http://2013.igem.org/wiki/index.php?title=Special:Contributions/Dustin&feed=atom&limit=50&target=Dustin&year=&month=2013.igem.org - User contributions [en]2024-03-29T01:24:33ZFrom 2013.igem.orgMediaWiki 1.16.5http://2013.igem.org/Team:Lethbridge/resultsTeam:Lethbridge/results2014-01-21T22:48:46Z<p>Dustin: corrected methods description</p>
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<BLOCKQUOTE><br />
=<font color="black">Results=<br />
<br />
<br />
<br />
<h2>Team Parts Sandbox</h2><br />
<center><br />
<table border="0" style="background-color:#FFF" width="100%" cellpadding="0" cellspacing="5"><br />
<tr><br />
<td><strong>Name</strong></td><br />
<td><strong>Type</strong></td><br />
<td><strong>Description</strong></td><br />
<td><strong>Designer</strong></td><br />
<td><strong>Length</strong></td><br />
<td><strong>Favorite Part</strong></td><br />
</tr><br />
<tr><br />
<td>[http://parts.igem.org/Part:BBa_K1210000 BBa_K1210000]</td><br />
<td>Device</td><br />
<td>LacI-CFP-PK401-YFP</td><br />
<td>Zak Stinson</td><br />
<td>1955</td><br />
<td><center>Yes</center></td><br />
</tr><br />
<tr><br />
<td>[http://parts.igem.org/Part:BBa_K1210001 BBa_K1210001]</td><br />
<td>RNA Part</td><br />
<td>PK401 Pseudoknot</td><br />
<td>Zak Stinson</td><br />
<td>93</td><br />
<td> </td><br />
</tr><br />
<tr><br />
<td>[http://parts.igem.org/Part:BBa_K1210002 BBa_K1210002]</td><br />
<td>Device</td><br />
<td>Enhanced Lumazine Synthase (ELS) Expression Construct</td><br />
<td>Harland Brandon</td><br />
<td>828</td><br />
<td> </td><br />
</tr><br />
<tr><br />
<td>[http://parts.igem.org/Part:BBa_K1210003 BBa_K1210003]</td><br />
<td>Device</td><br />
<td>Tagged dual coding test construct</td><br />
<td>Harland Brandon</td><br />
<td>1850</td><br />
<td> </td><br />
</tr><br />
</table><br />
<br />
</center><br />
<br />
<br><br />
<br />
<h2>Expression and Characterization of PK401 (BBa_K1210000)</h2><br />
<br />
<p><b>PK401 Overexpression</b></p><br />
<p>The PK401 construct was overexpressed to test and characterize the construct for frameshifting ability. To do this, cultures of E. coli DH5α containing either the PK401 plasmid or an empty control plasmid were grown from glycerol stocks overnight at 37°C in 50 mL LB media containing Kanamycin (50 µg/mL). These cultures were used the next day to inoculate 500 mL of LB media to a starting optical density at 600 nm (OD600) near 0.05. The OD600 was monitored and induced with 1 mM IPTG once reaching an OD600 of 0.6 (Fig. 1). Growth was monitored hourly, and equivalent amounts of cells were taken as samples for SDS-PAGE analysis up to 5 h after induction. The cultures were harvested by centrifuging at 5000 x g and shock frozen to store at -80°C.</p><br />
<br />
<center>[[File:ULeth2013_PK401-overexpression.png|600px]]</center><br />
<br />
<p><b>Figure 1. Growth curve of PK401 construct.</b> E. coli DH5α cells containing the PK401 plasmid or a control plasmid were grown at 37°C in LB media. The OD600 was monitored and the cultures were induced with 1 mM IPTG when the OD600 had reached 0.6. The cultures were grown for an additional 5 h, and a sample of 1 OD600 equivalent of cells were taken at each hour after induction.</p><br><br />
<br />
<p>The samples taken for SDS-PAGE analysis were pelleted and resuspended in 80 µL 0.1 M Tris-HCl pH 8.5 containing 5 M urea and 20 µL SDS-PAGE gel-loading buffer. The samples were then analyzed on a 12% SDS-PAGE and stained with Coomassie blue to confirm overexpression of the non-frameshifted and -1 frameshifted protein products from the PK401 construct (Fig. 2). The non-frameshifted product (CFP) has an expected size of 29 kD, and the -1 frameshifted product (a fusion protein of CFP-PK401-YFP) has an expected size of 60 kD. Bands of increasing intensity after induction with IPTG were seen at approximately 30 kD and 60 kD, corresponding to both the non-frameshifted and -1 frameshifted product. These same bands were seen in the uninduced samples, however this could be due to the expression being controlled by the pLacI promoter, which is known to give leaky expression.</p><br />
<br />
<p>To estimate the frameshifting frequency of PK401, the relative band intensities of the 60 kD protein were compared to that of the 29 kD protein. This resulted in a calculated frameshift efficiency of 21 ± 5%, indicating that 21% of the translated product was frameshifted into the -1 frame to produce the CFP-PK401-YFP fusion protein. This result correlates well with the previously reported frameshifting efficiency of 14 ± 2% for this pseudoknot (Tholstrup et al., 2011).</p><br />
<br />
<br><br />
<center>[[Image:ULeth2013_PK401-overexpressiongel.png|400px]]</center><br />
<br><br />
<br />
<p><b>Figure 2. Overexpression of non-frameshifted and -1 frameshifted protein products from the PK401 construct.</b> Equivalent amounts of cells at 0, 1, 3, and 5 h after induction with IPTG (PK401 +IPTG) were analyzed by 12% SDS-PAGE. The same time samples from the uninduced culture were also analyzed (PK401 –IPTG). Black boxes indicate bands of increasing intensity that migrated with an approximate molecular weight of 60 kD and 30 kD, corresponding to the -1 frameshifted CFP-PK401-YFP fusion product and the non-frameshifted CFP product, respectively.</p><br><br />
<br />
<p>The PK401 overexpression products were directly compared to the overexpression of CFP (BBa_K331033) and YFP (BBa_K331031) (Fig. 3). The smaller product from PK401 expression (non-frameshifted CFP) appears slightly larger in size than either CFP or YFP alone. This is likely due to the additional amino acids that are translated in the zero frame before encountering the stop codon after the pseudoknot.</p><br />
<br />
<br><br />
<center>[[Image:ULeth2013_PK401expression.png|400px]]</center><br />
<br><br />
<br />
<p><b>Figure 3. Comparison of PK401 over-expression products to CFP and YFP.</b> Equivalent amounts of cells from the expression of CFP (BBa_K331033), YFP (BBa_K331031), PK401 before (-IPTG) and after induction (+IPTG), and a non-expressing YFP construct (BBa_K331035) were analyzed by 12% SDS-PAGE. Black boxes indicate over-expressed protein products, including CFP and YFP at approximately 29 kD, the non-frameshifted CFP product from BBa_K1210000 around 30 kD, and -1 frameshifted CFP-PK401-YFP fusion product at 60 kD.</p><br><br />
<br />
<p><b>CFP and YFP Fluorescence from PK401 Overexpression</b></p><br />
<p>To confirm that the protein products seen in the overexpression of the PK401 construct corresponded to the expected fluorescent proteins, fluorescence spectra of the cell lysates from overexpression were measured. The cells from the overexpression were lysed using 1 mg/mL lysozyme and 12.5 mg/g sodium deoxycholate. DNase was added to degrade the DNA followed by centrifugation at 3000 x g for 30 min. The supernatant was collected and centrifuged further at 30 000 x g for 45 min. This supernatant was then used for fluorescence measurements.</p><br />
<br />
<p>The supernatants were diluted 100-fold in the buffer used for cell opening. Excitation and emission scans were measured for all samples in the CFP and YFP range in order to confirm the presence of both fluorescent proteins. CFP was excited at 430 nm, and emission was monitored from 445-650 nm. YFP was excited at 510 nm, and emission was monitored from 525-650 nm. These spectra were measured for the cell opening buffer, the induced and uninduced samples of the cells that contained the PK401 construct, the CFP and YFP cell lysates, as well as the cells containing the control plasmid.</p><br />
<br />
<p>The relative fluorescence intensity of CFP and YFP after direct excitation were compared to the relative fluorescence of the PK401 cell lysate (Fig. 4). The cell lysate from the PK401 overexpression show the characteristic emission spectrum of CFP upon excitation at 430 nm. There is an additional peak in the spectrum at 527 nm, which is the emission maximum of YFP. Direct excitation of YFP from the PK401 overexpression sample also showed the characteristic emission spectrum of YFP, indicating that both CFP and YFP were expressed in this sample. The fluorescence from YFP could only result from a frameshift during expression of the PK401 transcript, indicating that the pseudoknot worked as expected to induce a -1 frameshift during translation. </p><br />
<br />
<br><br />
<center>[[Image:ULeth2013_PK401-CFP-YFP_relative.png|600px]]</center><br />
<br />
<p><b>Figure 4. Relative fluorescence intensity of CFP, YFP, and P401 overexpression samples.</b> Cell lysates from the cultures used for overexpression were excited near the excitation maximum of CFP (at 430 nm) and the emission was monitored from 445-650 nm (K1210000, green; CFP, blue; YFP, yellow).</p><br><br />
<br />
<br />
<p>Upon excitation of the cell lysates from the control plasmid cultures at 430 nm, a very low fluorescence signal was observed that did not resemble the emission spectrum of CFP, and was likely due to other cellular components. Additionally, when the sample was excited at 510 nm, there was no detectable emission signal, indicating that YFP was not present in these samples. </p><br />
<br />
<br><br />
<center>[[Image:ULeth2013_PK401-FRET.png|600px]]</center><br />
<br />
<p><b>Figure 5. Maximum fluorescence intensity of overexpression cell lysates.</b> Intensities of cell lysates after excitation at 430 nm and monitoring at 475 nm (CFP fluorescence, blue bars) and excitation at 510 nm and emission at 527 nm (YFP fluorescence, yellow bars).</p><br><br />
<br />
