Team:TUDelft/Temperature design

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(Design phase I: Introducing an RNA thermometer into Escherichia coli)
 
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>> work in progress
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=Design phase I: <br /><br />Introducing an RNA thermometer into ''Escherichia coli''=
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[http://partsregistry.org/wiki/index.php?title=Part:BBa_K115001 BBa:K115001], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K115002 BBa:K115002],
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The design of artificial RNA thermometers is split in two phases. In the first phase, some known RNA thermometers are turned into BioBrick Standard Biological Parts, and in the [[Team:TUDelft/Temperature_design2|second phase]], RNA thermometers with different switching temperatures are designed. This page is devoted to the first design phase, in which the parts [http://partsregistry.org/wiki/index.php?title=Part:BBa_K115001 <code>BBa_K115001</code>], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K115002 <code>BBa_K115002</code>], and
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[http://partsregistry.org/wiki/index.php?title=Part:BBa_K115003 BBa:K115003],
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[http://partsregistry.org/wiki/index.php?title=Part:BBa_K115003 <code>BBa_K115003</code>] have been designed.
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[http://partsregistry.org/wiki/index.php?title=Part:BBa_K115008 BBa:K115008],
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[http://partsregistry.org/wiki/index.php?title=Part:BBa_K115009 BBa:K115009]
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=Introducing an RNA thermometer into e. Coli=
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==Three existing RNA thermometers==
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The first phase will be used to test if known RNA thermometers can be turned into standard biobricks and incorporated, iGEM style, into e. coli. A literature study is done to find RNA thermometers that are tested and proven to be working RNA thermometers.  
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The goal of the first phase is to test whether known RNA thermometers can be turned into a BioBrick Standard Biological Part and incorporated into ''Escherichia coli''. We selected three RNA thermometers from literature, one per RNA thermometer family. For each of these RNA thermometers it has been shown that they can be introduced into ''E. coli'', where they still function as a temperature sensitive switch.
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We selected three RNA thermometers (one per RNA thermometer family) that were retrieved from different organisms. A fourth working RNA thermometer attracted our attention because it was not retrieved from an organism, but a designed one. Unfortunately, as would come true later, this RNA thermometer cannot be turned into a biobrick.
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The first RNA thermometer is one of the Repressor Of heat-Shock gene Expression (ROSE) family and is retrieved from the organism ''Bradyrhizobium japonicum'' <span id="cite_ref_1">[[Team:TUDelft/Temperature_design#cite_note_1|[1]]]</span>. This RNA thermometer is switched off at 30°C and allows induction of translation by heating to 42°C. The second RNA thermometer is one of the FourU family and is retrieved from ''Salmonella''. This pathogenic microorganism expresses virulence proteins only inside a host <span id="cite_ref_2">[[Team:TUDelft/Temperature_design#cite_note_2|[2]]]</span>. An induction temperature of this switch should be around 37°C, the body temperature of the host. The third RNA thermometer is retrieved from the ''Listeria monocytogenes'' and belongs to the PrfA family. Translation is induced at 37°C <span id="cite_ref_3">[[Team:TUDelft/Temperature_design#cite_note_3|[3]]]</span>.
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===The three selected RNA thermometers===
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==From RNA thermometer to Standard Biological Part==
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The first RNA thermometer is one of the ROSE family and is retrieved from the organism Bradyrhizobium japonicum. Repressor Of heat-Shock gene Expression (ROSE) is the (conserved) mRNA sequence found in front of some prokaryotic heat-shock proteins <sup id="cite_ref-1"> [[Team:TUDelft/Temperature_design#cite_note-1 |[1]]]</sup>. Turning this into a biobrick should give an RNA thermometer that is switched off at 30 °C and allow induction of translation by heating to 42 °C.
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The second RNA thermometer is one of the FourU family and is retrieved from Salmonella. 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 <sup id="cite_ref-2"> [[Team:TUDelft/Temperature_design#cite_note-2 |[2]]]</sup>. Examples of microorganisms using this temperature induced virulence are ''Salmonella'' <sup id="cite_ref-3"> [[Team:TUDelft/Temperature_design#cite_note-3 |[3]]] </sup>, ''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 °C seems logical.
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[[Image:Overlap.png|thumb|200px|right|'''Figure 1:''' Overlap between the RNA thermometer and the protein coding part. These parts cannot be ligated this way because of the stop codon that is part of the scar.]]
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[[Image:Chopped.png|thumb|200px|right|'''Figure 2:''' The part of the RNA thermometer sequence that needs to be chopped of, in order to end up with a sequence in which the Shine Dalgarno region and the start codon of the protein coding part are separated by the desired six nucleotides.]]
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[[Image:ScarProblemss.png|200px|thumb|right|'''Figure 3:''' Introduction of the scar causes a change in the secondary structure of the RNA thermometer.]]
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[[Image:ScarProblem.png|thumb|200px|right|'''Figure 4:''' The original secondary structure is regained by alteration of the nucleotides opposite of the scar region.]]
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[[Image:Design1.png|thumb|200px|right|'''Figure 5:''' Three RNA thermometer parts. At the left, the secondary structures of the original sequences is given. At the right, the secondary structure of the parts, as they are added to the registry, are given. The blue dots around the nucleotides indicate the mutation that had to be made in order to adjust the secondary structure after introduction of the scar.]]
