Team:TUDelft/Temperature design2

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>> Work in progress
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=Design phase II: Changing the switching temperature of the RNA thermometer=
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=Parts Design II=
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Introducing existing RNA thermometers into ''E. coli'' provides us with a number of temperature sensitive switches, all with their own switching temperature. But this only provides a few switching temperatures, which are also relatively close to each other. In order to build a biothermometer, we needed more switching temperatures that have a bigger range. Note, however, that there will always be an upper bound in that the ''E. coli'' cannot survive temperatures above 40°C, and a lower bound in that the activity of the cell will drop at low temperatures.
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[http://partsregistry.org/wiki/index.php?title=Part:BBa_K115016 BBa:K115016], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K115017 BBa:K115017],
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This chapter presents a method that has been used to design RNA thermometer parts that should theoretically switch at a predefined temperature. The method used is based on the data analysis of which the results can be found on the [[Team:TUDelft/Temperature_analysis|analysis page]]. It has been used to design two new parts, one that should theoretically be switched on at 27°C ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K115016 <code>BBa_K115016</code>]), and two that should theoretically be switched on at 32°C ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K115017 BBa:K115017]).
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[http://partsregistry.org/wiki/index.php?title=Part:BBa_K115018 BBa:K115018],
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[http://partsregistry.org/wiki/index.php?title=Part:BBa_K115019 BBa:K115019]
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==Changing the temperature threshold==
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====Secondary structure stability and switching temperature====
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The principle of the RNA thermometer is based on base pairing between the nucleotides in the Shine Dalgarno region. At a certain temperature the RNA is folded into a structure in which base pairing nucleotides make the Shine Dalgarno region unreachable for the ribosome.  With a rise in temperature, the binding forces between the base pairing nucleotides decrease and above a certain threshold temperature the binding forces between the base pairs are to weak to hold the base pairing nucleotides together, causing the RNA to (partially) unfold. The binding between the base pairing nucleotides let loose, exposing the Shine-Dalgarno region and thereby enabling the ribosome to initiate translation.
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[[Image:HairpinStability.png|thumb|200px|right|'''Figure 1:''' '''A.''' A lot of G-C base pairs make a very stable hairpin (having a very low free energy). ''''B.''' The same hairpin structure with A-U base pairs is much less stable (having a higher free energy). '''C-E''' Bulge loops and internal loops destabilize the structure even further.]]
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When only looking at this principle, an RNA thermometer with a different temperature threshold can be designed by simply increasing or decreasing the binding forces of the base-pairing nucleotides in the Shine-Dalgarno region, shifting the temperature threshold to a higher or lower temperature respectively (fig). Increasing the binding forces could be achieved by incorporating base pairing C and G nucleotides, which bind relatively strong forming a stable helix. Decreasing the binding forces could be done by introducing less strong binding nucleotide base-pairs, such as A-U and G-U base pairs, forming a less stable helix.
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The switching temperature of the RNA thermometer depends on the stability of the hairpin structure that surrounds the Shine Dalgarno region (see also [Team:TUDelft/Temperature_analysis#Requirements_for_an_RNA_thermometer|Requirements for an RNA therometer]). This secondary structure will melt when it is exposed to a certain amount of heat. The more stable the hairpin structure is, the more heat will be needed to melt it, and the less stable the hairpin structure is, the less heat will be needed to melt it. In case of the RNA thermometer, this melting is needed to expose the Shine Dalgarno sequence which will cause the initiation of translation.
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But things are not that easy. At first, adding mutations in order to enforce or weaken the temperature sensitive region can also cause the RNA to fold into a completely different structure. This way it can lose its function as an RNA thermometer. Secondly, there are some constraints to the possible mutations; the start codon and the Shine Dalgarno sequence must off course remain unaltered and in case of a standard biobrick the scar must also be part of the temperature sensitive region (see the [https://2008.igem.org/Team:TUDelft/Temperature_design#Scar_chalenge scar problem]). Third, although it seems that only a thermosensitive hairpin is needed to have a functioning RNA thermometer (as described in the [https://2008.igem.org/Team:TUDelft/Temperature_analysis#What_is_needed_to_have_a_functioning_RNA_thermometer analysis section]) there could very well be more factors that are of influence on the functioning and temperature threshold of the RNA thermometer that are still unknown.
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In other words, a less stable hairpin structure will melt at a lower temperature than a more stable hairpin and thus, for the less stable hairpin, the translation will initiate at a lower temperature than the more stable one. This way we could theoretically shift the switching temperature to a lower or higher temperature by making the hairpin structure surrounding the SD region less or more stable respectively.
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== Design approach ==
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The stability of the hairpin structure depends on the base-pairing nucleotides within the secondary structure. For example, C-G base pairs are very strong, so a hairpin structure containing a lot of those base pairs will be very stable. A-U and U-G base pairs are less strong, so these can be used to destabilize the hairpin structure. The same can be achieved by incorporating bulge and internal loops within the hairpin structure (figure 1).
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=== Starting point ===
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====Constraints====