<p>When the peak intensities from each sample were analyzed individually (Fig. 5), more prominent fluorescence measurements were observed for the PK401 cell lysates than for the control cell lysates. Excitation at 430 nm resulted in a fluorescence emission at 475 nm, which is a characteristic emission maximum of CFP, for the PK401 samples, but not for the control samples. Additionally, direct excitation and emission from YFP was seen for the PK401 cell lysates, but no fluorescence emission was seen for the control samples after excitation at 510 nm. This further indicates that the PK401 pseudoknot induced a -1 frameshift during translation of the PK401 transcript to produce both CFP and YFP.</p><br><br />
<br />
<h2>Generating a Pseudoknot Library</h2><br />
<p><b>Generating a Library of Pseudoknots with Variable Frameshifting Frequencies</b></p><br />
<br />
<p>To make better use of the frameshifting capability of the pseudoknot, we are also generating a randomized library of pseudoknot sequences that can be characterized for frameshifting frequency. It is our goal to create a library of pseudoknots that have a range of frameshifting frequencies to better facilitate broad application of this part. To do this, the PK401 sequence will be mutagenized using error-prone PCR to generate a library of primers that will be used to amplify the plasmid containing the original sequence. The newly generated plasmids contained mutated PK401 sequences will be transformed into E. coli cells, sequenced, and characterized for their specific frameshifting efficiency using a construct such as BBa_K1210000.</p><br><br />
<br />
<p><b>Optimizing Conditions for the Error-Prone PCR</b></p><br />
<br />
<p>To generate a library with high variability of sequence, conditions for the error-prone PCR had to first be optimized. We wanted to disrupt the PCR experiment enough to produce a large variety of primer sequences, but not too much so that the PCR was no longer successful. To do this, we used a temperature gradient to determine the annealing temperature that would best facilitate the amplification of the expected product. Figure 6 shows the resulting PCR products from the temperature gradient reactions, using temperatures from 41.9-62.1°C. The expected size of the PCR product is 93 bp, which was seen in the first three reaction samples. Various unspecific PCR products were seen in the remaining reactions samples. For this reason, the temperature chosen for the subsequent error-prone PCRs was 43.6°C, the temperature used to produce the PCR product in lane 3.</p><br />
<br />
<br><br />
<center>[[Image:ULeth2013iGEM tempgradient.png|400px]]</center><br><br />
<br />
<p><b>Figure 6. Determining the ideal temperature for error-prone PCR reactions.</b> PCR reactions were performed using various annealing temperatures from 41.9-62.1°C (lanes 1-12). The primers used in the reaction flank the PK401 pseudoknot, making the expected product size 93 bp. The temperature from the reaction in lane 3 (43.6°C) was selected for subsequent error-prone PCRs.</p><br><br />
<br />
<p>Next, we modulated a few of the typical PCR conditions to induce errors during the reaction. First, we used Taq polymerase, which is known to have higher error rates than higher fidelity polymerases commonly used in PCRs. Second, we added MnCl2 to the reaction to further hinder the accuracy of the polymerase. Finally, different ratios of deoxynucleotides were used in the reactions, either by increasing the dGTP concentration or by using a higher concentration of both dTTP and dCTP. A number of these conditions worked to produce the expected PCR product at 93 bp (Fig. 7, lanes 1-4, 6). The conditions tested in lanes 1-4 included MnCl2 at concentrations of 0-0.45 mM, and lane 6 tested the effect of increasing dGTP concentrations to 1 mM while the other dNTPs were at a concentration of 0.2 mM.</p><br />
<br />
<br><br />
<center>[[Image:ULeth2013iGEM_errorpronepcr.png|400px]]</center><br><br />
<br />
<p><b>Figure 7. Error-prone PCR of PK401 sequence.</b> PCR conditions were adjusted to induce errors in the PK401 sequence. Reactions were performed with the following modified condtions: lanes 1-6 used MnCl2 concentrations from 0-0.65 mM, lanes 4-6 used dGTP concentrations that were increased to 0.7-1.2 mM, lane 7 used increased concentrations of dTTP and dCTP, lane 8 used increased concentrations of dGTP only, and lane 9 used 0.65 mM MnCl2 only.</p><br><br />
<br />
<p>Now that we have determined the conditions for the error-prone PCR, we will perform a large-scale amplification so that we can purify the resulting PK401 primers by extracting it from an agarose gel. The primers will then be used in a high fidelity PCR reaction to amplify the entire plasmid, which will create the library of PK401 sequences. The final steps will be to transform the final PCR products into E. coli cells, screen them for frameshifting frequency using a similar construct as BBa_K1210000, and sequence the pseudoknot before submitting the parts to the registry to complete the library of frameshifting elements.</p><br />
<br />
<br><h2>References</h2><br />
<p>Tholstrup, J., Oddershede, L. B., & Sørensen, M. A. (2011). mRNA pseudoknot structures can act as ribosomal roadblocks. Nucleic acids research, 40(1), 303-313.</p></div>Dustinhttp://2013.igem.org/Team:Lethbridge/resultsTeam:Lethbridge/results2014-01-21T22:48:01Z<p>Dustin: corrected mistake in figure caption</p>
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</body><br />
</html><br />
<br />
<BLOCKQUOTE><br />
=<font color="black">Results=<br />
<br />
<br />
<br />
<h2>Team Parts Sandbox</h2><br />
<center><br />
<table border="0" style="background-color:#FFF" width="100%" cellpadding="0" cellspacing="5"><br />
<tr><br />
<td><strong>Name</strong></td><br />
<td><strong>Type</strong></td><br />
<td><strong>Description</strong></td><br />
<td><strong>Designer</strong></td><br />
<td><strong>Length</strong></td><br />
<td><strong>Favorite Part</strong></td><br />
</tr><br />
<tr><br />
<td>[http://parts.igem.org/Part:BBa_K1210000 BBa_K1210000]</td><br />
<td>Device</td><br />
<td>LacI-CFP-PK401-YFP</td><br />
<td>Zak Stinson</td><br />
<td>1955</td><br />
<td><center>Yes</center></td><br />
</tr><br />
<tr><br />
<td>[http://parts.igem.org/Part:BBa_K1210001 BBa_K1210001]</td><br />
<td>RNA Part</td><br />
<td>PK401 Pseudoknot</td><br />
<td>Zak Stinson</td><br />
<td>93</td><br />
<td> </td><br />
</tr><br />
<tr><br />
<td>[http://parts.igem.org/Part:BBa_K1210002 BBa_K1210002]</td><br />
<td>Device</td><br />
<td>Enhanced Lumazine Synthase (ELS) Expression Construct</td><br />
<td>Harland Brandon</td><br />
<td>828</td><br />
<td> </td><br />
</tr><br />
<tr><br />
<td>[http://parts.igem.org/Part:BBa_K1210003 BBa_K1210003]</td><br />
<td>Device</td><br />
<td>Tagged dual coding test construct</td><br />
<td>Harland Brandon</td><br />
<td>1850</td><br />
<td> </td><br />
</tr><br />
</table><br />
<br />
</center><br />
<br />
<br><br />
<br />
<h2>Expression and Characterization of PK401 (BBa_K1210000)</h2><br />
<br />
<p><b>PK401 Overexpression</b></p><br />
<p>The PK401 construct was overexpressed to test and characterize the construct for frameshifting ability. To do this, cultures of E. coli DH5α containing either the PK401 plasmid or an empty control plasmid were grown from glycerol stocks overnight at 37°C in 50 mL LB media containing Kanamycin (100 µg/mL). These cultures were used the next day to inoculate 500 mL of LB media to a starting optical density at 600 nm (OD600) near 0.05. The OD600 was monitored and induced with 1 mM IPTG once reaching an OD600 of 0.6 (Fig. 1). Growth was monitored hourly, and equivalent amounts of cells were taken as samples for SDS-PAGE analysis up to 5 h after induction. The cultures were harvested by centrifuging at 5000 x g and shock frozen to store at -80°C.</p><br />
<br />
<center>[[File:ULeth2013_PK401-overexpression.png|600px]]</center><br />
<br />
<p><b>Figure 1. Growth curve of PK401 construct.</b> E. coli DH5α cells containing the PK401 plasmid or a control plasmid were grown at 37°C in LB media. The OD600 was monitored and the cultures were induced with 1 mM IPTG when the OD600 had reached 0.6. The cultures were grown for an additional 5 h, and a sample of 1 OD600 equivalent of cells were taken at each hour after induction.</p><br><br />
<br />
<p>The samples taken for SDS-PAGE analysis were pelleted and resuspended in 80 µL 0.1 M Tris-HCl pH 8.5 containing 5 M urea and 20 µL SDS-PAGE gel-loading buffer. The samples were then analyzed on a 12% SDS-PAGE and stained with Coomassie blue to confirm overexpression of the non-frameshifted and -1 frameshifted protein products from the PK401 construct (Fig. 2). The non-frameshifted product (CFP) has an expected size of 29 kD, and the -1 frameshifted product (a fusion protein of CFP-PK401-YFP) has an expected size of 60 kD. Bands of increasing intensity after induction with IPTG were seen at approximately 30 kD and 60 kD, corresponding to both the non-frameshifted and -1 frameshifted product. These same bands were seen in the uninduced samples, however this could be due to the expression being controlled by the pLacI promoter, which is known to give leaky expression.</p><br />
<br />
<p>To estimate the frameshifting frequency of PK401, the relative band intensities of the 60 kD protein were compared to that of the 29 kD protein. This resulted in a calculated frameshift efficiency of 21 ± 5%, indicating that 21% of the translated product was frameshifted into the -1 frame to produce the CFP-PK401-YFP fusion protein. This result correlates well with the previously reported frameshifting efficiency of 14 ± 2% for this pseudoknot (Tholstrup et al., 2011).</p><br />
<br />
<br><br />
<center>[[Image:ULeth2013_PK401-overexpressiongel.png|400px]]</center><br />
<br><br />
<br />
