwack: /* Design */

2007-12-06T20:05:55Z

‎Design

Erzwack: /* Design */

2007-12-06T19:59:35Z

‎Design

Erzwack: /* From Concept to Wet Lab */

2007-12-06T18:28:57Z

‎From Concept to Wet Lab

Erzwack: /* From Concept to Wet Lab */

2007-12-06T16:05:56Z

‎From Concept to Wet Lab

Erzwack: /* References */

2007-12-06T16:05:36Z

‎References

Erzwack: /* From Concept to Wet Lab */

2007-12-06T16:05:16Z

‎From Concept to Wet Lab

@@ Line 36: / Line 36: @@
 ==From Concept to Wet Lab==
-In wet lab, the crRNA sequence was placed in front of the GFP gene and introduced to cells.  Using flow cytometry, Isaacs et al. measured fluorescence of cells that had just the GFP gene, the GFP gene with crRNA upstream, the GFP gene with crRNA upstream and taRNA, and no GFP at all.  The crRNA decreased fluorescence to near basal levels.  When taRNA was present in the system, fluorescence increased by approximately a power of ten.  While the fluorescence did not equal fluorescence of cells with just GFP, the repression of gene expression with crRNA and return of expression with taRNA are strong enough to suggest the riboregulator system works (figure 12).
+In wet lab, the crRNA sequence was placed in front of the GFP gene and introduced to cells.  Using flow cytometry, Isaacs ''et al.'' measured fluorescence of cells that had just the GFP gene, the GFP gene with crRNA upstream, the GFP gene with crRNA upstream and taRNA, and no GFP at all.  The crRNA decreased fluorescence to near basal levels.  When taRNA was present in the system, fluorescence increased by approximately a power of ten.  While the fluorescence did not equal fluorescence of cells with just GFP, the repression of gene expression with crRNA and return of expression with taRNA are strong enough to suggest the riboregulator system works (figure 12).
 [[Image:experimental_taRNA.JPG]]

@@ Line 55: / Line 55: @@
 ==Modularity==
-While all of these experiments were done with the GFP gene, Isaacs et al. designed their riboregulator to be modular, capable of being used with any gene (Isaacs et al. 2004).  As the taRNA simply targets the crRNA and the crRNA can be placed in front of any gene, it can be considered modular.  The only caveat is that the crRNA construct added to the gene will need to contain the RBS unless the gene's RBS is close enough to the complement to bind to it.  Even small changes to a ribosomal binding site can can change the transcription rate of genes (Gardner et al. 2000).  If the original RBS is not close enough to the complement in the crRNA and you desire to keep the original transcriptional rate and level, you would have to redesign the crRNA and the taRNA as the complement to the RBS in the crRNA and the complement to the crRNA in the taRNA would need to be different.
+While all of these experiments were done with the GFP gene, Isaacs et al. designed their riboregulator to be modular, capable of being used with any gene (Isaacs et al. 2004).  As the taRNA simply targets the crRNA and the crRNA can be placed in front of any gene, it can be considered modular.  The above experiments were done with pBad and pLac controlling the production of taRNA.  As the system worked when the taRNA was under control of either promoter and the crRNA can be inserted upstream of any gene, this system is basically considered modular.
 ==Further Work==

← Older revision		Revision as of 20:10, 6 December 2007
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	'''Figure 12:''' The black curve represents fluorescence in cells that do not have a GFP gene (ie. the cells natural autofluorescence). The red curve represents fluorescence in cells that have GFP with crRNA upstream. The green curve represents fluorescence in cells that have GFP with crRNA upstream and produce the taRNA. The blue curve represents the fluorescence of cells that have normal GFP minus the cells' autofluorescence.		'''Figure 12:''' The black curve represents fluorescence in cells that do not have a GFP gene (ie. the cells natural autofluorescence). The red curve represents fluorescence in cells that have GFP with crRNA upstream. The green curve represents fluorescence in cells that have GFP with crRNA upstream and produce the taRNA. The blue curve represents the fluorescence of cells that have normal GFP minus the cells' autofluorescence.
−
−
−
−	These experiments were done with pBad and pLac controlling the production of taRNA. As the system worked when the taRNA was under control of either promoter and the crRNA can be inserted upstream of any gene, this system is considered modular.