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In order to design an RNA thermometer with a different temperature threshold an existing RNA thermometer is taken as a starting point (TODO: explain). The one chosen is a ROSE RNA thermometer from Bradyrhizobium japonicum USDA 110 ([http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&val=NC_004463 NC_004463]) residing at location [http://www.ncbi.nlm.nih.gov/projects/sviewer/?id=NC_004463.1&v=5784144-5784239 5784144-5784239] within the genome which is at the 5' side of the gene [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=1051455&ordinalpos=2&itool=EntrezSystem2.PEntrez.Gene.Gene_ResultsPanel.Gene_RVDocSum hspB]. The reason that a sequence of the ROSE family is chosen instead of one of the FourU family is that there are more ROSE than FourU sequences available which can be used for the analysis of the temperature sensitive part of the RNA thermometer. The reason that a sequence of the ROSE family is chosen instead of one of the PrfA family is that there are more papers that provide information about the working and effects of mutation on ROSE RNA thermometers than on PrfA RNA thermometers. There is no specific reason for the choice of this ROSE RNA thermometer, this is just a ROSE RNA thermometers taken from the [http://rfam.sanger.ac.uk/family?entry=rose Rfam database]. The figure below shows the secondary structure of the whole RNA thermometer and the temperature sensitive hairpin as predicted by [http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi RNAfold] together with a plot of the stacking energies compared to the stacking energies trend as found in the analysis (TODO: link).
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<gallery Caption="Design starting point">
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There are two things that have to be taken into account. Firstly, adding mutations in order to enforce or weaken the temperature sensitive region can also cause the RNA to fold into a completely different secondary structure. This way it can lose its function as an RNA thermometer. Secondly, there are some constraints to the possible mutations. The start codon and the Shine Dalgarno sequence must off course remain unaltered and in case of a standard biobrick the scar must also be part of the temperature sensitive hairpin.
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Image:Tudelft_design_starting_point.png | Full RNA thermometer
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Image:Tudelft_design_starting_point_hairpin.png | Temperature sensitive hairpin
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Image:Tudelft_design_starting_point_energystacking.png
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</gallery>
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=== Design requirements ===
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=Design starting point=
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Having a starting point we need a way to turn this RNA thermometer that functions at 37 degrees Celcius into an RNA thermometer that functions at a different temperature and that can be turned into a standard biobrick. It is not possible to just introduce mutations anywhere in order to, for example, make the temperature sensitive hairpin less stable which should theoretically lower the temperature threshold. There are certain restriction to the possible mutation in that there must be a start codon, a Shine Dalgarno sequence, and a scar present. Also the order and distances between these parts are predefined.
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Some other requirements are stated in order to simplify the design process and making it easier to predict expected temperature threshold. At first the sequence that precedes the thermosensitive hairpin is fixed. This is done because it is still unknown what the function and influence on the temperature threshold of these extra hairpins is. Altering this sequence would thus not be usefull because we cannot tell what the concequenses of these alterations would be. As a second requirement it is stated that the overal structure of the temperature sensitive structure should remain the same. This means that the length of the hairpin is fixed, as well as the location and the size of the loop at the end of the hairpin. This requirement is also stated in order to simplify the design process. This restriction greatly reduces the solution space compared to for example the situation where there is no restriction to the length of the hairpin. It also enables to make a better and more confident prediction of the functioning of the RNA thermometer. The analysis (reference or link) showed that mutations influence the translation efficiency but that the function as RNA thermometer can remain. It is unclear if the same would hold when for example the length of the temperature sensitive hairpin is changed.
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[[Image:Start.png|thumb|200px|right|'''Figure 2:''' The secondary structure of the RNA thermometer that has been used as starting point of the design process is displayed at the left. At the right the stability profile of the temperature sensitive hairpin is highlighted (light blue). Also the found stability trend (see [[Team:TUDelft/Temperature_analysis#Data_analysis|data analysis]]) and the specified boundaries (see [[Team:TUDelft/Temperature_design#Design_algorithm|design algorithm]]) are highlighted (dark blue). The grey lines are the stability profiles of the other 31 ROSE RNA thermometers from the [http://rfam.sanger.ac.uk Rfam database].]]
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Recapulating, the list of requirements is now as follows:
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Since we have a number of structures of which we know that they work as an RNA thermometer, the most logical option is to take one of those structures as the starting point for the design of an RNA thermometer with a different switching temperature. The one chosen is a ROSE RNA thermometer from ''Bradyrhizobium japonicum USDA 110'' (<code>NC_004463</code>) residing at location <code>5784144-5784239</code> within the genome, which is at the 5' side of the gene hspB. There was no specific reason for the choice of this ROSE RNA thermometer, this is just a ROSE RNA thermometer taken from the [http://rfam.sanger.ac.uk Rfam database].
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* The sequence before the temperature sensitive hairpin remains unaltered, so mutations are only allowed within the temperature sensitive hairpin.
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We will call this RNA thermometer a 37°C switch, while it actually reaches maximum translation several degrees higher. The reason that we call it a 37°C switch is because of the characteristic stability profile we found during the [[Team:TUDelft/Temperature_analysis#Data_analysis|analysis]], and which is calculated at 37°C. Having a 27°C switch would then mean that this is an RNA thermometer of which the stability profile at 27 degrees is similar to the trend as given in figure TODO\ref{figure:trend}. This switch should then theoretically be almost fully switched on at 27°C, as is the case for the 37°C version at 37°C.
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* The overall structure of the temperature sensitive hairpin must remain the same. This means that the loop at the end should be of the same size and at the same position and the length of the hairpin remains the same. There are no restrictions to the number and locations of internal loops and bulges within the temperature sensitive hairpin.
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* The start codon, SD sequence, and scar sequence are predefined sequences and are placed at fixed location within the structure.
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[[Image:Tudelft_design_template.png]]
