Team:TUDelft/Temperature overview

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=Temperature Overview=
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===Bacterial Thermometer===
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We want to create an RNA-based system that is able to have differential gene expression when temperature changes take place (comparable to the [https://2006.igem.org/Berkeley2006-RiboregulatorsMain Berkeley iGEM 2006] riboregulator lock/key part).
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[[Image:Rna_thermometer.png|200px|right|thumb|'''Figure 1:''' RNA thermometer]]
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There are several systems suggested in literature that are based on RNA secondary structure <span id="cite_ref_1">[[Team:TUDelft/Temperature_overview#cite_note_1|[1]]]</span>. The idea in general is that if the temperature drops below a certain temperature, the RNA will form stable base-pairs on the Shine-Dalgarno sequence, disabling the ribosome to bind. The base-pairing of this RNA region will block the expression of the protein encoded behind it (figure 1). In this way gene expression can be regulated on the RNA level by temperature.
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We want to create an RNA-based system that is able to have differential gene expression when temperature changes take place (kind of similar to the Berkeley iGEM 2006 - riboregulator lock/key part [http://parts.mit.edu/wiki/index.php/Berkeley2006-RiboregulatorsMain]).  
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At the beginning of the project, an [[Team:TUDelft/Temperature_analysis|analysis]] has been performed to get more insight into the functioning of an RNA thermometer. The design of the temperature sensitive switch is divided in two phases. In [[Team:TUDelft/Temperature_design|the first phase]] existing RNA thermometers have been turned into BioBrick Standard Biological Parts. In the [[Team:TUDelft/Temperature_design2|second phase]], an RNA thermometer has been redesigned in order to shift the switching temperature using the knowledge gained during the analysis.
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There are several systems suggested in literature that are based on RNA secondary structure. The idea in general is that if the temperature drops below a certain temperature, the RNA will form stable base-pairs on the Shine-Dalgarno sequence, disabling the ribosome to bind. The base-pairing of the RNA will impair the ability of the cell to express the protein encoded behind it. In this way gene expression can be regulated on the RNA level by temperature. The different ways that are described in literature are:
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Also some [[Team:TUDelft/Temperature_software|software]] tools have been developed during the project. The first tool, the [[Team:TUDelft/Temperature_software#Stability_Profile_Plotter|Stability Profile Plotter]], has been used during the analysis, to produce plots that characterize the stability of an RNA hairpin structure. The second tool, the [[Team:TUDelft/Temperature_software#RNA_Hairpin_Designer|RNA Hairpin Designer]], provides a (partial) implementation of the design approach as proposed in the second design phase. This tool can be used for the design of temperature sensitive RNA hairpins.
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====Virulence derived expression====
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The results of the labwork indicate that sonication of our samples, obtained the most reliable results. These indicated, as displayed [[Team:TUDelft/Temperature_results#Luciferase_Measurements|here]], that our strain K115035 shows the temperature induced switch-like behavior which was the aim of our project.
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Various pathogenic microorganisms express virulence proteins only inside a host. It has been shown in the 90's  this is induced by the increased temperature having effect on translation but not on transcription [http://jb.asm.org/cgi/reprint/175/24/7901]. Examples of microorganisms using this temperature induced virulence are ''Salmonella'' [http://www3.interscience.wiley.com/cgi-bin/fulltext/118542064/PDFSTART], ''Yersinia pestis'' or ''Listeria monocytogenes''. Of course we won't work with these virulent genes, only with the regulating mRNA sequences in front of them or their TF. An induction temperature of 37 degrees C seems logical.
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====(Designed)G-Quadruplex structures====
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==References==
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As described by Wieland et al. [http://www.sciencedirect.com/science?_ob=MImg&_imagekey=B6VRP-4P8YWVM-7-8&_cdi=6240&_user=499885&_orig=search&_coverDate=07%2F30%2F2007&_sk=999859992&view=c&wchp=dGLbVzz-zSkzS&md5=97741390ae4ff0d9a8c4d5f41beb0f1d&ie=/sdarticle.pdf] it is possible to design sequences that form tertiary structures occluding the RBS. A temperature range of 30 - 35 degrees C has been achieved.
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====ROSE====
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<ol class="references">
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''R''epressor ''O''f heat-''S''hock gene ''E''xpression (ROSE) is the (conserved) mRNA sequence found in front of some prokaryotic heat-shock proteins.[http://www.nature.com/emboj/journal/v25/n11/pdf/7601128a.pdf] Turning this into a biobrick should allow induction of translation by heating to 42 degrees C.
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<li id="cite_note_1"> [[Team:TUDelft/Temperature_overview#cite_ref_1 | ^]] Narberhaus F. mRNA-mediated detection of environmental conditions. ''Archives of Microbiology'', 178(6):404-410, 2002. [http://www.ncbi.nlm.nih.gov/pubmed/16680139 PMID:16680139]</li>
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</ol>
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Latest revision as of 17:54, 29 October 2008

Temperature Overview

We want to create an RNA-based system that is able to have differential gene expression when temperature changes take place (comparable to the Berkeley iGEM 2006 riboregulator lock/key part).

Figure 1: RNA thermometer

There are several systems suggested in literature that are based on RNA secondary structure [1]. The idea in general is that if the temperature drops below a certain temperature, the RNA will form stable base-pairs on the Shine-Dalgarno sequence, disabling the ribosome to bind. The base-pairing of this RNA region will block the expression of the protein encoded behind it (figure 1). In this way gene expression can be regulated on the RNA level by temperature.

At the beginning of the project, an analysis has been performed to get more insight into the functioning of an RNA thermometer. The design of the temperature sensitive switch is divided in two phases. In the first phase existing RNA thermometers have been turned into BioBrick Standard Biological Parts. In the second phase, an RNA thermometer has been redesigned in order to shift the switching temperature using the knowledge gained during the analysis.

Also some software tools have been developed during the project. The first tool, the Stability Profile Plotter, has been used during the analysis, to produce plots that characterize the stability of an RNA hairpin structure. The second tool, the RNA Hairpin Designer, provides a (partial) implementation of the design approach as proposed in the second design phase. This tool can be used for the design of temperature sensitive RNA hairpins.

The results of the labwork indicate that sonication of our samples, obtained the most reliable results. These indicated, as displayed here, that our strain K115035 shows the temperature induced switch-like behavior which was the aim of our project.

References

  1. ^ Narberhaus F. mRNA-mediated detection of environmental conditions. Archives of Microbiology, 178(6):404-410, 2002. [http://www.ncbi.nlm.nih.gov/pubmed/16680139 PMID:16680139]
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