@@ Line 36: / Line 36: @@
 ==From Concept to Wet Lab==
-In wet lab, the crRNA sequence was placed in front of the GFP gene and introduced to cells.  Using flow cytometry, Isaacs et al. measured fluorescence of cells that had just the GFP gene, the GFP gene with crRNA upstream, the GFP gene with crRNA upstream and taRNA, and no GFP at all.  The crRNA decreased fluorescence to near basal levels.  When taRNA was present in the system, fluorescence increased by approximately a power of ten.  While the fluorescence did not equal fluorescence of cells with just GFP, the repression of gene expression with crRNA and return of expression with taRNA are strong enough to suggest the riboregulator system works.
+In wet lab, the crRNA sequence was placed in front of the GFP gene and introduced to cells.  Using flow cytometry, Isaacs et al. measured fluorescence of cells that had just the GFP gene, the GFP gene with crRNA upstream, the GFP gene with crRNA upstream and taRNA, and no GFP at all.  The crRNA decreased fluorescence to near basal levels.  When taRNA was present in the system, fluorescence increased by approximately a power of ten.  While the fluorescence did not equal fluorescence of cells with just GFP, the repression of gene expression with crRNA and return of expression with taRNA are strong enough to suggest the riboregulator system works (figure 12).
 [[Image:experimental_taRNA.JPG]]
@@ Line 42: / Line 42: @@
 (Isaacs et al. 2004)
-'''Figure 12:''' The black curve represents fluoresnce in cells that do not have a GFP gene (ie. the cells natural autofluoresence).  The red curve represents fluoresence in cells that have GFP with crRNA upstream.  The green curve represents fluoresence in cells that have GFP with crRNA upstream and produce the taRNA.  The blue curve represents the fluorescence of cells that have normal GFP minus the cells' autofluoresence.
+'''Figure 12:''' The black curve represents fluorescence in cells that do not have a GFP gene (ie. the cells natural autofluorescence).  The red curve represents fluorescence in cells that have GFP with crRNA upstream.  The green curve represents fluorescence in cells that have GFP with crRNA upstream and produce the taRNA.  The blue curve represents the fluorescence of cells that have normal GFP minus the cells' autofluorescence.
@@ Line 49: / Line 49: @@
-Finally, riboregulators can control different genes in response to different stinuli by using different crRNA/taRNA pairs as the pairs are specific to each other.
+Finally, riboregulators can control different genes in response to different stimuli by using different crRNA/taRNA pairs as the pairs are specific to each other (figure 13).
 [[Image: Specific.JPG]]
@@ Line 55: / Line 55: @@
 (Isaacs et al. 2004)
-'''Figure 13:''' The graph shows both GFP fluorescence (black and white bars) when the ta-RNA promoter, pBad, is off (- arabinose) and on (+ arabinose).  All data is normalized to + arabinose GFP an RNA levels.  The low level GFP fluorescence when no arabinose is present shows that the efficiency of cr-RNA is not 100% but is still high.  As high GFP fluorescence is seen only when high amounts of the matching ta-RNA is present and not simply when any ta-RNA variant is present, this data shows that riboregulator pairs are specific and multiple pairs can be used to regulate multiple genes without fear that any ta-RNA produced would activated all the RNAs and not just its target RNA.
+'''Figure 13:''' The graph shows both GFP fluorescence (black and white bars) when the taRNA promoter, pBad, is off (- arabinose) and on (+ arabinose).  All data is normalized to + arabinose GFP and RNA levels.  The low level GFP fluorescence when no arabinose is present shows that the efficiency of crRNA is not 100% but is still high.  As high GFP fluorescence is seen only when high amounts of the matching taRNA is present and not simply when any taRNA variant is present, this data shows that riboregulator pairs are specific and multiple pairs can be used to regulate multiple genes without fear that any taRNA produced would activate all the RNAs and not just its target RNA.
 ==Modularity==