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Figure 2 shows the secondary structure of the chosen RNA thermometer as predicted by ''RNAfold''. Also the stability profile that is characteristic for this RNA thermometer at 37°C is shown in light blue. The thick dark blue line is the trend as found in the [[Team:TUDelft/Temperature_analysis#Data_analysis|data analysis]] and the thin dark blue lines are design boundaries which will be discussed later in this chapter. The stability profiles of the other 31 ROSE RNA thermometers from the Rfam database are shown in grey.
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These requirements are captured in figure x that can be seen as a template for the design of the temperature sensitive hairpin.
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=Design constraints=
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=== Design algorithm ===
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Having a starting point, we need a way to turn this RNA thermometer into one that switches at a different temperature, and that conforms to the Standard Biological Part standard. This can be achieved by increasing or decreasing the stability of the hairpin structure, as explained at the begin of this page. But, as also mentioned, there are certain constraints that has to be laid upon the design.
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[[Image:Tudelft_summed_unaligned_noloop_trend.png | thumb]]
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====Constraints on the sequence====
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The analysis of the temperature sensitive (TODO: link) hairpin of the known ROSE RNA thermometers has shown a trend in the stacking energies of the base pairs that is common to the different thermometers (figure). This trend observed for the stacking energies calculated at 37 degrees Celsius, the situation where the ROSE RNA thermometer is switched on (figure). As a convention we will call this a 37 degrees Celsius switch.
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Looking at the nucleotide sequence of the design, there is the constraint that there must be a start codon, a Shine Dalgarno sequence, and a scar present within the sequence. Note that this is the sequence after ligation of the RNA thermometer part to a protein coding part and that the scar and start codon are not part of the RNA thermometer part that we are designing. But since we are interested in the secondary structure of the end product we do have to take them into account. Also the order and distances between these regions are predefined (figure 3).
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To design a switch with a different temperature switching point, for example a 27 degrees Celsius switch, we assume that the stacking energies plot of this RNA thermometer, calculated at 27 degrees, should be similar to the trend we found for the 37 degrees Celsius switch (calculated at 37 degrees). This would mean that the stability of the temperature sensitive hairpin of the 27 degrees Celsius switch at 27 degrees Celsius is the same as the stability of the temperature sensitive hairpin of the 37 degrees Celsius switch at 37 degrees. 
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====Constraints on the secondary structure====
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A design can now be made by fitting the stacking energies plot (calculated at the desired temperature) of the designed hairpin to the found stacking energies trend, taking into account the mentioned requirements in the previous section.
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Extra constraints, which are laid upon the secondary structure of the designed RNA thermometer, are specified in order to ''1.'' simplify the design process; putting restrictions on, for example the length of the hairpin greatly reduces the solution space, making the problem easier to solve, and ''{2.'' to keep the secondary structure of the design as close as possible to the secondary structure of the original RNA thermometer. The [[Team:TUDelft/Temperature_analysis|analysis]] showed that mutations influence the translation efficiency without loosing its function as RNA thermometer. It is unclear if the same would hold when for example the length of the temperature sensitive hairpin is changed TODO\footnotemark. The more we change the secondary structure, the bigger the risk that it loses its function as an RNA thermometer.
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The design of a temperature sensitive hairpin that 'switches' at a certain temperature can be made using an algorithm that acts as follows:
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To keep the secondary structure as close as possible to the original RNA thermometer we state that mutations may only be applied to the temperature sensitive hairpin, the rest of the structure is fixed. For the temperature sensitive hairpin it is stated that the length of the hairpin, the size of the hairpin loop and the location of the hairpin loop must remain the same.
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# Choose a desired switching temperature.
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TODO\footnotetext{During the project a paper from \cite{neupert2008} has been published in which it is shown that the length of a temperature sensitive hairpin and the size of its hairpin loop can be varied without loosing the temperature sensitivity.}
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# Take a sequence with the predefined nucleotides (start codon, SD seqeunce, and the scar) present at the correct locations, the rest of the nucleotides can be filled at random.
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# Predict the secondary structure of the RNA thermometer (at the desired switching temperature) to check if it corresponds to the required structure. If not, go to step 2.
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# Compare the stacking energies plot (calculated for the desired switching temperature) of the temperature sensitive hairpin to the trend plot and calculate the distance between the two plots. If it is the smallest distance thus far, remember the sequence as the best one thus far and go back to step 2 to give it another try. Otherwise go back to step 2.
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In order to break this loop a conditional check can be added to the last step that ensures that the algorithm is stopped when the found distance is small enough, i.e. the fit is good enough.
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====List of constraints====
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'''Note''' The description of this algorithm is purely illustrative, only showing the basic principle. For the implementation of such an algorithm, a different approach is needed to make it more efficient.
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[[Image:Template.png|thumb|200px|right|'''Figure 3:''' A template for the design of a temperature sensitive hairpin. The grey dots will not be part of the final RNA thermometer part, but they have to be taken into account because they participate in the forming of the needed hairpin structure.]]
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It takes to much time to implement such an algorithm within the time frame of this project. It is also not yet shown that this design method produces working RNA thermometers, so it would be to early to spend a lot of time on the design of such an algorithm.
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Recapitulating, the list of design constraints is now as follows:
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== Design of a 27°C, 32°C, and 37°C switch ==