<p><b>Figure 2. Overexpression of non-frameshifted and -1 frameshifted protein products from the PK401 construct.</b> Equivalent amounts of cells at 0, 1, 3, and 5 h after induction with IPTG (PK401 +IPTG) were analyzed by 12% SDS-PAGE. The same time samples from the uninduced culture were also analyzed (PK401 –IPTG). Black boxes indicate bands of increasing intensity that migrated with an approximate molecular weight of 60 kD and 30 kD, corresponding to the -1 frameshifted CFP-PK401-YFP fusion product and the non-frameshifted CFP product, respectively.</p><br><br />
<br />
<p>The PK401 overexpression products were directly compared to the overexpression of CFP (BBa_K331033) and YFP (BBa_K331031) (Fig. 3). The smaller product from PK401 expression (non-frameshifted CFP) appears slightly larger in size than either CFP or YFP alone. This is likely due to the additional amino acids that are translated in the zero frame before encountering the stop codon after the pseudoknot.</p><br />
<br />
<br><br />
<center>[[Image:ULeth2013_PK401expression.png|400px]]</center><br />
<br><br />
<br />
<p><b>Figure 3. Comparison of PK401 over-expression products to CFP and YFP.</b> Equivalent amounts of cells from the expression of CFP (BBa_K331033), YFP (BBa_K331031), PK401 before (-IPTG) and after induction (+IPTG), and a non-expressing YFP construct (BBa_K331035) were analyzed by 12% SDS-PAGE. Black boxes indicate over-expressed protein products, including CFP and YFP at approximately 29 kD, the non-frameshifted CFP product from BBa_K1210000 around 30 kD, and -1 frameshifted CFP-PK401-YFP fusion product at 60 kD.</p><br><br />
<br />
<p><b>CFP and YFP Fluorescence from PK401 Overexpression</b></p><br />
<p>To confirm that the protein products seen in the overexpression of the PK401 construct corresponded to the expected fluorescent proteins, fluorescence spectra of the cell lysates from overexpression were measured. The cells from the overexpression were lysed using 1 mg/mL lysozyme and 12.5 mg/g sodium deoxycholate. DNase was added to degrade the DNA followed by centrifugation at 3000 x g for 30 min. The supernatant was collected and centrifuged further at 30 000 x g for 45 min. This supernatant was then used for fluorescence measurements.</p><br />
<br />
<p>The supernatants were diluted 100-fold in the buffer used for cell opening. Excitation and emission scans were measured for all samples in the CFP and YFP range in order to confirm the presence of both fluorescent proteins. CFP was excited at 430 nm, and emission was monitored from 445-650 nm. YFP was excited at 510 nm, and emission was monitored from 525-650 nm. These spectra were measured for the cell opening buffer, the induced and uninduced samples of the cells that contained the PK401 construct, the CFP and YFP cell lysates, as well as the cells containing the control plasmid.</p><br />
<br />
<p>The relative fluorescence intensity of CFP and YFP after direct excitation were compared to the relative fluorescence of the PK401 cell lysate (Fig. 4). The cell lysate from the PK401 overexpression show the characteristic emission spectrum of CFP upon excitation at 430 nm. There is an additional peak in the spectrum at 527 nm, which is the emission maximum of YFP. Direct excitation of YFP from the PK401 overexpression sample also showed the characteristic emission spectrum of YFP, indicating that both CFP and YFP were expressed in this sample. The fluorescence from YFP could only result from a frameshift during expression of the PK401 transcript, indicating that the pseudoknot worked as expected to induce a -1 frameshift during translation. </p><br />
<br />
<br><br />
<center>[[Image:ULeth2013_PK401-CFP-YFP_relative.png|600px]]</center><br />
<br />
<p><b>Figure 4. Relative fluorescence intensity of CFP, YFP, and P401 overexpression samples.</b> Cell lysates from the cultures used for overexpression were excited near the excitation maximum of CFP (at 430 nm) and the emission was monitored from 445-650 nm (K1210000, green; CFP, blue; YFP, yellow).</p><br><br />
<br />
<br />
<p>Upon excitation of the cell lysates from the control plasmid cultures at 430 nm, a very low fluorescence signal was observed that did not resemble the emission spectrum of CFP, and was likely due to other cellular components. Additionally, when the sample was excited at 510 nm, there was no detectable emission signal, indicating that YFP was not present in these samples. </p><br />
<br />
<br><br />
<center>[[Image:ULeth2013_PK401-FRET.png|600px]]</center><br />
<br />
<p><b>Figure 5. Maximum fluorescence intensity of overexpression cell lysates.</b> Intensities of cell lysates after excitation at 430 nm and monitoring at 475 nm (CFP fluorescence, blue bars) and excitation at 510 nm and emission at 527 nm (YFP fluorescence, yellow bars).</p><br><br />
<br />
<p>When the peak intensities from each sample were analyzed individually (Fig. 5), more prominent fluorescence measurements were observed for the PK401 cell lysates than for the control cell lysates. Excitation at 430 nm resulted in a fluorescence emission at 475 nm, which is a characteristic emission maximum of CFP, for the PK401 samples, but not for the control samples. Additionally, direct excitation and emission from YFP was seen for the PK401 cell lysates, but no fluorescence emission was seen for the control samples after excitation at 510 nm. This further indicates that the PK401 pseudoknot induced a -1 frameshift during translation of the PK401 transcript to produce both CFP and YFP.</p><br><br />
<br />
<h2>Generating a Pseudoknot Library</h2><br />
<p><b>Generating a Library of Pseudoknots with Variable Frameshifting Frequencies</b></p><br />
<br />
<p>To make better use of the frameshifting capability of the pseudoknot, we are also generating a randomized library of pseudoknot sequences that can be characterized for frameshifting frequency. It is our goal to create a library of pseudoknots that have a range of frameshifting frequencies to better facilitate broad application of this part. To do this, the PK401 sequence will be mutagenized using error-prone PCR to generate a library of primers that will be used to amplify the plasmid containing the original sequence. The newly generated plasmids contained mutated PK401 sequences will be transformed into E. coli cells, sequenced, and characterized for their specific frameshifting efficiency using a construct such as BBa_K1210000.</p><br><br />
<br />
<p><b>Optimizing Conditions for the Error-Prone PCR</b></p><br />
<br />
<p>To generate a library with high variability of sequence, conditions for the error-prone PCR had to first be optimized. We wanted to disrupt the PCR experiment enough to produce a large variety of primer sequences, but not too much so that the PCR was no longer successful. To do this, we used a temperature gradient to determine the annealing temperature that would best facilitate the amplification of the expected product. Figure 6 shows the resulting PCR products from the temperature gradient reactions, using temperatures from 41.9-62.1°C. The expected size of the PCR product is 93 bp, which was seen in the first three reaction samples. Various unspecific PCR products were seen in the remaining reactions samples. For this reason, the temperature chosen for the subsequent error-prone PCRs was 43.6°C, the temperature used to produce the PCR product in lane 3.</p><br />
<br />
<br><br />
<center>[[Image:ULeth2013iGEM tempgradient.png|400px]]</center><br><br />
<br />
<p><b>Figure 6. Determining the ideal temperature for error-prone PCR reactions.</b> PCR reactions were performed using various annealing temperatures from 41.9-62.1°C (lanes 1-12). The primers used in the reaction flank the PK401 pseudoknot, making the expected product size 93 bp. The temperature from the reaction in lane 3 (43.6°C) was selected for subsequent error-prone PCRs.</p><br><br />
<br />
<p>Next, we modulated a few of the typical PCR conditions to induce errors during the reaction. First, we used Taq polymerase, which is known to have higher error rates than higher fidelity polymerases commonly used in PCRs. Second, we added MnCl2 to the reaction to further hinder the accuracy of the polymerase. Finally, different ratios of deoxynucleotides were used in the reactions, either by increasing the dGTP concentration or by using a higher concentration of both dTTP and dCTP. A number of these conditions worked to produce the expected PCR product at 93 bp (Fig. 7, lanes 1-4, 6). The conditions tested in lanes 1-4 included MnCl2 at concentrations of 0-0.45 mM, and lane 6 tested the effect of increasing dGTP concentrations to 1 mM while the other dNTPs were at a concentration of 0.2 mM.</p><br />
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<center>[[Image:ULeth2013iGEM_errorpronepcr.png|400px]]</center><br><br />
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<p><b>Figure 7. Error-prone PCR of PK401 sequence.</b> PCR conditions were adjusted to induce errors in the PK401 sequence. Reactions were performed with the following modified condtions: lanes 1-6 used MnCl2 concentrations from 0-0.65 mM, lanes 4-6 used dGTP concentrations that were increased to 0.7-1.2 mM, lane 7 used increased concentrations of dTTP and dCTP, lane 8 used increased concentrations of dGTP only, and lane 9 used 0.65 mM MnCl2 only.</p><br><br />
<br />
<p>Now that we have determined the conditions for the error-prone PCR, we will perform a large-scale amplification so that we can purify the resulting PK401 primers by extracting it from an agarose gel. The primers will then be used in a high fidelity PCR reaction to amplify the entire plasmid, which will create the library of PK401 sequences. The final steps will be to transform the final PCR products into E. coli cells, screen them for frameshifting frequency using a similar construct as BBa_K1210000, and sequence the pseudoknot before submitting the parts to the registry to complete the library of frameshifting elements.</p><br />