@@ Line 17: / Line 17: @@
-The taRNA is free floating and regulated by an inducible promoter.  The inducible promoter allows the researcher to determine under what conditions translation will be allowed.  Besides the complement to the YUNR sequence, the taRNA contains a section of high complementarity to section of the crRNA that folds over to cover the RBS, which allows the taRNA to keep the crRNA sequestered from the RBS (figure 10).
+The taRNA is free floating and regulated by an inducible promoter.  The inducible promoter allows the researcher to determine under what conditions translation will be allowed.  Besides the complement to the YUNR sequence, the taRNA contains a section of high complementarity to the section of the crRNA that folds over to cover the RBS. This complementary sequence allows the taRNA to keep the crRNA sequestered from the RBS (figure 10).
 [[Image:TaRNA.JPG]]
@@ Line 23: / Line 23: @@
 (Isaacs et al. 2004)
-'''Figure 10:''' taRNA when not in contact with crRNA sequesters the complement to the crRNA as it is extremely close to the RBS sequence.  When the YUNR complement binds to the YUNR sequence in the cr-RNA, the binding of the crRNA to the rest of its complement in the taRNA begins, and the crRNA is pulled off the RBS (figure 11).
+'''Figure 10:''' taRNA when not in contact with crRNA sequesters the complement to the crRNA as it is extremely close to the RBS sequence.  If the complement to the crRNA was not sequestered until the taRNA comes in contact with the YUNR sequence, ribosomes could potentially bind to the taRNA and decrease the efficiency of translation in the cell.  When the YUNR complement binds to the YUNR sequence in the cr-RNA, the binding of the crRNA to the rest of its complement in the taRNA begins, and the crRNA is pulled off the RBS (figure 11).
@@ Line 32: / Line 32: @@
 (Isaacs et al. 2004)
-'''Figure 11:''' In a. the crRNA is bound to the RBS and blocks ribosomes from translating the mRNA into protein.  In b. the ta-RNA comes in contact with the YUNR sequence on the crRNA and begins to bind to the rest of the cr-RNA.  In c. the taRNA fully bound to the crRNA and peeled the crRNA off the RBS.  The RBS is now free for the ribosome to bind to and begin translation.
+'''Figure 11:''' a. the crRNA is bound to the RBS and blocks ribosomes from translating the mRNA into protein.  b. the taRNA comes in contact with the YUNR sequence on the crRNA and begins to bind to the rest of the crRNA.  c. the taRNA fully bound to the crRNA and peeled the crRNA off the RBS.  The RBS is now free for the ribosome to bind to and begin translation.
 ==From Concept to Wet Lab==