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* The sequence before the temperature sensitive hairpin remains unaltered, so mutations are only allowed within the temperature sensitive hairpin.
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* The overall structure of the temperature sensitive hairpin must remain the same. This means that the loop at the end should be of the same size and at the same position and the length of the hairpin remains the same. There are no restrictions to the number and locations of internal loops and bulge loops within the double helix region of the  temperature sensitive hairpin.
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* The start codon, SD sequence, and scar sequence are predefined sequences and are placed at fixed location within the hairpin structure.
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The design heuristic, as proposed in the previous section, is used to design three RNA thermometers, a 27°C, a 32°C, and a 37°C version (these are the temperatures at which the RNA thermometers are theoretically switched on). As also mentioned in the previous section, there is no algorithm to perform the optimization process, so the designs are made manually. [RNAfold] is used to predict the secondary structure at a certain temperature in the second step and [RNAeval] is used to calculate the stacking energies at a certain temperature in the third step. Fitting the plot is done visually by comparing the energy stacking plot of the design to the trend line as in the rightmost figures below, so there is no calculation method used to determine the distance between these two graphs. The termination of the design process is also not well defined. The sequence was altered until the stacking energy plot fitted the trend comparably to the differences between these two graphs for the existing 37°C RNA thermometers (figure). The resulting RNA thermometers are displayed below together with the plot of the stacking energies compared to the trend line.
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These constraints are also captured in figure 3 which can be seen as a template for the design of the temperature sensitive hairpin.
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<gallery Caption="27">
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==Design algorithm==
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Image:Tudelft_design27_0_at27degrees.png
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Image:Tudelft_design27_0_hairpin_at27degrees.png
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Image:Tudelft_design27_0_energystacking.png
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</gallery>
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<gallery Caption="32">
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Image:Tudelft_design32_0_at32degrees.png
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Image:Tudelft_design32_0_hairpin_at32degrees.png
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Image:Tudelft_design32_0_energystacking.png
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</gallery>
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<gallery Caption="37">
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Image:Tudelft_design37_0.png
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Image:Tudelft_design37_0_hairpin.png
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Image:Tudelft_design37_0_energystacking.png
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</gallery>
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== Temperature sensitive hairpin parts ==
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As specified in the previous section, mutations may only be applied within the temperature sensitive hairpin region. The temperature sensitive hairpin is therefore the only sequence that has to be designed and figure 3 serves as a template for this design.
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It is shown that an RNA thermometer also functions when it only consists of the temperature sensitive hairpin (ref Chowdhurry). The results of the experiment indicate that the temperature threshold shifts upward in case of the temperature sensitive hairpin only version. To test this, the designed parts are also added as a temperature sensitive hairpin version only.
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The data analysis of the known ROSE RNA thermometers has shown a trend within the stability profiles of the temperature sensitive hairpins (figure TODO\ref{figure:trend}). This trend has been observed for the stability profiles calculated at 37°C, and as already mentioned those RNA thermometers will therefore be called  37°C switches.
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If these short hairpins happen to be enough to get a working RNA thermometer, this would be a nice starting point for a third design phase in which these small parts are further optimized. For us, there's not enough time left to do this, but it could be a good starting point for a next years iGEM project.
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To design a switch with a different temperature switching point, for example a 27°C switch, we assume that the stability profile of this RNA thermometer, calculated at 27°C, should be similar to the trend we found for the 37°C switch (calculated at 37°C). In other words, we would like the stability of the temperature sensitive hairpin of the 27°C switch at 27°C, to be the same as the stability of the temperature sensitive hairpin of the 37°C switch at 37°C.
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== References ==
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====Algorithm====
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<ol class="references">
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<li id="cite_note-1">[[Team:TUDelft/Temperature_design#cite_ref-1 | ^ ]] De Smit M H, Van Duijn J (1990). "Secondary structure of the ribosome binding site determines translation efficiency: A quantitative analysis". PNAS, 1990-10, vol.87, no.19, 7668-7672. [http://www.ncbi.nlm.nih.gov/pubmed/2217199 PMID:2217199]</li>
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<li> [[Team:TUDelft/Temperature_design# | ^]] 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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Such a design can be made by fitting the stability profile (calculated at the desired temperature) of the hairpin to the found stability profile trend, taking into account the mentioned constraints in the previous section. The design of a temperature sensitive hairpin that switches at a certain temperature (which is given as input to the algorithm) can be made using an algorithm that acts as follows TODO\footnotemark:
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<li> [[Team:TUDelft/Temperature_design# | ^]] 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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TODO\footnotetext{Note that the description of this algorithm is purely illustrative, only showing the principle. For the implementation of such an algorithm, a different approach is needed to make it more efficient (see also section \ref{section:RNAHairpinDesigner}).}
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<li> [[Team:TUDelft/Temperature_design# | ^]] Nocker A, Hausherr T, Balsiger S, Krstulovic N, Hennecke H, Narberhaus F (2001). "A mRNA-based thermosensor controls expression of rhizobial heat shock genes". Nucleic Acids Research, 2001 December 1, 29(23):4800-4807. [http://www.ncbi.nlm.nih.gov/pubmed/11726689 PMID:11726689] </li>
 