<br />
<br><h2>References</h2><br />
<p>Tholstrup, J., Oddershede, L. B., & Sørensen, M. A. (2011). mRNA pseudoknot structures can act as ribosomal roadblocks. Nucleic acids research, 40(1), 303-313.</p></div>Dustinhttp://2013.igem.org/Team:Lethbridge/attributionsTeam:Lethbridge/attributions2013-12-06T22:19:52Z<p>Dustin: </p>
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<p><center>Courtesy of University of Lethbridge Advancement</p></center><br />
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=<font color="black">Attributions=<br />
All planning and wet lab work was performed by the undergraduate members of our iGEM team.<br />
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<h2>Contributions</h2><br />
<p><b>Dr. Gaymon Bennett</b> (Fred Hutchinson Cancer Research Center)</p><br />
<p><b>Dr. Karmella Haynes</b> (Arizona State University)</p> <br />
<p><b>Edward You</b> (FBI, Weapons of Mass Destruction Directorate)</p> <br />
<p><b>Public Health Agency of Canada</b></p> <br />
<p><b>Angela Birnbaum</b> (Harvard University)</p> <br />
<p><b>Dallas Thomas</b> (University of Lethbridge, Lethbridge Agriculture and Agri-Food Canada)</p> <br />
<p><b>Dr. Jeffrey Fischer</b> (University of Calgary)</p> <br />
<p><b>Sutherland Dubé, Dipankar Goyal, [http://seventhousandplus.weebly.com/index.html Kelsey Kristensen], Richard McLean, Fan Mo, Justin Vigar,</b> and <b>Anthony Vuong</b> (University of Lethbridge)</p><br />
<p>The Department of Chemistry and Biochemistry, the Alberta RNA Research and Training Institute, the Kothe lab, Selinger lab, and Wieden lab at the University of Lethbridge for providing us with equipment and laboratory space.<br />
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<h2>Sponsors</h2><br />
<p>We would also like to acknowledge our sponsors for the following support of the 2013 Lethbridge iGEM team.</p><br><br />
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<p><b>1. Platinum - $5000+ or gift in kind</b></p><br />
<p>Logo on front of team shirts, large logo on scientific poster, large logo on team wiki and verbal recognition during team project presentations/media interviews.</p><br />
<p><b>2. Gold - $2000-$4999 or gift in kind</b></p><br />
<p>Logo on team shirts, medium logo on scientific poster, medium logo on team wiki and written recognition at end of team project presentations.</p><br />
<p><b>3. Silver - $1000-$1999 or gift in kind</b></p><br />
<p>Medium logo on scientific poster, medium logo on team wiki and written recognition at end of team project presentations.</p><br />
<p><b>4. Bronze - <$999 or gift in kind</b></p><br />
<p>Small logo on scientific poster, small logo on team wiki and written recognition at end of team project presentations.</p></div>Dustinhttp://2013.igem.org/Team:Lethbridge/teamTeam:Lethbridge/team2013-10-29T02:28:06Z<p>Dustin: </p>
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<h1>Team</h1><br />
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<h2>Suneet Kharey </h2><br />
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<td><p position="right">Hello, my name is Suneet (sun-eat). This is my first year in iGEM, I've always been intrigued by synthetic biology so I'm grateful for this opportunity. In my free time (which is currently non-existent) I enjoy learning new languages (currently at 3), painting, buying more Doc Martens, and pointing out obscure constellations in the night sky. Currently I am learning French and teaching myself how to play the Harmonica. </p></td></tr><br />
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<h2>Graeme Glaister</h2><br />
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<td><p position="right">I am a fourth year B.Sc. Neuroscience student, planning on pursuing a Master’s degree. This is my first year in iGEM. I grew up in Fort McMurray but moved to Lethbridge in Jr. High. The reason I chose to participate in iGEM is so that I could have the unique experience (for an undergrad) of participating in the planning process for an experiment as well as the necessary lab work. I eagerly look forward to continuing on with iGEM in 2014.</p></td></tr><br />
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<h2>Zak Stinson</h2><br />
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<td><p position="right">Hey, I'm Zak, and I am an iGEMmer. I thought I was almost free of my fascination with genetic technologies when I transferred to the neuroscience program at the U of L in 2011 from biology at the U of R, but I lapsed right back when I learned that iGEM existed. I thought I had refocused when I began doing behavioural experiments with rats, but immediately searched out and executed a synthetic biology project designing a system for reporting gene activity in the brains of live animals. After completing my B.Sc. in neuroscience this summer I will likely continue to design synthetic biology projects in the M.Sc. neuroscience program at the U of L as I am dangerously hooked. So I guess I'll talk to you all next year!</p></td></tr><br />
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<h2>Dustin Smith</h2><br />
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<td><p position="right">Hello, my name is Dustin Smith. I was born and raised in Lethbridge, and I am now a Master’s student majoring in Biochemistry. I’m happy to be back with the University of Lethbridge iGEM team for a third year, and as always it has been a great experience. Outside of school my favorite activities are ice hockey, snowboarding, and lifting.</p></td></tr><br />
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<h2>Jenna Friedt</h2><br />
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<td><p position="right">Hello, my name is Jenna and I am one of the graduate student advisors for the 2013 Lethbridge iGEM team. Since I’m convinced I was born in the wrong era, I’m currently looking for assistance in inventing a time machine to send me back to the ‘50s, where I would happily fit in with the bubblegum rockers and poodle skirt-wearing youth of the day. Until that happens, I’ll settle for nights on the patio listening to the Beatles, reading a good book, and watching Audrey Hepburn movies.</p></td></tr><br />
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<h2>Harland Brandon</h2><br />
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<td><p position="right">Harland is an avid biochemist pursuing his Masters degree. In his spare time he enjoys building and creating new things, as such it was only natural that he continued with his interest in iGEM. Having been around the competition for four years now he has decided to take a backseat role in guiding and mentoring the schools iGEM team. When you don't find him in lab or at the university he can most likely be found at his computer playing or enjoying a fine strong Scottish beverage.</p></td></tr><br />
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<h2>Supervisor: Hans-Joachim Wieden</h2><br />
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<td><p position="right">Originally from Germany I moved to Canada in 2005 to start a research group on the structure and function of the bacterial protein synthesis machinery, a cellular process targeted by over 50% of the known antibiotics. I am intrigued by the molecular design and function of this essential bio-nanomachine. I try to unravel the underlying design principles in order to enable the rational design and engineering of novel bio-nanomachines. I am essentially asking the question if such novel bio-machines can be constructed from simple and fundamental principles or are these assemblies just too complex. Well and that’s why it was extremely easy to rope me into doing iGEM.</p></td></tr><br />
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<h1>Human Practices</h1><br />
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<h2>Biosecurity and DNA Synthesis</h2><br />
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<h3><b>Current Sequence Screening Methods</b></h3><br />
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<p style="color:black"> In the last 5 years, there has been increased recognition of the powers of gene synthesis. It is now easy and affordable to look up genetic sequences for nearly any organism, design an expression construct, and order that gene from a synthesis company. This allows for the creative projects we see each year at the iGEM jamborees, but it also allows those with malevolent intentions and adequate knowledge to easily order genes that may pose a hazard to others. </p><br />
<p style="color:black"> The recognition of this potential has led members of governments and large synthesis companies to try and establish a framework for screening these synthesis orders to ensure that potentially hazardous sequences stay in the hands of those who would use them for legitimate research purposes. This effort to regulate the gene synthesis industry has largely come from within. In the late 2000's, both Europe's International Association of Synthetic Biology (IASP) and North America's International Gene Synthesis Consortium (IGSC) put forth reports on the state of synthesis order screening as well as a set of best practices to follow [1-2]. These bodies are made up of individuals from the major gene synthesis companies in each region as well as experts from major universities.</p><br />