@@ Line 8: / Line 8: @@
 ==Design==
-The design itself has two components: a short cis-repressed RNA sequence (crRNA) that is inserted upstream of the gene and a transactivating RNA sequence (taRNA) that targets the crRNA and is not attached to the target.  The crRNA sequence contains two fundamental components: the complement of the ribosomal binding site (RBS) and a pyrimidine-uracil-nucleotide-purine (YUNR) sequence (figure 9).  When not interacting with the taRNA, the complement of the RBS binds to the RBS, causing the crRNA to loop and block the ribosome's access to the RBS.  When the RBS is blocked, translation does not occur; the gene expression is off.  The YUNR sequence has a complement on the taRNA.  When the taRNA finds a crRNA, the interaction with the YUNR sequence begins pulling the crRNA off the RBS.
+The design itself has two components: a short cis-repressed RNA sequence (crRNA) that is inserted upstream of the gene and a transactivating RNA sequence (taRNA) that targets the crRNA and is free floating.  The crRNA sequence contains two fundamental components: the complement of the ribosomal binding site (RBS) and a pyrimidine-uracil-nucleotide-purine (YUNR) sequence (figure 9).  When not interacting with the taRNA, the complement of the RBS binds to the RBS, causing the crRNA to loop and block the ribosome's access to the RBS.  When the RBS is blocked, translation does not occur; the gene expression is off.  The YUNR sequence has a complement on the taRNA.  When the taRNA finds a crRNA, the interaction with the YUNR sequence begins pulling the crRNA off the RBS (figure 11).
 [[Image:CrRNA.JPG ]]
@@ Line 14: / Line 14: @@
 (Isaacs et al. 2004)
-'''Figure 9:''' Cis repressing RNA, cr-RNA, sequence is inserted upstream of the RBS.  Part of the cr-RNA (red) complements the RBS and a few bases on either side.  This section of the cr-RNA is not an exact complement; thus, the cr-RNA can be peeled off the RBS by the trans activating RNA, ta-RNA.  The complementary sequence to YUNR is found in the ta-RNA and begins peeling off the cr-RNA by binding to it (figure 11).
+'''Figure 9:''' Cis repressing RNA, crRNA, sequence is inserted upstream of the RBS.  Part of the crRNA (red) complements the RBS and a few bases on either side.  This section of the crRNA is not an exact complement; thus, the crRNA can be peeled off the RBS by the trans activating RNA, taRNA.  The complementary sequence to YUNR is found in the ta-RNA and begins peeling off the crRNA by binding to it (figure 11).
@@ Line 23: / Line 23: @@
 (Isaacs et al. 2004)
-'''Figure 10:''' ta-RNA when not in contact with cr-RNA sequesters the complement to the cr-RNA as it is extremely close to the RBS sequence.  When the YUNR complement binds to the YUNR sequence in the cr-RNA, the binding of the cr-RNA to the rest of its complement in the ta-RNA begins, and the cr-RNA is pulled off the RBS (figure 11).
+'''Figure 10:''' taRNA when not in contact with crRNA sequesters the complement to the crRNA as it is extremely close to the RBS sequence.  When the YUNR complement binds to the YUNR sequence in the cr-RNA, the binding of the crRNA to the rest of its complement in the taRNA begins, and the crRNA is pulled off the RBS (figure 11).
@@ Line 32: / Line 32: @@
 (Isaacs et al. 2004)
-'''Figure 11:''' In a. the cr-RNA is bound to the RBS and blocks ribosomes from translating the mRNA into protein.  In b. the ta-RNA comes in contact with the YUNR sequence on the cr-RNA and begins to bind to the rest of the cr-RNA.  In c. the ta-RNA fully bound to the cr-RNA and peeled the cr-RNA off the RBS.  The RBS is now free for the ribosome to bind to and begin translation.
+'''Figure 11:''' In a. the crRNA is bound to the RBS and blocks ribosomes from translating the mRNA into protein.  In b. the ta-RNA comes in contact with the YUNR sequence on the crRNA and begins to bind to the rest of the cr-RNA.  In c. the taRNA fully bound to the crRNA and peeled the crRNA off the RBS.  The RBS is now free for the ribosome to bind to and begin translation.
 ==From Concept to Wet Lab==

← Older revision		Revision as of 18:28, 6 December 2007
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	'''Figure 13:''' The graph shows both GFP fluorescence (black and white bars) when the ta-RNA promoter, pBad, is off (- arabinose) and on (+ arabinose). All data is normalized to + arabinose GFP an RNA levels. The low level GFP fluorescence when no arabinose is present shows that the efficiency of cr-RNA is not 100% but is still high. As high GFP fluorescence is seen only when high amounts of the matching ta-RNA is present and not simply when any ta-RNA variant is present, this data shows that riboregulator pairs are specific and multiple pairs can be used to regulate multiple genes without fear that any ta-RNA produced would activated all the RNAs and not just its target RNA.		'''Figure 13:''' The graph shows both GFP fluorescence (black and white bars) when the ta-RNA promoter, pBad, is off (- arabinose) and on (+ arabinose). All data is normalized to + arabinose GFP an RNA levels. The low level GFP fluorescence when no arabinose is present shows that the efficiency of cr-RNA is not 100% but is still high. As high GFP fluorescence is seen only when high amounts of the matching ta-RNA is present and not simply when any ta-RNA variant is present, this data shows that riboregulator pairs are specific and multiple pairs can be used to regulate multiple genes without fear that any ta-RNA produced would activated all the RNAs and not just its target RNA.