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<li> [[Team:TUDelft/Temperature_design# | ^]] Balsiger S, Ragaz C, Baron C, Narberhaus F. "Replicon-specific regulation of small heat shock genes in Agrobacterium tumefaciens". Journal of Bacteriology, October 2004, p. 6824-6829, Vol.186, No.20. [http://www.ncbi.nlm.nih.gov/pubmed/15466035 PMID:15466035]</li>
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* Take a sequence with the predefined nucleotides (start codon, SD sequence, and the scar) present at the correct locations, the rest of the nucleotides can be filled at random.
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* Predict the secondary structure of the RNA thermometer (at the desired switching temperature) to check if it corresponds to the required structure. If not, go to step one.
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* Compare the stability profile plot (calculated for the desired switching temperature) of the temperature sensitive hairpin to the trend plot. If it stays within the boundaries that are set at a certain distance form the trend line, a good enough design is found and the algorithm is terminated. Otherwise go back to step one to give it another try.
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<li> [[Team:TUDelft/Temperature_design# | ^]] 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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[[Image:Boundaries.png|thumb|200px|right|'''Figure 4:''' Boundaries are set at a distance of 2.0 ''kcal/mol'' from the trend line. A designed temperature sensitive hairpin must have a stability profile that stays within the given boundaries.]]
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<li> [[Team:TUDelft/Temperature_design# | ^]] 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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In order to break this optimization algorithm, a conditional check is added to the last step that stops the algorithm when the stability profile of the design fits the trend line good enough. This has been done by specifying boundaries that reside at a certain distance from the trend line (figure 4). The design process is stopped as soon as the stability profile of the design stays completely within the boundaries. Looking at the plot in which the trend line has been observed (figure TODO\ref{figure:trend}) a boundary at a distance of 2.0 ''kcal/mol' from the trend line seems reasonable.
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<li> [[Team:TUDelft/Temperature_design# | ^]] Chowdhury S, Ragaz C, Kreuger E, and Narberhaus F (2003). "Temperature-controlled Structural Alterations of an RNA Thermometer". J. Biol. Chem.,November 28, 2003, Vol. 278, Issue 48, 47915-47921. [http://www.ncbi.nlm.nih.gov/pubmed/12963744 PMID:12963744] </li>
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====Manual approach====
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Because of the restricted time of the project, there was no time to wait for a software implementation of the design algorithm. Therefore a number of parts have been designed manually. The manual design has been done using a less naive approach than the given algorithm. Taking random nucleotides for the nineteen positions that are free to choose is not efficient. The number of possible sequences is enormous while only a small fraction of these will fold into the desired secondary structure.
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For the manual approach the total sequence of the RNA thermometer from figure 2 has been taken as starting point. Instead of randomly choosing nucleotides for all the positions that may be mutated, those positions are considered one by one to see if the double helix can be destabilized through mutations of the nucleotides at these positions. For example, at a position where there is a G-C base pair of which the C may be mutated, the C can be mutated to a U, which results in a less stable hairpin. Also a mutation to G or A would be possible in order to incorporate an internal loop, which destabilizes the double helix even further.
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After each mutation, the secondary structure has been predicted, using ''RNAfold'', to check if it still folds into the desired structure. When it does, the stability profile has been plotted, using ''RNAeval'' and the [[Team:TUDelft/Temperature_software#Stability_Profile_Plotter|Stability Profile Plotter]], to check if it stays within the trend boundaries.
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==Result==
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[[Image:Design2.png|thumb|200px|right|'''Figure 5:''' The secondary structures of the RNA thermometers are given at the left. The top one is the original RNA thermometer that has been used as starting point. The bottom two are the designed RNA thermometers, one with a theoretical switching point at 32\textdegree C and one with a theoretical switching point at 27\textdegree C. The stability profiles of the temperature sensitive hairpins, calculated at three different temperatures, are given at the right. The colored stability profiles shows the stability at the theoretical switching temperature. The aim of the design process was to get this colored line within the given trend boundaries.]]
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[[Image:Design2 1.png|thumb|200px|right|'''Figure 6:''' The stability profiles of the original and the two designed RNA thermometers, calculated at their own theoretical switching temperature. This shows that each RNA thermometer has a similar stability at its theoretical switching temperature.]]
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This manual approach has been used to design two RNA thermometers, a 27°C, and a 32°C version (<code>BBa_K115016</code>} and <code>BBa_K115017</code>). The secondary structures of the resulting RNA thermometers are shown in figure 5, together with the stability profiles and the trend line.
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The stability profiles of the temperature sensitive hairpins off all three RNA thermometers (calculated at their switching temperature) are also displayed in one plot in figure 6. This shows that each of them has a similar stability at their own theoretical switching temperature.
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====Temperature sensitive hairpin parts====
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The only sequence that has to be designed is the sequence of the temperature sensitive hairpin. The rest of the structure is kept intact to stay as close as possible to the secondary structure of the original RNA thermometer that is used as starting point. It has been shown by Chowdhury2003} that a temperature sensitive hairpin on itself, so without the rest of the ROSE structure, also works as a temperature sensitive switch.
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Three temperature sensitive hairpins are added to the registry to test if these short sequences can also be used as temperature sensitive switch. These are the temperature sensitive hairpins of the ROSE RNA thermometer as designed in the first design phase (\texttt{BBa\_K115001}), and the temperature sensitive hairpins of the 27\textdegree C and 32\textdegree C switch as designed in the second design phase (\texttt{BBa\_K115016} and \texttt{BBa\_K115017}). This are the parts \texttt{BBa\_K115020}, \texttt{BBa\_K115019}, and \texttt{BBa\_K115018} respectively.
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%If these short hairpins happen to be enough to get a working RNA thermometer, this would be a nice starting point for a third design phase in which these small parts are further optimized. For us, there's not enough time left to do this, but it could be a good starting point for a next years iGEM project.%wiki only
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== References ==
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<ol>
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<li id="cite_note_1"> [[Team:TUDelft/Temperature_design2#cite_ref_1 | ^]] Saheli Chowdhury, Curdin Ragaz, Emma Kreuger, and Franz Narberhaus. Temperature-controlled Structural Alterations of an RNA Thermometer. ''The Journal of Biological Chemistry'', 278(48):47915-47921, 2003. [http://www.ncbi.nlm.nih.gov/pubmed/12963744 PMID:12963744] </li>
</ol>
</ol>
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Revision as of 20:56, 26 October 2008