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The third RNA thermometer is retrieved from the Listeria monocytogenes and belongs to the PrfA family. The switching temperature is at 37 degrees Celsius <sup id="cite_ref-4"> [[Team:TUDelft/Temperature_design#cite_note-4 |[4]]]</sup>.
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Turning a protein coding gene into a Standard Biological Part is straightforward. If the DNA sequence contains any of the forbidden restriction sites, the only thing that has to be done is to remove those. Turning the RNA thermometer sequences into Standard Biological Parts is less straightforward. The reason for this is that the start codon of the protein coding part, following the RNA thermometer, is also part of the RNA thermometer sequence as found in literature ([https://2008.igem.org/Image:Overlap.png figure 1]).
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Properties of the 3 used DNA pieces...
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====Getting the right sequence====
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<div id="v_toggle"">
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A resulting sequence after ligation of two parts will contain a scar in between the two ligated parts. If both the RNA thermometer sequence and the protein coding sequence are kept intact, the resulting sequence after ligation will be the one shown at the bottom of [https://2008.igem.org/Image:Overlap.png figure 1]. The resulting sequence contains two start codons of which only the leftmost one will act as initiation point for the translation. This is because of its distance to the Shine Dalgarno sequence, which should be six or seven nucleotides.
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===Artificial RNA thermometer based on G-quadruplex===
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</div>
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<div id=vertical_slide>
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Unfortunately there is a problem with this resulting sequence. Because of the shift of the start codon to the 5' side, some extra amino acids will be added to the protein. This could already by a problem, but even worse, the scar contains a stop codon. This will cause the translation process to be terminated even before the protein coding region is reached.
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A fourth working RNA thermometer found in literature is an artificial one <sup id="cite_ref-5"> [[Team:TUDelft/Temperature_design#cite_note-5 |[5]]]</sup>. It is based on a special tertiary (3D) structure in which an RNA stretch can fold. This structure also occludes the Shine Dalgarno region and thereby blocks the translation. Above a certain threshold temperature the structure becomes unstable and the Shine-Dalgarno becomes exposed, enabling the ribosome to bind to the RNA and initiate the translation process.
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Although this artificial RNA thermometer is very interesting, it turned out to be impossible make a biobrick out of it. As can be read in the design part (link) the scar (the result of the ligation of the RNA thermometer part with the protein coding part) has to be introduced in the RNA thermometer. Unfortunately the introduction of the scar changes the structure that makes the part temperature sensitive. Therefor this G-quadruplex RNA thermometer is not taken into account any further.
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Still it remains very interesting, because it shows that an artificial thermometer can be made using only the structure of the RNA.
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</div>
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===Scar problem===
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Another option would be to chop off the 3' end side of the RNA thermometer sequence. In order to get a gap of six nucleotides between the SD and the start codon, the RNA thermometer sequence has to be chopped of after the SD sequence as shown in [https://2008.igem.org/Image:Chopped.png figure 2]. The resulting sequence after ligation contains a SD and a start codon separated by six nucleotides, the same as the distance between them in the original sequence of the RNA thermometer.
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Turning the RNA thermometer sequences into standard biobricks is not as straightforward as adding a prefix and suffix to the found sequences. The reason for this is that the start codon of the protein coding part following the RNA thermometer is also part of the RNA thermometer sequence (figure ... sequences met atg overlap). The problem with this approach is that the ligation of the two parts results in a sequence containing two start codons with in between the scar (figure ... wrong situation sticking together two parts). The first of these start codon (the one on the RNA thermometer) will act as the initiation point for the translation, because of its distance to the Shine Dalgarno sequence. At first some extra amino acids will be added to the protein, but even worse, the protein will not be translated at all. This is because of the scar that contains a stop codon. So the translation will already end before the protein coding region is reached.
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====Getting the right secondary structure====
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[[Image:Tudelft_img_partsDesign_1_0.png]]
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But a new problem arises when we look at the [[Team:TUDelft/Temperature_analysis#RNA_secondary_structure_and_free_energy|secondary structure]] of this new sequence. As can be seen in [https://2008.igem.org/Image:ScarProblemss.png figure 3], the secondary structure of the new sequence (after ligation to a protein coding part) is different compared to the secondary structure of the original sequence of the RNA thermometer, while this secondary structure is essential for the temperature sensitivity.
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To solve this problem we have to alter the sequence of the RNA thermometer. Alteration of the protein coding part is of course no option because we are aiming for a standard part that can be combined with any other standardized protein coding part. So these parts, that always start with a start codon should be kept intact. The simple solution is to chop of the last part of the RNA thermometer sequence, which will be replaced by the scar and the start of the protein coding part after ligation (figure ... solution without scar adaption).
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Through the change of the nucleotides in the scar region, the base pairing with the opposite base pairs is lost. The original secondary structure can be regained by changing the nucleotides opposite the scar region, so that the nucleotides in that region form base pairs again ([https://2008.igem.org/Image:ScarProblem.png figure 4]).
