Revision as of 15:55, 12 October 2016

Results

Improvement of a previous part, BBa_K13372001

As a part of the characterization of a previous existing Biobrick Part, we have chosen the [http://parts.igem.org/Part:BBa_K1372001 BBa_K13372001] biobrick from the Paris-Saclay 2014 project This is not a lemon. It was designed to mimic the ripening of a lemon in E. coli by a salycilate-inducible expression of a suppressor tRNA.

The Paris Saclay 2014 team chose to use chromoproteins to express these colours in E. coli. Chromoproteins are reflective proteins that contain a pigmented prosthetic group and do not need to be excited to be seen. They fused a yellow chromoprotein with a blue one in order to display a green color. This construction is referred as the green fusion chromoprotein. In order to make the bacteria ripe like a real lemon, they decided to take advantage of the fusion protein’s design by using a translational suppression system. They added an amber codon (stop codon) within the linker separating the yellow and the blue chromoproteins genes. Therefore, the suppressor tRNA will suppress amber codon allowing the translation of the green fusion chromoprotein in presence of salicylate. Conversely, the down regulation of the suppressor tRNA in absence of salicylate will allow bacteria switch from green to yellow, thus simulating the ripening of a real lemon. This system is referred to as the colour switch system.

summary of the lemon ripening project

The tRNA used is the supD suppressor tRNA. It has been placed under control of a salicylate inducible promoter Psal. Its role is to suppress the introduced amber codon. The nahR gene encodes a transcriptional regulator that is induced by salicylate and thus binds nah or Psal promoters. In presence of high salicylate concentration in the agar media, supD will be expressed and so the green fusion chromoprotein: bacteria will display a green color. However, as bacteria grow into agar, less salicylate will remain available into the media. Thus, the decrease of the nahR-salicylate complex amount within bacteria will lead to supD downregulation through time. In turn, decrease of supD amount will result in less codon readthrough and so less translation of the green fusion protein and more translation of the yellow chromoprotein. As a result, bacteria will gradually change from green to yellow.

explanatory diagram of the lemon ripening

Characterization

In order to characterize the biobrick, the color switch system (BBa_K13372001) was tested on three different constructions:

TAA: LacZ and Luc coding sequences in the same open reading frame, separated with an ochre stop codon.
TQ: LacZ and Luc coding sequences in the same open reading frame.
TAG: LacZ and Luc coding sequences in the same open reading frame, separated with an amber stop codon.

Each condition was tested under three different salicylate concentrations. In order to achieve that, both measurements of Beta-Galactosidase and Luciferase activities were performed on bacteria cultures.

The experiment was conducted on three sets of cultures of bacteria:

Remarque : attention on doit parler du plasmide contenant BBa_K13372001 + on a pas dit avant pour pcl qui s’appelle d’ailleurs pcl99

TAA: BL21|BBa_K1372001 and pcl_TAA
TQ: BL21|BBa_K1372001 and pcl_Tq
TAG: BL21|BBa_K1372001 and pcl_TAG

Each of those sets of culture were incubated with three different salicylate concentrations: 0, 30µM and 1mM.

Each construction (TAA, TQ or TAG) was tested on 3 different clones (clones 1, 2 or 3), with 3 different salicylate concentrations (0, 30µM or 1mM), with in addition a negative control sample.

Pcl_TAA construction contains two TAA stop codon within the green fusion chromoprotein linker. This codon is not recognized by the supD suppressor t-RNA, thus no blue fluorescence is expected. pcl_Tq construction does not contain any stop codon, thus the green fusion chromoprotein construction should entirely been transcripted and emit both yellow and blue fluorescence. pcl_TAG contains the TAG codon recognized by supD suppressor t-RNA. Thus the transcription and the blue fluorescence of AeBlue should be inducible by salicylate. The luciferase luminescence is expected to vary depending to the different constructions conditions and to salicylate concentrations, instead of the Beta Galactosidase activity, which will remain constant. Thus luciferase data were normalized with those from Beta Galactosidase and our results are expressed as the Luciferase/Beta-Galactosidase activity. This ratio is then independent of the level of transcription, initiation or mRNA stability.

T--Paris Saclay--activity Luc Gal Tq fonction salicylate.PNG

The Tq plasmid does not contain any stop codon between LacZ and Luc. Thus, no matter the salicylate concentration, both Luciferase and Beta Galactosidase activities are supposed to be detected. As expected a high level of Luciferase/bGal activity is observed, but the ratio decreases when salicylate concentration increases. Indeed, both activities of Luciferase and bGal drop from 30µM of salicylate, but luciferase activity was more affected by the salicylate than bGal one. One may hypothesis that salicylate inhibits differently both reporter proteins activities, with a stronger inhibition of luciferase activity. We cannot determine whether this inhibition is due to a physiological consequence onto bacteria metabolism or occures after protein extraction.