Contents

Design phase II: Changing the switching temperature of the RNA thermometer

Introducing existing RNA thermometers into E. coli provides us with a number of temperature sensitive switches, all with their own switching temperature. But this only provides a few switching temperatures, which are also relatively close to each other. In order to build a biothermometer, we needed more switching temperatures that have a bigger range. Note, however, that there will always be an upper bound in that the E. coli cannot survive temperatures above 40°C, and a lower bound in that the activity of the cell will drop at low temperatures.

This chapter presents a method that has been used to design RNA thermometer parts that should theoretically switch at a predefined temperature. The method used is based on the data analysis of which the results can be found on the analysis page. It has been used to design two new parts, one that should theoretically be switched on at 27°C ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K115016 BBa_K115016]), and two that should theoretically be switched on at 32°C ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K115017 BBa:K115017]).

Secondary structure stability and switching temperature

Figure 1: A. A lot of G-C base pairs make a very stable hairpin (having a very low free energy). 'B. The same hairpin structure with A-U base pairs is much less stable (having a higher free energy). C-E Bulge loops and internal loops destabilize the structure even further.

The switching temperature of the RNA thermometer depends on the stability of the hairpin structure that surrounds the Shine Dalgarno region (see also [Team:TUDelft/Temperature_analysis#Requirements_for_an_RNA_thermometer|Requirements for an RNA therometer]). This secondary structure will melt when it is exposed to a certain amount of heat. The more stable the hairpin structure is, the more heat will be needed to melt it, and the less stable the hairpin structure is, the less heat will be needed to melt it. In case of the RNA thermometer, this melting is needed to expose the Shine Dalgarno sequence which will cause the initiation of translation.

In other words, a less stable hairpin structure will melt at a lower temperature than a more stable hairpin and thus, for the less stable hairpin, the translation will initiate at a lower temperature than the more stable one. This way we could theoretically shift the switching temperature to a lower or higher temperature by making the hairpin structure surrounding the SD region less or more stable respectively.

The stability of the hairpin structure depends on the base-pairing nucleotides within the secondary structure. For example, C-G base pairs are very strong, so a hairpin structure containing a lot of those base pairs will be very stable. A-U and U-G base pairs are less strong, so these can be used to destabilize the hairpin structure. The same can be achieved by incorporating bulge and internal loops within the hairpin structure (figure 1).

Constraints

There are two things that have to be taken into account. Firstly, adding mutations in order to enforce or weaken the temperature sensitive region can also cause the RNA to fold into a completely different secondary structure. This way it can lose its function as an RNA thermometer. Secondly, there are some constraints to the possible mutations. The start codon and the Shine Dalgarno sequence must off course remain unaltered and in case of a standard biobrick the scar must also be part of the temperature sensitive hairpin.

Design starting point

Figure 2: The secondary structure of the RNA thermometer that has been used as starting point of the design process is displayed at the left. At the right the stability profile of the temperature sensitive hairpin is highlighted (light blue). Also the found stability trend (see data analysis) and the specified boundaries (see design algorithm) are highlighted (dark blue). The grey lines are the stability profiles of the other 31 ROSE RNA thermometers from the [http://rfam.sanger.ac.uk Rfam database].

