Team:VictoriaBC/project/biothermometer

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  E. coli Bio-Thermometer   Introduction            A biological thermometer would ideally be a simple, accurate and easily observable register of the organism&rsquo;s environment. A basic version is comprised of E. coli cells that respond to differences in temperature by producing different fluorescent proteins. It is essentially cells that can appear blue or green or red depending on the temperature they are exposed to.           The TU Delft 2008 team has produced a number of RNA thermometers, with varying levels of effectiveness. They retrieved natural RNA thermometers from three species, then sequenced and redesigned them to test for temperature variability range. They have produced temperature-sensitive RNA biobricks that include the ribosome binding site and can prevent the SD sequence for the next part from being read. This is useful because it provides temperature control for any output.   Figure 1. General Temperature-Sensitive RNA Thermometer <p style="line-height: 150%;" class="MsoBodyText"><span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><img src="/f/Rna_thermometer.png" /> <span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> <p style="line-height: 150%;" class="MsoBodyText"><span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">          Combining this with activators and repressors produces temperature-dependent responses. For the system to turn reporters on and off, multiple activators and repressors are used to provide the different states. For instance, going from a low to a high temperature, the lowest state (cyan), must be turned off and stay off; the middle state (green) must be both turned on and then off, and the highest state (red), must be turned on only during high temperatures. Ideally only one colour would be present at any stable state. <p style="text-indent: 36pt; line-height: 150%;" class="MsoBodyText"><span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">Using two of the RNA thermometers developed by TU Delft 2008, we can produce a system with three different states spaced along a temperature gradient. Below 32 oC, cyan fluorescence protein (CFP) is produced under the control of a LacI promoter. Both the LuxR activator for green fluorescence protein (GFP) and the LasR activator for red fluorescence protein (RFP), are unable to be translated at this low temperature, and thus the system glows blue. Above 32 oC but before reaching 37 oC, two things change. The LacI repressor can now be translated thereby shutting CFP off. As well, the activator for GFP is produced changing the system to green. After 37 oC, the cI repressor controlling GFP, is translated stopping production of GFP, and the LasR activator is also produced and turns on the RFP genes for the final red state. <span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> <p style="line-height: 150%;" class="MsoBodyText"> <span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">Figure 2. Below 32 oC – Cyan Active <p style="line-height: 150%;" class="MsoBodyText"><span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><img width="700" height="204" style="border: 0px solid rgb(0, 0, 0); margin: 0px;" src="/f/30.jpg" /> <p style="line-height: 150%;" class="MsoBodyText"><span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">          CFP is produced with the LacI promoter, but neither GFP nor RFP are activated yet. <span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> <p style="line-height: 150%;" class="MsoBodyText"> <span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">Figure 3. Between 32 oC and 37 oC – Green Active <p style="line-height: 150%;" class="MsoBodyText"><span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><img width="700" height="204" style="border: 0px solid rgb(0, 0, 0); margin: 0px;" src="/f/32.jpg" /> <p style="line-height: 150%;" class="MsoBodyText"><span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">          The LacI promoter for CFP is blocked and GFP is activated by the LuxR activator, but RFP is still not activated. <span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">Figure 4. Above 37 oC – Red Active <p style="line-height: 150%;" class="MsoBodyText"><span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><img width="700" height="204" style="border: 0px solid rgb(0, 0, 0); margin: 0px;" src="/f/37.jpg" /> <p style="line-height: 150%;" class="MsoBodyText"><span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">          Both CFP and GFP are blocked by repressors and the LasR activator has started RFP production. <span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> <p style="line-height: 150%;" class="MsoBodyText"> <span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">Parts </b> <span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">  </b> <p style="line-height: 150%;" class="MsoBodyText"> <span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">Figure 5. CFP Plasmid <p style="line-height: 150%;" class="MsoBodyText"><span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><img width="700" height="272" style="border: 0px solid rgb(0, 0, 0); margin: 0px;" src="/f/cfpplasmid.jpg" /> <p style="text-indent: 36pt; line-height: 150%;" class="MsoBodyText"><span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">The CFP genes on this plasmid are under control of LacI, while the LacI repressor is under control of a constitutive promoter and a hairpin loop that opens at 32 oC. CFP is produced until the temperature reaches 32 oC, whereupon