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[[Image:Tudelft_img_partsDesign_2_0.png]]
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==Result==
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But this results in a new problem. The alteration of the RNA thermometer sequence also changes its secondary structure and that will also affect its function as temperature sensor (figure ... change of secondary structure). To resolve this we made some extra alteration in the RNA thermometer sequence at the opposite position of the scar in order to regain the original secondary structure.
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The example in the previous section has shown how the FourU RNA thermometer sequence that is retrieved from ''Salmonella'' can be turned into a Standard Biological Part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K115002 <code>BBa_K115002</code>]). The same method has also been used for the design of the other two RNA thermometer parts that are based on sequences from ''Bradyrhizobium japonicum''  and ''Listeria monocytogenes''. This has resulted in the two new parts: [http://partsregistry.org/wiki/index.php?title=Part:BBa_K115001 <code>BBa_K115001</code>] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K115003 <code>BBa_K115003</code>] respectively. The secondary structures of the original sequences and of the designed sequences (after ligation to a protein coding part) are shown in [https://2008.igem.org/Image:Design1.png figure 5].
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We applied this procedure to three of the four RNA thermometers that we got from the literature. We used the Vienna RNAfold webserver to predict the secondary structure of the original RNA thermometer and the altered sequence. We regained the original secondary structure using a trial and error approach adding different nucleotides opposite of the added scar...
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An alternative solution would be to combine the RNA thermometer sequence and the protein coding sequence into one part, instead of having a separate part for both sequences. The downside of this solution is that a new part has to be added to the registry for each protein that needs to be regulated by temperature. Conforming the iGEM vision it is desired to make a separate temperature sensitive part that can be reused in combination with any protein coding part without going over the same design process over and over again. That is why separate temperature sensitive parts have been designed.
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The fourth RNA thermometer, the artificial one based on the G-quadruplex structure, will loose its temperature sensitivity when the scar is introduced into the sequence. It happens to replace multiple G nucleotides that make up the G-quadruplex structure which is responsible for the temperature sensitivity.
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==References==
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Having designed sequences that have the same secondary structure does not guarantee the same functionality. The pairing nucleotides around the Shine Dalgarno region are changed, so the temperature needed to expose the Shine Dalgarno sequence will probably be changed. Tests will have to point out what the switching temperature of the RNA thermometer parts will be. This is of course if the parts work at all. There are probably a lot of factors (more than the predicted secondary structure can tell us) that play a role in the temperature sensing process, also many that we don't now of. Even one mutation can already lead ...
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===References===
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<ol class="references">
<ol class="references">
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<li id="cite_note-1"> [[Team:TUDelft/Temperature_design#cite_ref-1 | ^]] Chowdhurry S, Maris C, Allain F H T, Narberhaus F (2006). "Molecular basis for temperature sensing by an RNA thermometer". The EMBO Journal, 2006, 25, 2487–2497. [http://www.ncbi.nlm.nih.gov/pubmed/16710302 PMID:16710302] </li>
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<li id="cite_note_1"> [[Team:TUDelft/Temperature_design#cite_ref_1 | ^]] Saheli Chowdhury, Christophe Maris, Frédéric H-T Allain, and Franz Narberhaus. Molecular basis for temperature sensing by an RNA thermometer. ''The EMBO Journal'', 25:2487–2497, 2006. [http://www.ncbi.nlm.nih.gov/pubmed/16710302 PMID:16710302] </li>
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<li id="cite_note-2"> [[Team:TUDelft/Temperature_design#cite_ref-2 | ^]] Hoe N P, Goguen J D (1993). "Temperature sensing in Yersinia pestis: Translation of the LcrF activator protein is thermally regulated". J Bacteriol, 1993 December, 175(24), 7901-7909. [http://www.ncbi.nlm.nih.gov/pubmed/7504666 PMID:7504666] </li>
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<li id="cite_note-3"> [[Team:TUDelft/Temperature_design#cite_ref-3 | ^]] Waldminghaus T, Heidrich N, Branti S, Narberhaus F (2007). "FourU: a novel type of RNA thermometer in Salmonella". Molecular Microbiology, Volume 65, Issue 2, 413-424. [http://www.ncbi.nlm.nih.gov/pubmed/17630972 PMID:17630972]</li>
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<li id="cite_note-4"> [[Team:TUDelft/Temperature_design#cite_ref-4 | ^]] Johansson J, Mandin P, Renzoni A, Chiaruttinni C, Springer M, Cossart P. "An RNA thermosensor controls expression of virulance genes in Listeria monocytogenes". Cell , Volume 110 , Issue 5 , 551-561. [http://www.ncbi.nlm.nih.gov/pubmed/12230973 PMID:12230973] </li>
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<li id="cite_note_2"> [[Team:TUDelft/Temperature_design#cite_ref_2 | ^]] Torsten Waldminghaus, Nadja Heidrich, Sabine Brantl, and Franz Narberhaus. FourU: a novel type of RNA thermometer in Salmonella. ''Molecular Microbiology'', 65(2):413-424, 2007. [http://www.ncbi.nlm.nih.gov/pubmed/17630972 PMID:17630972]</li>
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<li id="cite_note-5"> [[Team:TUDelft/Temperature_design#cite_ref-5 | ^]] Wieland M, Hartig J (2007). "RNA Quadruplex-Based Modulation of Gene Expression". Chemistry & Biology, Volume 14, Issue 7, 757-763. [http://www.ncbi.nlm.nih.gov/pubmed/17656312 PMID:17656312]</li>
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<li id="cite_note_3"> [[Team:TUDelft/Temperature_design#cite_ref_3 | ^]] J . Johansson, P . Mandin, A . Renzoni, C . Chiaruttini, M . Springer, and P . Cossart. An RNA thermosensor controls expression of virulance genes in Listeria monocytogenes. ''Cell'' , 110(5):551-561, 2002. [http://www.ncbi.nlm.nih.gov/pubmed/12230973 PMID:12230973] </li>
</ol>
</ol>
{{Template:TUDelftiGEM2008_sidebar}}
{{Template:TUDelftiGEM2008_sidebar}}