Since we have a number of structures of which we know that they work as an RNA thermometer, the most logical option is to take one of those structures as the starting point for the design of an RNA thermometer with a different switching temperature. The one chosen is a ROSE RNA thermometer from Bradyrhizobium japonicum USDA 110 (NC_004463) residing at location 5784144-5784239 within the genome, which is at the 5' side of the gene hspB. There was no specific reason for the choice of this ROSE RNA thermometer, this is just a ROSE RNA thermometer taken from the [http://rfam.sanger.ac.uk Rfam database].

We will call this RNA thermometer a 37°C switch, while it actually reaches maximum translation several degrees higher. The reason that we call it a 37°C switch is because of the characteristic stability profile we found during the analysis, and which is calculated at 37°C. Having a 27°C switch would then mean that this is an RNA thermometer of which the stability profile at 27 degrees is similar to the trend as given in figure TODO\ref{figure:trend}. This switch should then theoretically be almost fully switched on at 27°C, as is the case for the 37°C version at 37°C.

Figure 2 shows the secondary structure of the chosen RNA thermometer as predicted by RNAfold. Also the stability profile that is characteristic for this RNA thermometer at 37°C is shown in light blue. The thick dark blue line is the trend as found in the data analysis and the thin dark blue lines are design boundaries which will be discussed later in this chapter. The stability profiles of the other 31 ROSE RNA thermometers from the Rfam database are shown in grey.

Design constraints

Having a starting point, we need a way to turn this RNA thermometer into one that switches at a different temperature, and that conforms to the Standard Biological Part standard. This can be achieved by increasing or decreasing the stability of the hairpin structure, as explained at the begin of this page. But, as also mentioned, there are certain constraints that has to be laid upon the design.

Constraints on the sequence

Looking at the nucleotide sequence of the design, there is the constraint that there must be a start codon, a Shine Dalgarno sequence, and a scar present within the sequence. Note that this is the sequence after ligation of the RNA thermometer part to a protein coding part and that the scar and start codon are not part of the RNA thermometer part that we are designing. But since we are interested in the secondary structure of the end product we do have to take them into account. Also the order and distances between these regions are predefined (figure 3).

Constraints on the secondary structure

Extra constraints, which are laid upon the secondary structure of the designed RNA thermometer, are specified in order to 1. simplify the design process; putting restrictions on, for example the length of the hairpin greatly reduces the solution space, making the problem easier to solve, and {2. to keep the secondary structure of the design as close as possible to the secondary structure of the original RNA thermometer. The analysis showed that mutations influence the translation efficiency without loosing its function as RNA thermometer. It is unclear if the same would hold when for example the length of the temperature sensitive hairpin is changed TODO\footnotemark. The more we change the secondary structure, the bigger the risk that it loses its function as an RNA thermometer.

To keep the secondary structure as close as possible to the original RNA thermometer we state that mutations may only be applied to the temperature sensitive hairpin, the rest of the structure is fixed. For the temperature sensitive hairpin it is stated that the length of the hairpin, the size of the hairpin loop and the location of the hairpin loop must remain the same.

TODO\footnotetext{During the project a paper from \cite{neupert2008} has been published in which it is shown that the length of a temperature sensitive hairpin and the size of its hairpin loop can be varied without loosing the temperature sensitivity.}

List of constraints

Figure 3: A template for the design of a temperature sensitive hairpin. The grey dots will not be part of the final RNA thermometer part, but they have to be taken into account because they participate in the forming of the needed hairpin structure.

Recapitulating, the list of design constraints is now as follows:

  • The sequence before the temperature sensitive hairpin remains unaltered, so mutations are only allowed within the temperature sensitive hairpin.
  • The overall structure of the temperature sensitive hairpin must remain the same. This means that the loop at the end should be of the same size and at the same position and the length of the hairpin remains the same. There are no restrictions to the number and locations of internal loops and bulge loops within the double helix region of the temperature sensitive hairpin.
  • The start codon, SD sequence, and scar sequence are predefined sequences and are placed at fixed location within the hairpin structure.

These constraints are also captured in figure 3 which can be seen as a template for the design of the temperature sensitive hairpin.

Design algorithm

As specified in the previous section, mutations may only be applied within the temperature sensitive hairpin region. The temperature sensitive hairpin is therefore the only sequence that has to be designed and figure 3 serves as a template for this design.

The data analysis of the known ROSE RNA thermometers has shown a trend within the stability profiles of the temperature sensitive hairpins (figure TODO\ref{figure:trend}). This trend has been observed for the stability profiles calculated at 37°C, and as already mentioned those RNA thermometers will therefore be called 37°C switches.

To design a switch with a different temperature switching point, for example a 27°C switch, we assume that the stability profile of this RNA thermometer, calculated at 27°C, should be similar to the trend we found for the 37°C switch (calculated at 37°C). In other words, we would like the stability of the temperature sensitive hairpin of the 27°C switch at 27°C, to be the same as the stability of the temperature sensitive hairpin of the 37°C switch at 37°C.