<p style="color:black">Both groups outline a very similar approach to screening these orders for legitimacy. This entails a two part approach that first compares the ordered sequence to sequences on a list of known bio-hazardous agents and second, verifies the legitimacy of the customer and their intended use of the final product. In both reports the sequence screening utilizes existing pathogen databases, such as the US Select Agents and Toxins List or the Australia Group List as well as internal pathogen databases, and BLASTs the submitted sequence against these regulated ones. This first step in screening is conducted automatically. If there is a similarity between the submitted sequence and one of the sequences on these lists that exceeds the specified threshold, human investigation is used to further characterize the sequence [2].</p><br />
<p style="color:black">Customer screening is arguably the most important aspect of the current gene synthesis security strategies. It is possible that ordering sequences that could be considered hazardous is necessary for research applications and adequate customer screening could determine if this sequence was going to someone at a research facility for legitimate use. European and North American groups recommend collection of the name, mailing address, and institutional affiliation of the customer to ensure that they are individuals working in verifiable positions within companies or academic institutions [1-2]. This information is then independently verified and checked against a number of national and international lists of individuals of concern, such as the US Specially Designated Nationals list.</p><br />
<p style="color:black">While these protocols are put forth by consortium members in both Europe and North America, as well as there being a set of guidelines published by the US Department of Health and Human Services, all of these measures are voluntary [3]. There are no penalties to synthesis companies that do not screen the sequences or customers they deal with, outside of restrictions on international shipment of dual-use goods. This lack of legal regulation has the potential to allow dangerous sequences into the hands of malevolent individuals if any company decides to loosen their security criteria in order to save time or money in processing an order. </p><br />
<br />
<h3><b>Potential Weaknesses of Current Screening Procedures</b></h3><br />
<br />
<p style="color:black">Although companies included in the IASB and IGSC adhere to the regulations of the Code of Conduct for Best Practices in Gene Synthesis or the Harmonized Screening Protocol, respectively [1-2], these protocols have a few potential weaknesses. Both of these protocols require that all synthesis orders are at minimum screened against a regulated pathogen database. However, these lists are by no means complete and there is a chance that potentially hazardous sequences can be ordered and synthesized without any efforts made to investigate the source of the order. This is currently one of the major weaknesses of screening protocols, and efforts are being made to compile a list of data from organisms on the Select Agents list, the Australia Group List, and other national lists of regulated pathogens. Once complete, this list will provide a more comprehensive database of potential pathogenic and toxic organism sequences as a step toward higher biosecurity.</p><br />
<p style="color:black">The following are a few other weaknesses associated with current screening protocols. First, the IASB requires its member companies to screen orders of a minimum 200 base pairs in length, but there is also the potential of larger sequences being ordered as a series of short oligonucleotide sequences, from one company or multiple companies, that could bypass the screening process entirely. Though it can be more difficult to get direct database hits for shorter sequences, including these types of orders in the screening procedure is still feasible and may only require extra processing time for human investigation for these database matches. Second, though a legitimate customer can be approved for ordering hazardous sequences, the synthesis company cannot be sure of the final end user. There is no way to ensure that the customer does not ship the product to a third-party user that has not been investigated. Finally, and almost the most concerning weakness of current screening protocol, is the accountability of DNA synthesis companies. While most of the larger synthesis companies are members of the IASB or IGSC, complying to the standards mandated by these groups is still only a voluntary practice. There are no regulations in place that require a synthesis company to screen their orders for hazardous sequences or to follow-up with customer investigations of suspicious orders [4]. Even for orders that do not give a direct match to a hazardous sequence, any additional steps to associate function with the sequence is at the discretion of the company. Minshull and Wagner (representing DNA2.0 and GENEART) suggest that synthesis companies should be subject to routine “tests” of their screening protocols by their respective government bodies to ensure that they are complying to screening protocols and using the most up-to-date screening databases [5].</p><br />
<br />
<h3><b>How elements of our project were used to examine synthesis screening procedures</b></h3><br />
<br />
<p style="color:black">Our project involves the characterization of pseudoknot RNA secondary structural motifs. These motifs can be used to express dual-coding gene sequences to give protein products whose expression can be regulated by the pseudoknot’s ability to induce ribosomal frameshifting. This method of coding can allow for the expression of a protein which may be encoded by fragments in alternating reading frames. This technology adds another level of complexity in terms of screening for controlled sequences, in that the protein produced from a synthesized construct may not be the product of translating a gene in one continuous reading frame.</p><br />
<p style="color:black">It was our goal to investigate the ability of DNA synthesis companies to identify hazardous sequences in their screening procedures in the presence of frameshifting elements. A series of hazardous sequences containing intervening pseudoknots were designed and tested by two of the leading synthesis companies in North America in their standard screening procedures. These constructs contained all the necessary components to form a dangerous protein product, with DNA segments allocated into different reading frames and successively frameshifted using pseudoknots. The results from this screening test indicate that the current screening methods are successful at identifying hazardous sequences that had been “hidden” in multiple reading frames. The companies expressed their support of our efforts to investigate loopholes and problems in current screening procedures with regards to this new type of technology.</p><br />
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<h3><b>Possible Methods for Bypassing Screening</h3></b><br />
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<p style="color:black"><b>Codon redundancy</p></b><br />
<p style="color:black">Codon redundancy in the genetic code refers to having multiple codons that code for a single amino acid. This redundancy allows for the DNA sequence of a protein to be changed without altering the resulting amino acid sequence. By utilizing codon redundancy, bioterrorists could drastically change the known DNA sequence of a harmful virus or protein. Fortunately, synthesis companies scan both the DNA and protein sequence of sequences submitted for synthesis, and in this way would still be able to identify a harmful sequence that had been changed using codon redundancy. However, this method in conjunction with others, such as frameshifting elements or those others listed below, could potentially be used to bypass the DNA and amino acid sequence screening performed by synthesis companies.</p><br />
<br />
<p style="color:black"><b>Utilizing conservative and non-conservative regions of proteins</p></b><br />
<p style="color:black">Homologous proteins are those that are derived from the same ancestor; however, the two proteins do not have to share 100% amino acid identity. Multiple sequence alignments of amino acid sequences of homologous proteins from different organisms can be used to identify functionally important residues in a protein by indicating which residues are absolutely conserved, semi-conserved, and non-conserved. This would allow an individual to alter a controlled protein sequence by changing all or some of the conserved and semi-conserved residues to residues with similar physiochemical properties. In addition, all or some of the non-conserved residues could be substituted with essentially any other amino acid without risking loss of the protein’s function. This method, in combination with utilizing codon redundancy, would allow for more drastic alterations to be made to both the DNA and protein sequence from a pathogenic organism that could bypass screening procedures.</p><br />
<br />
<p style="color:black"><b>Using “custom” tRNAs</p></b><br />
<p style="color:black">A more complicated means for bypassing screening procedures by decoupling protein sequence from function would be to use a highly engineered system with non-canonical tRNAs. An organism could be designed that uses engineered amino acyl-tRNA synthetases that recognize non-cognate tRNAs and therefore aminoacylate the tRNA with the incorrect amino acid. By using this alternative genetic code in the engineered organism, the DNA sequence from a pathogenic organism could be altered in an almost indistinguishable way while still producing the protein of interest.</p><br />