Latest revision as of 07:59, 29 October 2008

Contents

Design phase I:

Introducing an RNA thermometer into Escherichia coli

The design of artificial RNA thermometers is split in two phases. In the first phase, some known RNA thermometers are turned into BioBrick Standard Biological Parts, and in the second phase, RNA thermometers with different switching temperatures are designed. This page is devoted to the first design phase, in which the parts [http://partsregistry.org/wiki/index.php?title=Part:BBa_K115001 BBa_K115001], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K115002 BBa_K115002], and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K115003 BBa_K115003] have been designed.

Three existing RNA thermometers

The goal of the first phase is to test whether known RNA thermometers can be turned into a BioBrick Standard Biological Part and incorporated into Escherichia coli. We selected three RNA thermometers from literature, one per RNA thermometer family. For each of these RNA thermometers it has been shown that they can be introduced into E. coli, where they still function as a temperature sensitive switch.

The first RNA thermometer is one of the Repressor Of heat-Shock gene Expression (ROSE) family and is retrieved from the organism Bradyrhizobium japonicum [1]. This RNA thermometer is switched off at 30°C and allows induction of translation by heating to 42°C. The second RNA thermometer is one of the FourU family and is retrieved from Salmonella. This pathogenic microorganism expresses virulence proteins only inside a host [2]. An induction temperature of this switch should be around 37°C, the body temperature of the host. The third RNA thermometer is retrieved from the Listeria monocytogenes and belongs to the PrfA family. Translation is induced at 37°C [3].