the LacI repressor is now produced and stops production of CFP. This plasmid is the most likely to work. <span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">Figure 6. GFP Plasmid <p style="line-height: 150%;" class="MsoBodyText"><span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><img width="700" height="311" style="border: 0px solid rgb(0, 0, 0); margin: 0px;" src="/f/gfpplasmid.jpg" /> <p style="line-height: 150%;" class="MsoBodyText"><span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">          A LuxR/lambda cI promoter regulates GFP production, while a constitutive promoter controls both the LuxR activator and the cI repressor, also regulated by a 32 oC hairpin loop and a 37 oC hairpin loop respectively. Before reaching 32 oC, the LuxR activator is not produced and thus GFP is not produced until that temperature. When the temperature reaches 37 oC, the cI repressor is now present and stops GFP production. <span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> <p style="line-height: 150%;" class="MsoBodyText"> <span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">Figure 7. RFP Plasmid <p style="line-height: 150%;" class="MsoBodyText"><span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><img width="700" height="213" style="border: 0px solid rgb(0, 0, 0); margin: 0px;" src="/f/rfpplasmid.jpg" /> <p style="line-height: 150%;" class="MsoBodyText"><span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">          RFP production is controlled by a LasR promoter, and the LasR and LasI activators are controlled by a constitutive promoter and a 37 oC hairpin loop. This means that RFP is only turned on after reaching 37 oC, when the activators are able to be produced. <span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> <p style="line-height: 150%;" class="MsoBodyText"> <span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">Table 1. Temperature Responsive Regulators <span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> <p style="line-height: 150%;" class="MsoBodyText"> <span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">Figure 8. Temperature-Sensitive Hairpin Loops <p style="line-height: 150%;" class="MsoBodyText"><span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><img src="/f/BBa_K115017_ss.png" />  <img src="/f/BBa_K115001_ss.png" /> <p style="line-height: 150%;" class="MsoBodyText"><span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">(<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K115020">BBa_K115020</a> is the hairpin loop of <a title="Part:BBa K115001" href="http://partsregistry.org/Part:BBa_K115001">BBa_K115001</a>.) <span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> <p style="text-indent: 36pt; line-height: 150%;" class="MsoBodyText"><span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K115017">BBa_K115017</a>, and <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K115020">BBa_K115020</a> were chosen from TU Delft 2008&rsquo;s different thermometers because the first was shown to be the most successful with a clear response when heated above 32 oC; and the second is intended to be indicative over 37 oC, which holds interest as the human body temperature. <span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> <p style="line-height: 150%;" class="MsoBodyText"> <span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">Table 2. Flourescent Protein Reporters <span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> <p style="line-height: 150%;" class="MsoBodyText"><span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">          Fluorescence proteins were chosen because the intention of the project is to have a visible display of the temperature felt by the cells. In addition, this type of response is assayable and simple. <span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">Table 3. Promoters and Regulators <span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> <p style="line-height: 150%;" class="MsoBodyText"> <span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">Table 4. General Parts <span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> <p style="line-height: 150%;" class="MsoBodyText"> <span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">Table 5. Pre-combined <span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> <p style="line-height: 150%;" class="MsoBodyText"> <span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">Assembly </b> <p style="text-indent: 36pt; line-height: 150%;" class="MsoBodyText"><span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">3A assembly is designed to use three-way ligation and both positive and negative selection to minimise incorrect assemblies. 3A assembly stands for three antibiotic assembly, as the three starting plasmids all have different antibiotic resistance markers. The construction vector (red) contains ccdB (black block arrow) and the EcoRI and PstI restriction sites. The blue plasmid contains the prefix part (pink), and the green plasmid contains the suffix part (yellow). These two plasmids can have the same or different resistance markers, as long as they are distinct from the construction vector. All these BioBrick parts are standardised to contain these restriction sites and an antibiotic resistance marker making this an appropriate method for the whole project. There are also an assortment of construction vectors with different antibiotic resistance markers enabling simple changes from part to part as only up to three antibiotics will be necessary at one time. <p style="text-indent: 36pt; line-height: 150%;" class="MsoBodyText"><span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">EcoRI (E) and PstI (P) are used to cut the construction vector, removing the ccdB gene and leaving E and P open ends. EcoRI (E) and SpeI (S) are used to cut the prefix plasmid, and XbaI (X) and PstI (P) are used to cut the suffix plasmid. These pieces of DNA can then be ligated together and transformed. A mixed site (M) is left between the combined parts. A plate supplemented with the red antibiotic is used to grow the transformed cells. For the red vector, a ccdB resistant strain such as DB3.1 must be used. <span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> <p style="line-height: 150%;" class="MsoBodyText"> <span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">Figure 9. 