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<p style="color:black"><b>Do-it-yourself synthesis</p></b><br />
<p style="color:black">As time progresses, the cost of a DNA synthesizer is getting more affordable to research labs and independent users. Initially this may seem like a good thing, but there are tremendous dangers that are associated with this development. Directly bypassing screening procedures by not requiring the services of synthesis companies allows the owner of the DNA synthesizer unrestricted access to synthesize whatever sequence they choose. This would make any techniques to bypass the screening methods of synthesis companies obsolete. As a result, there may need to be regulations put in place to limit or restrict the access of DNA synthesizers. This can be done for example by requiring the owner to upload any sequences they synthesize to a governing body that will scan them for harmful sequences, or by installing software that will screen sequences prior to allowing them to be synthesized. A combination of these two methods as well as additional advances in screening procedures is crucial to ensure the safety of the general public.</p><br />
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<h3><b>Changes recommended for screening protocols</h3></b><br />
<br />
<p style="color:black">Though commendable biosecurity efforts have been put forward by major international synthesis companies, these groups are aware that standard protocols may not be enough to mitigate the risk of the synthesis and delivery of hazardous sequences. In the IASB Code of Conduct for Best Practices in Gene Synthesis, all member companies are mandated to take part in ongoing efforts to refine and improve the current screening technologies by establishing a review committee to update and expand the Code of Conduct as new or changing threats emerge, maintain open communication with member companies through the exchange of research and literature searches, and regularly collaborating on best practices and new screening ideas [2]. While these practices are important for synthesis companies to implement, DNA synthesis is becoming less expensive and more accessible by non-professionals. According to Minshull and Wagner “[a]nyone who is sufficiently motivated could synthesize the gene for a toxin or even an entire viral genome using readily available reagents and without ever going near a specialized synthesizer” [5]. With molecular biology equipment becoming available through avenues such as E-Bay and other online dealers, individuals with limited molecular biology experience could soon realistically synthesize their own DNA sequences in the next few years [4]. Screening protocols could thereafter become obsolete. Until then, further steps are required to assure the public, government, and research community that biosecurity is being upheld to the highest standards possible. This may involve expanding the use of online forums, such as VIREP (Virulence Factor Information Repository), to allow researchers to deposit and access information about genes and organisms. Additionally, government regulations may need to be implemented that require all synthesis companies to adhere to standard practices and implement human investigation of suspicious orders [6]. This may best be achieved through the integration of both the IASB and IGSC protocols into an industry-wide Code of Conduct.</p><br />
<br />
<h2>Testing the System</h2><br />
<h3><b>Learning to Be Bad</b></h3><br />
<p style="color:black">This year, we focused on the implications our frameshifting project might have on biosecurity. In thinking about the ways our pseudoknots could be used to do new, exciting things in synthetic biology, we came up with a use that is more frightening than exciting. Bioterrorism.</p><br />
<br />
<p style="color:black">The idea is this: There are guidelines put forward by a number of industry groups on how DNA synthesis orders should be screened to ensure no biohazardous sequences get into the hands of the wrong people. The standard protocol for screening sequences involves taking the submitted DNA sequences and translating all six reading frames, then using BLAST to compare the DNA and amino acid sequences to those of organisms on a list of controlled agents.</p><br />
<br />
<p style="color:black">Our pseudoknot enables the ribosome to switch frames mid-translation, essentially splitting the entire protein amongst as many reading frames as there are pseudoknots. If someone were to split a protein from the Ebola virus into small fragments distributed across the reading frames, could they bypass this initial automatic screening step? </p><br />
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<h3><b>Putting our White Hats On</b></h3><br />
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<p style="color:black">To investigate this potential for abuse of our project, we worked together with major North American synthesis companies to see if we could try and fool their screening methods using our frameshifting elements. <br />
We designed and submitted sequences with vary coding changes and coding fragment sizes between the sequences for our PK401 pseudoknot to the synthesis companies we had partnered with. There is a full description of the sequences and a link to the raw data files below.</p><br />
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<center><table width="800px"; border="1px"; border-color="black"><br />
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<td><b>Sequence ID Number</td><br />
<td><b>Sequence Origin</td><br />
<td><b>Total Length (bp)</td><br />
<td><b>Codon Changes (%)</td><br />
<td><b>Length between PK (bp)</td><br />
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<td>1</td><br />
<td>CFP</td><br />
<td>966</td><br />
<td>25</td><br />
<td>180</td><br />
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<td>2</td><br />
<td>Staph-ORF</td><br />
<td>1869</td><br />
<td>0</td><br />
<td>210</td><br />
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<td>3</td><br />
<td>Ricin</td><br />
<td>2392</td><br />
<td>25</td><br />
<td>198</td><br />
</tr><br />
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<td>4</td><br />
<td>Staph-ORF</td><br />
<td>2450</td><br />
<td>25</td><br />
<td>102</td><br />
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<td>5</td><br />
<td>CFP</td><br />
<td>966</td><br />
<td>16</td><br />
<td>210</td><br />
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<td>6</td><br />
<td>CFP</td><br />
<td>966</td><br />
<td>0</td><br />
<td>180</td><br />
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<td>7</td><br />
<td>Staph-ORF</td><br />
<td>2450</td><br />
<td>0</td><br />
<td>102</td><br />
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<br />
<tr><br />
<td>8</td><br />
<td>Ricin</td><br />
<td>2392</td><br />
<td>0</td><br />
<td>198</td><br />
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<td>9</td><br />
<td>Ebola Matrix Protein</td><br />
<td>1031</td><br />
<td>0</td><br />
<td>0</td><br />
</tr><br />
<br />
<tr><br />
<td>10</td><br />
<td>CFP</td><br />
<td>966</td><br />
<td>16</td><br />
<td>180</td><br />
</tr><br />
<br />
<tr><br />
<td>11</td><br />
<td>Staph-ORF</td><br />
<td>2450</td><br />
<td>0</td><br />
<td>102</td><br />
</tr><br />
<br />
<tr><br />
<td>12</td><br />
<td>CFP</td><br />
<td>966</td><br />
<td>0</td><br />
<td>210</td><br />
</tr><br />
<br />
<tr><br />
<td>13</td><br />
<td>Staph-ORF</td><br />
<td>1869</td><br />
<td>20</td><br />
<td>210</td><br />
</tr><br />
<br />
<tr><br />
<td>14</td><br />
<td>Ricin</td><br />
<td>3139</td><br />
<td>25</td><br />
<td>99</td><br />
</tr><br />
<br />
<tr><br />
<td>15</td><br />
<td>Staph-ORF</td><br />
<td>1869</td><br />
<td>25</td><br />
<td>210</td><br />
</tr><br />
<br />
<tr><br />
<td>16</td><br />
<td>CFP</td><br />
<td>966</td><br />
<td>25</td><br />
<td>210</td><br />
</tr><br />
<br />
<tr><br />
<td>17</td><br />
<td>Ricin</td><br />
<td>3139</td><br />
<td>0</td><br />
<td>99</td><br />
</tr><br />
</table></center><br />
<br><br />
<a href="https://static.igem.org/mediawiki/2013/c/c5/Lethbridge_iGEM_2013_Screening_Sequences.txt"> Click here for sequences used </a><br />
<br />
<br />
<h3><b>Can We Sleep at Night?</b></h3><br />
<br />
<p style="color:black">These sequences were sent to the companies and screened for hazardous elements. One company managed to detect all of the “threats” on the first level of screening. According to them, their next steps would be to do a review of the “threat” sequences using a group of human experts while interviewing the customer to determine their background, shipping and payment information, and the intended use of the synthesized DNA.</p><br />
<br />
<p style="color:black">Another company simply analyzed the sequences to determine if they could actually construct the DNA if we were to order it. All of the sequences containing the pseudoknot elements were flagged as containing high repeats, but sequence 9, the Ebola matrix protein with no pseudoknot elements, was determined to be ready for synthesis. They did not make it clear whether or not there would be another level of screening to determine the origin of the sequences. </p><br />
<br />
<p style="color:black">Based on these results, current industry standard screening protocols appear to be sufficient to detect biosecurity threats, even with codon changes and the distribution of coding sequences amongst many reading frames. What is still a cause for concern is the strictness with which these protocols are applied. There is no legal requirement to execute biosecurity screens on DNA synthesis orders; all of the proposed protocols are currently voluntary guidelines. This could allow companies to relax their security protocols and may increase the potential of a serious bioterrorism threat coming to fruition. </p><br />