		+	==Modularity==

	While all of these experiments were done with the GFP gene, Isaacs et al. designed their riboregulator to be modular, capable of being used with any gene (Isaacs et al. 2004). As the taRNA simply targets the crRNA and the crRNA can be placed in front of any gene, it can be considered modular. The only caveat is that the crRNA construct added to the gene will need to contain the RBS unless the gene's RBS is close enough to the complement to bind to it. Even small changes to a ribosomal binding site can can change the transcription rate of genes (Gardner et al. 2000). If the original RBS is not close enough to the complement in the crRNA and you desire to keep the original transcriptional rate and level, you would have to redesign the crRNA and the taRNA as the complement to the RBS in the crRNA and the complement to the crRNA in the taRNA would need to be different.		While all of these experiments were done with the GFP gene, Isaacs et al. designed their riboregulator to be modular, capable of being used with any gene (Isaacs et al. 2004). As the taRNA simply targets the crRNA and the crRNA can be placed in front of any gene, it can be considered modular. The only caveat is that the crRNA construct added to the gene will need to contain the RBS unless the gene's RBS is close enough to the complement to bind to it. Even small changes to a ribosomal binding site can can change the transcription rate of genes (Gardner et al. 2000). If the original RBS is not close enough to the complement in the crRNA and you desire to keep the original transcriptional rate and level, you would have to redesign the crRNA and the taRNA as the complement to the RBS in the crRNA and the complement to the crRNA in the taRNA would need to be different.

← Older revision		Revision as of 16:05, 6 December 2007
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−	While all of these experiments were done with the GFP gene, Isaacs et al. designed their riboregulator to be modular, capable of being used with any gene (Isaacs et al. 2004). As the taRNA simply targets the crRNA and the crRNA can be placed in front of any gene, it can be considered modular. The only caveat is that the crRNA construct added to the gene will need to contain the RBS unless the gene's RBS is close enough to the complement to bind to it. Even small changes to a ribosomal binding site can can change the transcription rate of genes (Gardner et al. ~~2004~~). If the original RBS is not close enough to the complement in the crRNA and you desire to keep the original transcriptional rate and level, you would have to redesign the crRNA and the taRNA as the complement to the RBS in the crRNA and the complement to the crRNA in the taRNA would need to be different.	+	While all of these experiments were done with the GFP gene, Isaacs et al. designed their riboregulator to be modular, capable of being used with any gene (Isaacs et al. 2004). As the taRNA simply targets the crRNA and the crRNA can be placed in front of any gene, it can be considered modular. The only caveat is that the crRNA construct added to the gene will need to contain the RBS unless the gene's RBS is close enough to the complement to bind to it. Even small changes to a ribosomal binding site can can change the transcription rate of genes (Gardner et al. 2000). If the original RBS is not close enough to the complement in the crRNA and you desire to keep the original transcriptional rate and level, you would have to redesign the crRNA and the taRNA as the complement to the RBS in the crRNA and the complement to the crRNA in the taRNA would need to be different.

	==Further Work==		==Further Work==

← Older revision		Revision as of 16:05, 6 December 2007
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	==References==		==References==
		+
		+
		+	Gardner, T.S., Cantor, C.R., and Collins, J.J. Construction of a genetic toggle switch in Eschreichia coli. Nature (2000) 403: 339-342.

	Isaacs FJ, et al. Engineered riboregulators enable post-transcriptional control of gene expression. Nat Biotechnol. (2004) 22:841-47.		Isaacs FJ, et al. Engineered riboregulators enable post-transcriptional control of gene expression. Nat Biotechnol. (2004) 22:841-47.