Algorithm

Such a design can be made by fitting the stability profile (calculated at the desired temperature) of the hairpin to the found stability profile trend, taking into account the mentioned constraints in the previous section. The design of a temperature sensitive hairpin that switches at a certain temperature (which is given as input to the algorithm) can be made using an algorithm that acts as follows TODO\footnotemark:

TODO\footnotetext{Note that the description of this algorithm is purely illustrative, only showing the principle. For the implementation of such an algorithm, a different approach is needed to make it more efficient (see also section \ref{section:RNAHairpinDesigner}).}


  • Take a sequence with the predefined nucleotides (start codon, SD sequence, and the scar) present at the correct locations, the rest of the nucleotides can be filled at random.
  • Predict the secondary structure of the RNA thermometer (at the desired switching temperature) to check if it corresponds to the required structure. If not, go to step one.
  • Compare the stability profile plot (calculated for the desired switching temperature) of the temperature sensitive hairpin to the trend plot. If it stays within the boundaries that are set at a certain distance form the trend line, a good enough design is found and the algorithm is terminated. Otherwise go back to step one to give it another try.
Figure 4: Boundaries are set at a distance of 2.0 kcal/mol from the trend line. A designed temperature sensitive hairpin must have a stability profile that stays within the given boundaries.

In order to break this optimization algorithm, a conditional check is added to the last step that stops the algorithm when the stability profile of the design fits the trend line good enough. This has been done by specifying boundaries that reside at a certain distance from the trend line (figure 4). The design process is stopped as soon as the stability profile of the design stays completely within the boundaries. Looking at the plot in which the trend line has been observed (figure TODO\ref{figure:trend}) a boundary at a distance of 2.0 kcal/mol' from the trend line seems reasonable.

Manual approach

Because of the restricted time of the project, there was no time to wait for a software implementation of the design algorithm. Therefore a number of parts have been designed manually. The manual design has been done using a less naive approach than the given algorithm. Taking random nucleotides for the nineteen positions that are free to choose is not efficient. The number of possible sequences is enormous while only a small fraction of these will fold into the desired secondary structure.

For the manual approach the total sequence of the RNA thermometer from figure 2 has been taken as starting point. Instead of randomly choosing nucleotides for all the positions that may be mutated, those positions are considered one by one to see if the double helix can be destabilized through mutations of the nucleotides at these positions. For example, at a position where there is a G-C base pair of which the C may be mutated, the C can be mutated to a U, which results in a less stable hairpin. Also a mutation to G or A would be possible in order to incorporate an internal loop, which destabilizes the double helix even further.

After each mutation, the secondary structure has been predicted, using RNAfold, to check if it still folds into the desired structure. When it does, the stability profile has been plotted, using RNAeval and the Stability Profile Plotter, to check if it stays within the trend boundaries.

Result

Figure 5: The secondary structures of the RNA thermometers are given at the left. The top one is the original RNA thermometer that has been used as starting point. The bottom two are the designed RNA thermometers, one with a theoretical switching point at 32\textdegree C and one with a theoretical switching point at 27\textdegree C. The stability profiles of the temperature sensitive hairpins, calculated at three different temperatures, are given at the right. The colored stability profiles shows the stability at the theoretical switching temperature. The aim of the design process was to get this colored line within the given trend boundaries.
Figure 6: The stability profiles of the original and the two designed RNA thermometers, calculated at their own theoretical switching temperature. This shows that each RNA thermometer has a similar stability at its theoretical switching temperature.

This manual approach has been used to design two RNA thermometers, a 27°C, and a 32°C version (BBa_K115016} and BBa_K115017). The secondary structures of the resulting RNA thermometers are shown in figure 5, together with the stability profiles and the trend line.

The stability profiles of the temperature sensitive hairpins off all three RNA thermometers (calculated at their switching temperature) are also displayed in one plot in figure 6. This shows that each of them has a similar stability at their own theoretical switching temperature.

Temperature sensitive hairpin parts

The only sequence that has to be designed is the sequence of the temperature sensitive hairpin. The rest of the structure is kept intact to stay as close as possible to the secondary structure of the original RNA thermometer that is used as starting point. It has been shown by Chowdhury2003} that a temperature sensitive hairpin on itself, so without the rest of the ROSE structure, also works as a temperature sensitive switch.

Three temperature sensitive hairpins are added to the registry to test if these short sequences can also be used as temperature sensitive switch. These are the temperature sensitive hairpins of the ROSE RNA thermometer as designed in the first design phase (\texttt{BBa\_K115001}), and the temperature sensitive hairpins of the 27\textdegree C and 32\textdegree C switch as designed in the second design phase (\texttt{BBa\_K115016} and \texttt{BBa\_K115017}). This are the parts \texttt{BBa\_K115020}, \texttt{BBa\_K115019}, and \texttt{BBa\_K115018} respectively.

%If these short hairpins happen to be enough to get a working RNA thermometer, this would be a nice starting point for a third design phase in which these small parts are further optimized. For us, there's not enough time left to do this, but it could be a good starting point for a next years iGEM project.%wiki only

References

  1. ^ Saheli Chowdhury, Curdin Ragaz, Emma Kreuger, and Franz Narberhaus. Temperature-controlled Structural Alterations of an RNA Thermometer. The Journal of Biological Chemistry, 278(48):47915-47921, 2003. [http://www.ncbi.nlm.nih.gov/pubmed/12963744 PMID:12963744]
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