In order to read the next results, we calculated a readthrough percentage, by doing a ratio between luciferase/bGal from TAA or TAG constructs and luciferase/bGal from TQ. In this way, we obtain a readthrough percentage.

T--Paris Saclay--activity Luc Gal TAA fonction salicylate.PNG

In TAA condition, regardless of the salicylate concentration, there is no significant Luciferase activity, so the ratio remains very low at any concentrations. We conclude that supD suppressor tRNA is very specific of the TAG codon and has no impact on the TAA stop codon.

T--Paris Saclay--activity Luc Gal TAG fonction salicylate.PNG

In TAG condition we can see an increase of stop codon readthrough activity with the increase of the salicylate concentration. In comparison to the results obtained with the TAA construction, the readthrough level increases similarly to the concentration of salicylate. This indicates that TAG stop codon is efficiently readthrough in presence of supD tRNA, allowing the production of a significant amount of luciferase.

In conclusion, the Psal promoter is fully inducible by salicylate and the suppressor tRNA is functional to suppress the TAG codon. These experiments demonstrate that BBa_K1372001 is fully functional.

@@ Line 1: / Line 1: @@
-{{Team:Paris_Saclay/project_header|titre=Description}}
+{{Team:Paris_Saclay/project_header|titre=Results}}
-=Overview=
+=Improvement of a previous part, BBa_K13372001=
-Recent observations lead to the idea that genes, not in the same operon but spatially close, are highly co-transcribed, even in the absence of regulatory factors at their promoter regions.
+As a part of the characterization of a previous existing Biobrick Part, we have chosen the [http://parts.igem.org/Part:BBa_K1372001 BBa_K13372001] biobrick from the [https://2014.igem.org/Team:Paris_Saclay/Project Paris-Saclay 2014 project '''This is not a lemon''']. It was designed to mimic the ripening of a lemon in ''E. coli'' by a salycilate-inducible expression of a suppressor tRNA.
-The iGEM Paris-Saclay project aims to study the effects of DNA topology on gene expression in ''E. coli'' by answering this question:
+The Paris Saclay 2014 team chose to use chromoproteins to express these colours in ''E. coli''. Chromoproteins are reflective proteins that contain a pigmented prosthetic group and do not need to be excited to be seen. They fused a yellow chromoprotein with a blue one in order to display a green color. This construction is referred as the green fusion chromoprotein. In order to make the bacteria ripe like a real lemon, they decided to take advantage of the fusion protein’s design by using a translational suppression system. They added an amber codon (stop codon) within the linker separating the yellow and the blue chromoproteins genes. Therefore, the suppressor tRNA will suppress amber codon allowing the translation of the green fusion chromoprotein in presence of salicylate. Conversely, the down regulation of the suppressor tRNA in absence of salicylate will allow bacteria switch from green to yellow, thus simulating the ripening of a real lemon. This system is referred to as the colour switch system.
-'''Does bringing a strong promoter closer to a weak promoter influences the expression level of genes located downstream?'''
+[[File:T--Paris_Saclay--project_2014_1.jpeg.png|400px|thumb|centre|summary of the lemon ripening project]]
-We have designed a new tool based on the CRISPR/Cas9 system to bring two specific DNA regions closer. This system is composed of two different dCas9s fused with each part of the FRB / FKBP12 dimerization system. Each dCas9 will target a specific DNA sequence, one on the chromosome and one on a plasmid, whereas the dimerization system will promote the joining of the two dCas9s when rapalog is added.
+The tRNA used is the supD suppressor tRNA. It has been placed under control of a salicylate inducible promoter Psal. Its role is to suppress the introduced amber codon. The nahR gene encodes a transcriptional regulator that is induced by salicylate and thus binds nah or Psal promoters. In presence of high salicylate concentration in the agar media, supD will be expressed and so the green fusion chromoprotein: bacteria will display a green color. However, as bacteria grow into agar, less salicylate will remain available into the media. Thus, the decrease of the nahR-salicylate complex amount within bacteria will lead to supD downregulation through time. In turn, decrease of supD amount will result in less codon readthrough and so less translation of the green fusion protein and more translation of the yellow chromoprotein. As a result, bacteria will gradually change from green to yellow.
-In order to assess whether or not this system works, we have also designed a new tool to visualize the interaction between both dCas9s. This tool is composed of a split GFP attached to two dCas9s. These two small GFP tags will interact with the complementary GFP detector only if the two dCas9s are closed enough to interact.
+[[File:T--Paris_Saclay--project_2014_2.jpeg|400px|thumb|centre|explanatory diagram of the lemon ripening]]
-<html><div style="text-align:center;"><video width="600" controls>
+==Characterization==
-<source src="https://static.igem.org/mediawiki/2016/5/5e/T--Paris_Saclay--100916_Film.mp4" type='video/mp4'/>