3A Assembly <p style="line-height: 150%;" class="MsoBodyText"><span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><img src="/f/TUDelft3Amixture.png" /> <p style="line-height: 150%;" class="MsoBodyText"><span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">          Digested parts before ligation. <p style="line-height: 150%;" class="MsoBodyText"> <span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">Figure 10. 3A Assembly <p style="line-height: 150%;" class="MsoBodyText"><span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><img src="/f/3AAssembly.gif" /> <p style="line-height: 150%;" class="MsoBodyText"><span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">          Purified plasmid fragments ligated into the construction vector. <span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> <p style="line-height: 150%;" class="MsoBodyText"> <span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">Methods </b> <p style="text-indent: 36pt; line-height: 150%;" class="MsoBodyText"><span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">This procedure is taken from the open wetware page: <a href="http://openwetware.org/wiki/Synthetic_Biology:BioBricks/3A_assembly">http://openwetware.org/wiki/Synthetic_Biology:BioBricks/3A_assembly</a> <span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> <p style="line-height: 150%;" class="MsoBodyText"><span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">Construction Procedure <p style="margin-left: 0cm; text-indent: 0cm; line-height: 150%;" class="MsoBodyText"><span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> 1.<span style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;">                  <span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><a title="Miniprep" href="http://openwetware.org/wiki/Miniprep">Miniprep</a> the two parts and construction plasmid. e.g. <p style="margin-left: 36pt; text-indent: -18pt; line-height: 150%;" class="MsoBodyText">  &Oslash;<span style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;">   <span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">Prefix part <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K137029">BBa_K137029</a> comes on a <a href="http://partsregistry.org/wiki/index.php?title=Part:pSB1A2">pSB1A2</a> plasmid with ampicillin resistance <p style="margin-left: 36pt; text-indent: -18pt; line-height: 150%;" class="MsoBodyText"> &Oslash;<span style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;">   <span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">Suffix part <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K115017">BBa_K115017</a> comes on a <a href="http://partsregistry.org/wiki/index.php?title=Part:pSB1AT3">pSB1AT3</a> plasmid with ampicillin and tetracycline resistance <p style="margin-left: 36pt; text-indent: -18pt; line-height: 150%;" class="MsoBodyText"> &Oslash;<span style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;">   <span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">Construction vector <a href="http://partsregistry.org/wiki/index.php?title=Part:pSB1C3">pSB1C3</a> is a plasmid backbone with chloramphenicol resistance <p style="margin-left: 0cm; text-indent: 0cm; line-height: 150%;" class="MsoBodyText"><span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> 2.<span style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;">                  <span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><a title="Knight:Restriction Digest" href="http://openwetware.org/wiki/Knight:Restriction_Digest">Digest</a> the two parts and destination vector with the following enzymes <p style="margin-left: 72pt; text-indent: -18pt; line-height: 150%;" class="MsoBodyText"> &middot;<span style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;">          <span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">Prefix part (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K137029">BBa_K137029</a>) with EcoRI and SpeI <p style="margin-left: 72pt; text-indent: -18pt; line-height: 150%;" class="MsoBodyText"> &middot;<span style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;">          <span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">Suffix part (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K115017">BBa_K115017</a>) with XbaI and PstI <p style="margin-left: 72pt; text-indent: -18pt; line-height: 150%;" class="MsoBodyText"> &middot;<span style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;">          <span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">Construction vector (<a href="http://partsregistry.org/wiki/index.php?title=Part:pSB1C3">pSB1C3</a>) with EcoRI and PstI <p style="margin-left: 0cm; text-indent: 0cm; line-height: 150%;" class="MsoBodyText"><span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> 3.