From RNA thermometer to Standard Biological Part

Figure 1: Overlap between the RNA thermometer and the protein coding part. These parts cannot be ligated this way because of the stop codon that is part of the scar.
Figure 2: The part of the RNA thermometer sequence that needs to be chopped of, in order to end up with a sequence in which the Shine Dalgarno region and the start codon of the protein coding part are separated by the desired six nucleotides.
Figure 3: Introduction of the scar causes a change in the secondary structure of the RNA thermometer.
Figure 4: The original secondary structure is regained by alteration of the nucleotides opposite of the scar region.
Figure 5: Three RNA thermometer parts. At the left, the secondary structures of the original sequences is given. At the right, the secondary structure of the parts, as they are added to the registry, are given. The blue dots around the nucleotides indicate the mutation that had to be made in order to adjust the secondary structure after introduction of the scar.

Turning a protein coding gene into a Standard Biological Part is straightforward. If the DNA sequence contains any of the forbidden restriction sites, the only thing that has to be done is to remove those. Turning the RNA thermometer sequences into Standard Biological Parts is less straightforward. The reason for this is that the start codon of the protein coding part, following the RNA thermometer, is also part of the RNA thermometer sequence as found in literature (figure 1).

Getting the right sequence

A resulting sequence after ligation of two parts will contain a scar in between the two ligated parts. If both the RNA thermometer sequence and the protein coding sequence are kept intact, the resulting sequence after ligation will be the one shown at the bottom of figure 1. The resulting sequence contains two start codons of which only the leftmost one will act as initiation point for the translation. This is because of its distance to the Shine Dalgarno sequence, which should be six or seven nucleotides.

Unfortunately there is a problem with this resulting sequence. Because of the shift of the start codon to the 5' side, some extra amino acids will be added to the protein. This could already by a problem, but even worse, the scar contains a stop codon. This will cause the translation process to be terminated even before the protein coding region is reached.

Another option would be to chop off the 3' end side of the RNA thermometer sequence. In order to get a gap of six nucleotides between the SD and the start codon, the RNA thermometer sequence has to be chopped of after the SD sequence as shown in figure 2. The resulting sequence after ligation contains a SD and a start codon separated by six nucleotides, the same as the distance between them in the original sequence of the RNA thermometer.

Getting the right secondary structure

But a new problem arises when we look at the secondary structure of this new sequence. As can be seen in figure 3, the secondary structure of the new sequence (after ligation to a protein coding part) is different compared to the secondary structure of the original sequence of the RNA thermometer, while this secondary structure is essential for the temperature sensitivity.

Through the change of the nucleotides in the scar region, the base pairing with the opposite base pairs is lost. The original secondary structure can be regained by changing the nucleotides opposite the scar region, so that the nucleotides in that region form base pairs again (figure 4).

Result

The example in the previous section has shown how the FourU RNA thermometer sequence that is retrieved from Salmonella can be turned into a Standard Biological Part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K115002 BBa_K115002]). The same method has also been used for the design of the other two RNA thermometer parts that are based on sequences from Bradyrhizobium japonicum and Listeria monocytogenes. This has resulted in the two new parts: [http://partsregistry.org/wiki/index.php?title=Part:BBa_K115001 BBa_K115001] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K115003 BBa_K115003] respectively. The secondary structures of the original sequences and of the designed sequences (after ligation to a protein coding part) are shown in figure 5.

An alternative solution would be to combine the RNA thermometer sequence and the protein coding sequence into one part, instead of having a separate part for both sequences. The downside of this solution is that a new part has to be added to the registry for each protein that needs to be regulated by temperature. Conforming the iGEM vision it is desired to make a separate temperature sensitive part that can be reused in combination with any protein coding part without going over the same design process over and over again. That is why separate temperature sensitive parts have been designed.

References

  1. ^ Saheli Chowdhury, Christophe Maris, Frédéric H-T Allain, and Franz Narberhaus. Molecular basis for temperature sensing by an RNA thermometer. The EMBO Journal, 25:2487–2497, 2006. [http://www.ncbi.nlm.nih.gov/pubmed/16710302 PMID:16710302]
  2. ^ Torsten Waldminghaus, Nadja Heidrich, Sabine Brantl, and Franz Narberhaus. FourU: a novel type of RNA thermometer in Salmonella. Molecular Microbiology, 65(2):413-424, 2007. [http://www.ncbi.nlm.nih.gov/pubmed/17630972 PMID:17630972]
  3. ^ J . Johansson, P . Mandin, A . Renzoni, C . Chiaruttini, M . Springer, and P . Cossart. An RNA thermosensor controls expression of virulance genes in Listeria monocytogenes. Cell , 110(5):551-561, 2002. [http://www.ncbi.nlm.nih.gov/pubmed/12230973 PMID:12230973]