<br />
<p style="color:black">In order to make sure that the act of releasing the results of our study did not pose a security threat in itself, we consulted with Edward You. Edward is a representative of the FBI’s WMD department. Many of the industry guidelines for screening call for collaboration between the synthesis companies and government agencies responsible for responding to bioterrorism threats.</p><br />
<br />
<a href="https://static.igem.org/mediawiki/2013/9/9d/Lethbridge_iGEM_2013_Screening_Sequences_October_25.txt"> Click here for sequences used </a><br />
<br />
<h3><b>References</h3></b><br />
<br />
<p style="color:black">[1] International Gene Synthesis Consortium. Harmonized screening protocol: gene sequence & customer screening to promote biosecurity. http://www.genesynthesisconsortium.org/wp-content/uploads/2012/02/IGSC-Harmonized-Screening-Protocol1.pdf (2009).</p><br />
<p style="color:black">[2] International Association Synthetic Biology. Code of conduct for best practices in gene synthesis. http://www.ia-sb.eu/tasks/sites/synthetic-biology/assets/File/pdf/iasb_code_of_conduct_final.pdf (2009). </p><br />
<p style="color:black">[3] U.S. Department of Health and Human Services. Screening Framework Guidance for Providers of Synthetic DoublesStranded DNA. http://www.phe.gov/Preparedness/legal/guidance/syndna/Documents/syndna-guidance.pdf (2010).</p><br />
<p style="color:black">[4] Maurer S. M., Fischer M., Schwer H., Stähler C., Stähler P., & Bernauer H. S. Working paper: making commercial biology safer: what the gene synthesis industry has learned about screening customers and orders. http://gspp.berkeley.edu/iths/Maurer_IASB_Screening.pdf (2009). </p><br />
<p style="color:black">[5] Minshull J. & Wagner, R. Nat. Biotechnol. 27, 800-801 (2009).</p><br />
<p style="color:black">[6] Fischer M. & Maurer S. M. Nat. Biotechnol. 28, 20-22 (2010).</p><br />
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<div></div>Dustinhttp://2013.igem.org/Team:Lethbridge/human_practicesTeam:Lethbridge/human practices2013-10-29T01:42:30Z<p>Dustin: </p>
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<h1>Human Practices</h1><br />
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<h2>Biosecurity and DNA Synthesis</h2><br />
<br />
<h3><b>Current Sequence Screening Methods</b></h3><br />
<br />
<p style="color:black"> In the last 5 years, there has been increased recognition of the powers of gene synthesis. It is now easy and affordable to look up genetic sequences for nearly any organism, design an expression construct, and order that gene from a synthesis company. This allows for the creative projects we see each year at the iGEM jamborees, but it also allows those with malevolent intentions and adequate knowledge to easily order genes that may pose a hazard to others. </p><br />
<p style="color:black"> The recognition of this potential has led members of governments and large synthesis companies to try and establish a framework for screening these synthesis orders to ensure that potentially hazardous sequences stay in the hands of those who would use them for legitimate research purposes. This effort to regulate the gene synthesis industry has largely come from within. In the late 2000's, both Europe's International Association of Synthetic Biology (IASP) and North America's International Gene Synthesis Consortium (IGSC) put forth reports on the state of synthesis order screening as well as a set of best practices to follow [1-2]. These bodies are made up of individuals from the major gene synthesis companies in each region as well as experts from major universities.</p><br />
<p style="color:black">Both groups outline a very similar approach to screening these orders for legitimacy. This entails a two part approach that first compares the ordered sequence to sequences on a list of known bio-hazardous agents and second, verifies the legitimacy of the customer and their intended use of the final product. In both reports the sequence screening utilizes existing pathogen databases, such as the US Select Agents and Toxins List or the Australia Group List as well as internal pathogen databases, and BLASTs the submitted sequence against these regulated ones. This first step in screening is conducted automatically. If there is a similarity between the submitted sequence and one of the sequences on these lists that exceeds the specified threshold, human investigation is used to further characterize the sequence [2].</p><br />
<p style="color:black">Customer screening is arguably the most important aspect of the current gene synthesis security strategies. It is possible that ordering sequences that could be considered hazardous is necessary for research applications and adequate customer screening could determine if this sequence was going to someone at a research facility for legitimate use. European and North American groups recommend collection of the name, mailing address, and institutional affiliation of the customer to ensure that they are individuals working in verifiable positions within companies or academic institutions [1-2]. This information is then independently verified and checked against a number of national and international lists of individuals of concern, such as the US Specially Designated Nationals list.</p><br />
<p style="color:black">While these protocols are put forth by consortium members in both Europe and North America, as well as there being a set of guidelines published by the US Department of Health and Human Services, all of these measures are voluntary [3]. There are no penalties to synthesis companies that do not screen the sequences or customers they deal with, outside of restrictions on international shipment of dual-use goods. This lack of legal regulation has the potential to allow dangerous sequences into the hands of malevolent individuals if any company decides to loosen their security criteria in order to save time or money in processing an order. </p><br />
<br />
<h3><b>Potential Weaknesses of Current Screening Procedures</b></h3><br />
<br />
<p style="color:black">Although companies included in the IASB and IGSC adhere to the regulations of the Code of Conduct for Best Practices in Gene Synthesis or the Harmonized Screening Protocol, respectively [1-2], these protocols have a few potential weaknesses. Both of these protocols require that all synthesis orders are at minimum screened against a regulated pathogen database. However, these lists are by no means complete and there is a chance that potentially hazardous sequences can be ordered and synthesized without any efforts made to investigate the source of the order. This is currently one of the major weaknesses of screening protocols, and efforts are being made to compile a list of data from organisms on the Select Agents list, the Australia Group List, and other national lists of regulated pathogens. Once complete, this list will provide a more comprehensive database of potential pathogenic and toxic organism sequences as a step toward higher biosecurity.</p><br />
<p style="color:black">The following are a few other weaknesses associated with current screening protocols. First, the IASB requires its member companies to screen orders of a minimum 200 base pairs in length, but there is also the potential of larger sequences being ordered as a series of short oligonucleotide sequences, from one company or multiple companies, that could bypass the screening process entirely. Though it can be more difficult to get direct database hits for shorter sequences, including these types of orders in the screening procedure is still feasible and may only require extra processing time for human investigation for these database matches. Second, though a legitimate customer can be approved for ordering hazardous sequences, the synthesis company cannot be sure of the final end user. There is no way to ensure that the customer does not ship the product to a third-party user that has not been investigated. Finally, and almost the most concerning weakness of current screening protocol, is the accountability of DNA synthesis companies. While most of the larger synthesis companies are members of the IASB or IGSC, complying to the standards mandated by these groups is still only a voluntary practice. There are no regulations in place that require a synthesis company to screen their orders for hazardous sequences or to follow-up with customer investigations of suspicious orders [4]. Even for orders that do not give a direct match to a hazardous sequence, any additional steps to associate function with the sequence is at the discretion of the company. Minshull and Wagner (representing DNA2.0 and GENEART) suggest that synthesis companies should be subject to routine “tests” of their screening protocols by their respective government bodies to ensure that they are complying to screening protocols and using the most up-to-date screening databases [5].</p><br />
<br />
<h3><b>How elements of our project were used to examine synthesis screening procedures</b></h3><br />
<br />
<p style="color:black">Our project involves the characterization of pseudoknot RNA secondary structural motifs. These motifs can be used to express dual-coding gene sequences to give protein products whose expression can be regulated by the pseudoknot’s ability to induce ribosomal frameshifting. This method of coding can allow for the expression of a protein which may be encoded by fragments in alternating reading frames. This technology adds another level of complexity in terms of screening for controlled sequences, in that the protein produced from a synthesized construct may not be the product of translating a gene in one continuous reading frame.</p><br />