<span style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;">                  <span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">Purify the restriction digest. <p style="margin-left: 72pt; text-indent: -18pt; line-height: 150%;" class="MsoBodyText"> &middot;<span style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;">          <span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">For parts &lt; 200 bp in length, do a <a title="Knight:Micropure EZ and Microcon purification" href="http://openwetware.org/wiki/Knight:Micropure_EZ_and_Microcon_purification">Knight:Micropure EZ and Microcon purification</a> (or alternatively an <a title="Ethanol precipitation of small DNA fragments" href="http://openwetware.org/wiki/Ethanol_precipitation_of_small_DNA_fragments">Ethanol precipitation of small DNA fragments</a>). All other parts and the construction vector can be purified using a <a title="http://www1.qiagen.com/HB/QIAquickSpin" href="http://www1.qiagen.com/HB/QIAquickSpin">QIAquick PCR purification kit</a> or <a title="http://www1.qiagen.com/literature/handbooks/PDF/DNACleanupAndConcentration/MinElute/1027886_HB_QQ_MinElute_0604.pdf|" href="http://www1.qiagen.com/literature/handbooks/PDF/DNACleanupAndConcentration/MinElute/1027886_HB_QQ_MinElute_0604.pdf%7C">Qiagen Minelute PCR Purification Kit</a> (elutes in a smaller volume giving greater concentration of the DNA). <p style="margin-left: 0cm; text-indent: 0cm; line-height: 150%;" class="MsoBodyText"><span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> 4.<span style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;">                  <span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><a title="Knight:DNA ligation using T4 DNA ligase" href="http://openwetware.org/wiki/Knight:DNA_ligation_using_T4_DNA_ligase">Ligate</a> the two parts and construction vector together. (<a title="Endy:DNA ligation using T4 DNA ligase" href="http://openwetware.org/wiki/Endy:DNA_ligation_using_T4_DNA_ligase">Endy lab protocol</a>) <p style="margin-left: 0cm; text-indent: 0cm; line-height: 150%;" class="MsoBodyText"><span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> 5.<span style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;">                  <span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><a title="TOP10 chemically competent cells" href="http://openwetware.org/wiki/TOP10_chemically_competent_cells">Transform</a> the ligation product. (See also <a title="Electroporation" href="http://openwetware.org/wiki/Electroporation">Electroporation</a> or <a title="Transforming chemically competent cells" href="http://openwetware.org/wiki/Transforming_chemically_competent_cells">another chemical transformation</a>.) <p style="margin-left: 0cm; text-indent: 0cm; line-height: 150%;" class="MsoBodyText"><span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> 6.<span style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;">                  <span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">Analyze the transformation with <a title="Knight:Colony PCR" href="http://openwetware.org/wiki/Knight:Colony_PCR">single colony PCR</a> followed by <a title="Knight:Agarose gel electrophoresis" href="http://openwetware.org/wiki/Knight:Agarose_gel_electrophoresis">agarose gel electrophoresis</a>. <p style="margin-left: 72pt; text-indent: -18pt; line-height: 150%;" class="MsoBodyText"> &middot;<span style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;">          <span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">In rolling, large scale assembly, this step is omitted. <p style="margin-left: 0cm; text-indent: 0cm; line-height: 150%;" class="MsoBodyText"><span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> 7.<span style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;">                  <span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><a title="Miniprep" href="http://openwetware.org/wiki/Miniprep">Miniprep</a> clones that generated a band of the appropriate size. <p style="margin-left: 0cm; text-indent: 0cm; line-height: 150%;" class="MsoBodyText"><span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> 8.<span style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;">                  <span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><a title="MIT:Sequencing BioBrick DNA" href="http://openwetware.org/wiki/MIT:Sequencing_BioBrick_DNA">Sequence</a> the clone. <p style="margin-left: 0cm; text-indent: 0cm; line-height: 150%;" class="MsoBodyText"><span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> 9.<span style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;">                  <span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">Repeat for all of the parts to form three complete plasmids. <p style="margin-left: 0cm; text-indent: 0cm; line-height: 150%;" class="MsoBodyText"><span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> 10.