+In order to characterize the biobrick, the color switch system (BBa_K13372001) was tested on three different constructions:
-</video></div></html>
+* ''TAA'': LacZ and Luc coding sequences in the same open reading frame, separated with an ochre stop codon.
+* ''TQ'': LacZ and Luc coding sequences in the same open reading frame.
+* ''TAG'': LacZ and Luc coding sequences in the same open reading frame, separated with an amber stop codon.
+Each condition was tested under three different salicylate concentrations. In order to achieve that, both measurements of Beta-Galactosidase and Luciferase activities were performed on bacteria cultures.
-=Introduction=
+The experiment was conducted on three sets of cultures of bacteria:
-==The transcription and its regulation==
+<b><span style="color:red">Remarque : attention on doit parler du plasmide contenant BBa_K13372001 + on a pas dit avant pour pcl qui s’appelle d’ailleurs pcl99</span></b>
+* ''TAA'': BL21|BBa_K1372001 and pcl_TAA
-Transcription is the first step of the Central dogma of molecular biology. It is the process in which an RNA molecule is created based on the sequence of a DNA template. In this case the template is a gene, a region of DNA whose RNAs can be translated into proteins, which perform different functions within the cell.
+* ''TQ'': BL21|BBa_K1372001 and pcl_Tq
-Because the environment of bacteria is constantly changing, cells have to adapt. A part of adaptation consists in using different proteins present at different times in the cell. These changes can be the result of transcriptional regulation.
+* ''TAG'': BL21|BBa_K1372001 and pcl_TAG
+Each of those sets of culture were incubated with three different salicylate concentrations: 0, 30µM and 1mM.
-Sequences called promoters are small DNA sequences upstream of genes. RNA polymerase is the protein that performs transcription by binding to promoters before starting transcription. RNA polymerase is composed of several parts, each of them called a “subunit”. In bacteria, it’s one of these subunits that binds first to the promoter. It is called “σ-factor” '''[Fig. 1]'''.
+Each construction (TAA, TQ or TAG) was tested on 3 different clones (clones 1, 2 or 3), with 3 different salicylate concentrations (0, 30µM or 1mM), with in addition a negative control sample.
-[[File:T--Paris_Saclay--fig1.png|650px|center|]]
+Pcl_TAA construction contains two TAA stop codon within the green fusion chromoprotein linker. This codon is not recognized by the supD suppressor t-RNA, thus no blue fluorescence is expected.
+pcl_Tq construction does not contain any stop codon, thus the green fusion chromoprotein construction should entirely been transcripted and emit both yellow and blue fluorescence.
+pcl_TAG contains the TAG codon recognized by supD suppressor t-RNA. Thus the transcription and the blue fluorescence of AeBlue should be inducible by salicylate.
+The luciferase luminescence is expected to vary depending to the different constructions conditions and to salicylate concentrations, instead of the Beta Galactosidase activity, which will remain constant. Thus luciferase data were normalized with those from Beta Galactosidase and our results are expressed as the Luciferase/Beta-Galactosidase activity. This ratio is then independent of the level of transcription, initiation or mRNA stability.
-But other factors can be necessary to begin transcription or to prevent it, especially when the promoter isn’t strong enough to keep the σ-factor attached to it. All of the proteins that can bind to DNA are called DNA binding protein. The binding of a multisubunit RNA polymerase or general transcription factors to a specialized transcription promoter DNA sequence is an essential step in initiating DNA transcription in all organisms (Friedman 2013).
+[[File:T--Paris_Saclay--activity_Luc_Gal_Tq_fonction_salicylate.PNG|400px|thumb|centre|]]
-Numerous molecules and factors are involved in the mechanism of transcription and can be involved in its regulation.  Transcription factors are proteins that enhance or prevent transcription; other factors such as small molecules, DNA sequence properties, and chromosome structure also play a role even though these mechanisms are diverse and often poorly understood (Browning 2002) <b>[Fig. 2]</b>.
+The Tq plasmid does not contain any stop codon between LacZ and Luc. Thus, no matter the salicylate concentration, both Luciferase and Beta Galactosidase activities are supposed to be detected.
+As expected a high level of Luciferase/bGal activity is observed, but the ratio decreases when salicylate concentration increases. Indeed, both activities of Luciferase and bGal drop from 30µM of salicylate, but luciferase activity was more affected by the salicylate than bGal one.
+One may hypothesis that salicylate inhibits differently both reporter proteins activities, with a stronger inhibition of luciferase activity. We cannot determine whether this inhibition is due to a physiological consequence onto bacteria metabolism or occures after protein extraction.
-[[File:T--Paris_Saclay--fig2_overview.png|650px|center|]]
+In order to read the next results, we calculated a readthrough percentage, by doing a ratio between luciferase/bGal from TAA or TAG constructs and luciferase/bGal from TQ. In this way, we obtain a readthrough percentage.