<span style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;">              <span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">Assemble all three plasmids together to produce cells with all three responses. <span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> <p style="text-indent: 36pt; line-height: 150%;" class="MsoBodyText"><span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">The antibiotic resistance of the different parts helps to ensure that only the construction vector backbone plasmid is selected for, while the ccdB gene prevents unaltered construction vectors from continuing. These plasmids would be finally assembled together in a single cell. It may be advantageous to switch from high copy number assembly plasmid backbones, used while combining the parts, to low or medium copy backbones for the final product. <p style="text-indent: 36pt; line-height: 150%;" class="MsoBodyText"><span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">The individual plasmids should be tested first on their own to determine their individual effectiveness. This would also allow us to more easily form BioBrick parts for these systems. The working parts would then be combined to produce cells with multiple temperature reactions. <span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> <p style="line-height: 150%;" class="MsoBodyText"><span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">Testing Procedure <p style="margin-left: 36pt; text-indent: -18pt; line-height: 150%;" class="MsoBodyText"><span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> 1.<span style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;">      <span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">Grow each plasmid individually in cultures at 30 oC, 35 oC, and 38 oC (nine samples). <p style="margin-left: 36pt; text-indent: -18pt; line-height: 150%;" class="MsoBodyText"><span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> 2.<span style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;">      <span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">Grow the combined cells in cultures at 30 oC, 35 oC, and 38 oC (three samples). <p style="margin-left: 36pt; text-indent: -18pt; line-height: 150%;" class="MsoBodyText"><span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> 3.<span style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;">      <span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">Grow control cultures to account for system biases. <p style="margin-left: 36pt; text-indent: -18pt; line-height: 150%;" class="MsoBodyText"><span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> 4.<span style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;">      <span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">Assay for fluorescence proteins. <span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> <p style="line-height: 150%;" class="MsoBodyText"> <span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">Discussion </b> <p style="text-indent: 36pt; line-height: 150%;" class="MsoBodyText"><span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">The largest issue that might be a problem is that the 37 oC RNA thermometer part (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K115020">BBa_K115020</a> ) has not been shown to be effective. If it is in fact ineffective this would mean that only the CFP plasmid would work entirely correctly. The GFP plasmid would either never be repressed (still useful by producing a working change in temperature at 32 oC), or would be always repressed (no GFP ever produced). The RFP plasmid would also suffer similarly, either always on or always off, serving no purpose. In this situation, we would want to assemble a system involving only the 32 oC dependent aspects; this system would only have one change, from blue to green at 32 oC. <p style="text-indent: 36pt; line-height: 150%;" class="MsoBodyText"><span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">Other factors that might hinder the overall visible result include, if the fluorescence proteins take too long to show visible results; if the old fluorescence proteins take too long to disappear after a change in temperature; if leakiness in the promoters leaves too much background colour, muddying the indicator; and if the repressors and activators do not produce a great enough change in fluorescence to be observable. Depending on these factors the system may be most accurate when intended for use at only one temperature – a situation that would then have proof of being at a constant temperature within a certain range as long as the expressed colour was relatively pure. Mixed or muddy colouring would indicate inconstant temperature. <span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> <p style="line-height: 150%;" class="MsoBodyText"> <span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">Conclusion </b> <p style="line-height: 150%;" class="MsoBodyText"><span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">          This presents a procedure for developing a bacterial thermometer with three distinct states using two RNA temperature switches. All parts are from the iGem Parts Registry and are suitable to assemble and use together. The 3A assembly method is appropriate for these parts and can be implemented with a combination of relatively standard processes and techniques. <span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> <span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">References </b> <p style="line-height: 150%;" class="MsoBodyText"> <span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><a href="http://partsregistry.org/Main_Page">http://partsregistry.org/Main_Page</a> <p style="line-height: 150%;" class="MsoBodyText"><span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><a href="http://2008.igem.org/Team:TUDelft">http://2008.igem.org/Team:TUDelft</a> <p style="line-height: 150%;" class="MsoBodyText"><span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><a href="http://openwetware.org/wiki/Synthetic_Biology:BioBricks/3A_assembly">http://openwetware.org/wiki/Synthetic_Biology:BioBricks/3A_assembly</a> <span style="font-size: 11pt; line-height: 150%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">