-==DNA binding proteins==
+[[File:T--Paris_Saclay--activity_Luc_Gal_TAA_fonction_salicylate.PNG|400px|thumb|centre|]]
-These DNA binding proteins are able to bind to DNA with different strengths also called affinities. Some proteins bind DNA with the same affinity regardless of the DNA sequence, others can only be recruited by specific sequences. Four mechanisms by which a DNA binding protein finds its target sequence are known: sliding, when the protein slides along DNA <b>[Fig. 4a]</b>; hopping, when the protein unbinds a DNA segment and binds to another one <b>[Fig. 4b]</b>; intersegment transfer, when the protein binds two different segments of DNA before releasing one; and looping, which consists on the binding of two DNA segments leading to a loop (Bustamante 1999, Bondos 2015) <b>[Fig. 4c,d]</b>. Among these, only the one-dimensional sliding is independent of spatial organization in the chromosome, that’s why the structural organization of genomes is very important for transcriptional regulation.
-[[File:T--Paris_Saclay--fig3_overview.png|650px|center|]]
-==Competition for binding within a cell==
+In TAA condition, regardless of the salicylate concentration, there is no significant Luciferase activity, so the ratio remains very low at any concentrations.
+We conclude that supD suppressor tRNA is very specific of the TAG codon and has no impact on the TAA stop codon.
-These four mechanisms allow for dynamic binding of proteins to their target sequences, despite the fact that these proteins are not present in large concentrations. For example, the number of genes in <i>Escherichia coli</i>  is about 5 000 and the number of active RNA polymerases is about 200 per cell, of which  140 only transcribe 2% of the genes leaving 60 polymerases for the other 4995 genes.
+[[File:T--Paris_Saclay--activity_Luc_Gal_TAG_fonction_salicylate.PNG|400px|thumb|centre|]]
-Thus, there is tight competition between genes to get transcribed: competition between the same σ-factor for several promoters, competition between σ-factors for the rest of the RNA polymerase or competition between a σ-factor and other DNA binding proteins. These competitions are fundamental in the mechanism of transcriptional regulation. They are often allowed or prevented by remodeling of the chromosome topology (Haugen 2008) [Fig. 4].
-[[File:T--Paris_Saclay--fig4_overview.png|650px|center|]]
+In TAG condition we can see an increase of stop codon readthrough activity with the increase of the salicylate concentration.
+In comparison to the results obtained with the TAA construction, the readthrough level increases similarly to the concentration of salicylate.
+This indicates that TAG stop codon is efficiently readthrough in presence of supD tRNA, allowing the production of a significant amount of luciferase.
+In conclusion, the Psal promoter is fully inducible by salicylate and the suppressor tRNA is functional to suppress the TAG codon. These experiments demonstrate that BBa_K1372001 is fully functional.
-That’s why, the link between genome topology and gene regulation is a major research topic in the subject of transcription. Although the mechanism of global gene regulation by changes in DNA organization are well established, the fine tuning of transcription is particularly hard to study and not very well understood.  Considering this gap in knowledge, new studies have to be conducted without a priori in order to determine if fine tuning of the DNA topology takes place during transcriptional regulation and if so, how does such regulation work.
-==Transcription initiation==
-Regulation can occur at every step on the pathway to gene expression, but transcription initiation is probably the most frequently regulated step (Haugen 2008). It is the result of
-# The recognition of a promoter by a σ-factor;
-# The recruitment of the other parts of the RNA polymerase;
-# The formation of an open complex, thus the unwinding of DNA from either side of the promoter;
-# The beginning of RNA synthesis and finally;
-# The release of the σ-factor (Browning 2002).
-The first step, in which the σ-factor binds to the promoter, can limit the transcription rate. Moreover one σ-factor can lead to the beginning of transcription of a gene, be released and lead to the beginning of the transcription of an other one. That’s why we think that spatially close promoters can act on each others, for example by doubling the number of sequences which can recruit a σ-factor in a certain area. A strong promoter could recruit a σ-factor and after its release, this σ-factor would have a better probability of being recruited to a weaker promoter. Conversely, a strong promoter could have a better chance of recruiting a σ-factor leading to “starving” of the weaker promoters within the same area.
-=Question=
-To understand transcriptional regulation, we have to study chromosome topology in 4-dimensions (3D + time) and its effect on the recruitment of DNA binding proteins. In this project, we try to answer to a fundamental question: are proximal promoters influencing each other’s transcriptional rates? '''[Fig. 5]'''.
-=References=
 {{Team:Paris_Saclay/project_footer}}

Difference between revisions of "Team:Paris Saclay/Description"

Revision as of 15:55, 12 October 2016

Results

Improvement of a previous part, BBa_K13372001

Characterization