<p style="color:black">It was our goal to investigate the ability of DNA synthesis companies to identify hazardous sequences in their screening procedures in the presence of frameshifting elements. A series of hazardous sequences containing intervening pseudoknots were designed and tested by two of the leading synthesis companies in North America in their standard screening procedures. These constructs contained all the necessary components to form a dangerous protein product, with DNA segments allocated into different reading frames and successively frameshifted using pseudoknots. The results from this screening test indicate that the current screening methods are successful at identifying hazardous sequences that had been “hidden” in multiple reading frames. The companies expressed their support of our efforts to investigate loopholes and problems in current screening procedures with regards to this new type of technology.</p><br />
<br />
<h3><b>Possible Methods for Bypassing Screening</h3></b><br />
<br />
<p style="color:black"><b>Codon redundancy</p></b><br />
<p style="color:black">Codon redundancy in the genetic code refers to having multiple codons that code for a single amino acid. This redundancy allows for the DNA sequence of a protein to be changed without altering the resulting amino acid sequence. By utilizing codon redundancy, bioterrorists could drastically change the known DNA sequence of a harmful virus or protein. Fortunately, synthesis companies scan both the DNA and protein sequence of sequences submitted for synthesis, and in this way would still be able to identify a harmful sequence that had been changed using codon redundancy. However, this method in conjunction with others, such as frameshifting elements or those others listed below, could potentially be used to bypass the DNA and amino acid sequence screening performed by synthesis companies.</p><br />
<br />
<p style="color:black"><b>Utilizing conservative and non-conservative regions of proteins</p></b><br />
<p style="color:black">Homologous proteins are those that are derived from the same ancestor; however, the two proteins do not have to share 100% amino acid identity. Multiple sequence alignments of amino acid sequences of homologous proteins from different organisms can be used to identify functionally important residues in a protein by indicating which residues are absolutely conserved, semi-conserved, and non-conserved. This would allow an individual to alter a controlled protein sequence by changing all or some of the conserved and semi-conserved residues to residues with similar physiochemical properties. In addition, all or some of the non-conserved residues could be substituted with essentially any other amino acid without risking loss of the protein’s function. This method, in combination with utilizing codon redundancy, would allow for more drastic alterations to be made to both the DNA and protein sequence from a pathogenic organism that could bypass screening procedures.</p><br />
<br />
<p style="color:black"><b>Using “custom” tRNAs</p></b><br />
<p style="color:black">A more complicated means for bypassing screening procedures by decoupling protein sequence from function would be to use a highly engineered system with non-canonical tRNAs. An organism could be designed that uses engineered amino acyl-tRNA synthetases that recognize non-cognate tRNAs and therefore aminoacylate the tRNA with the incorrect amino acid. By using this alternative genetic code in the engineered organism, the DNA sequence from a pathogenic organism could be altered in an almost indistinguishable way while still producing the protein of interest.</p><br />
<br />
<p style="color:black"><b>Do-it-yourself synthesis</p></b><br />
<p style="color:black">As time progresses, the cost of a DNA synthesizer is getting more affordable to research labs and independent users. Initially this may seem like a good thing, but there are tremendous dangers that are associated with this development. Directly bypassing screening procedures by not requiring the services of synthesis companies allows the owner of the DNA synthesizer unrestricted access to synthesize whatever sequence they choose. This would make any techniques to bypass the screening methods of synthesis companies obsolete. As a result, there may need to be regulations put in place to limit or restrict the access of DNA synthesizers. This can be done for example by requiring the owner to upload any sequences they synthesize to a governing body that will scan them for harmful sequences, or by installing software that will screen sequences prior to allowing them to be synthesized. A combination of these two methods as well as additional advances in screening procedures is crucial to ensure the safety of the general public.</p><br />
<br />
<h3><b>Changes recommended for screening protocols</h3></b><br />
<br />
<p style="color:black">Though commendable biosecurity efforts have been put forward by major international synthesis companies, these groups are aware that standard protocols may not be enough to mitigate the risk of the synthesis and delivery of hazardous sequences. In the IASB Code of Conduct for Best Practices in Gene Synthesis, all member companies are mandated to take part in ongoing efforts to refine and improve the current screening technologies by establishing a review committee to update and expand the Code of Conduct as new or changing threats emerge, maintain open communication with member companies through the exchange of research and literature searches, and regularly collaborating on best practices and new screening ideas [2]. While these practices are important for synthesis companies to implement, DNA synthesis is becoming less expensive and more accessible by non-professionals. According to Minshull and Wagner “[a]nyone who is sufficiently motivated could synthesize the gene for a toxin or even an entire viral genome using readily available reagents and without ever going near a specialized synthesizer” [5]. With molecular biology equipment becoming available through avenues such as E-Bay and other online dealers, individuals with limited molecular biology experience could soon realistically synthesize their own DNA sequences in the next few years [4]. Screening protocols could thereafter become obsolete. Until then, further steps are required to assure the public, government, and research community that biosecurity is being upheld to the highest standards possible. This may involve expanding the use of online forums, such as VIREP (Virulence Factor Information Repository), to allow researchers to deposit and access information about genes and organisms. Additionally, government regulations may need to be implemented that require all synthesis companies to adhere to standard practices and implement human investigation of suspicious orders [6]. This may best be achieved through the integration of both the IASB and IGSC protocols into an industry-wide Code of Conduct.</p><br />
<br />
<h2>Testing the System</h2><br />
<h3><b>Learning to Be Bad</b></h3><br />
<p style="color:black">This year, we focused on the implications our frameshifting project might have on biosecurity. In thinking about the ways our pseudoknots could be used to do new, exciting things in synthetic biology, we came up with a use that is more frightening than exciting. Bioterrorism.</p><br />
<br />
<p style="color:black">The idea is this: There are guidelines put forward by a number of industry groups on how DNA synthesis orders should be screened to ensure no biohazardous sequences get into the hands of the wrong people. The standard protocol for screening sequences involves taking the submitted DNA sequences and translating all six reading frames, then using BLAST to compare the DNA and amino acid sequences to those of organisms on a list of controlled agents.</p><br />
<br />
<p style="color:black">Our pseudoknot enables the ribosome to switch frames mid-translation, essentially splitting the entire protein amongst as many reading frames as there are pseudoknots. If someone were to split a protein from the Ebola virus into small fragments distributed across the reading frames, could they bypass this initial automatic screening step? </p><br />
<br />
<h3><b>Putting our White Hats On</b></h3><br />
<br />
<p style="color:black">To investigate this potential for abuse of our project, we worked together with major North American synthesis companies to see if we could try and fool their screening methods using our frameshifting elements. <br />
We designed and submitted sequences with vary coding changes and coding fragment sizes between the sequences for our PK401 pseudoknot to the synthesis companies we had partnered with. There is a full description of the sequences and a link to the raw data files below.</p><br />
<br />
<center><table width="800px"; border="1px"; border-color="black"><br />
<tr><br />
<td><b>Sequence ID Number</td><br />
<td><b>Sequence Origin</td><br />
<td><b>Total Length (bp)</td><br />
<td><b>Codon Changes (%)</td><br />
<td><b>Length between PK (bp)</td><br />
</tr><br />
<br />
<tr><br />
<td>1</td><br />
<td>CFP</td><br />
<td>966</td><br />
<td>25</td><br />
<td>180</td><br />
</tr><br />
<br />
<tr><br />
<td>2</td><br />
<td>Staph-ORF</td><br />
<td>1869</td><br />
<td>0</td><br />
<td>210</td><br />
</tr><br />
<br />
<tr><br />
<td>3</td><br />
<td>Ricin</td><br />
<td>2392</td><br />
<td>25</td><br />
<td>198</td><br />
</tr><br />
<br />
<tr><br />
<td>4</td><br />
<td>Staph-ORF</td><br />
<td>2450</td><br />
<td>25</td><br />
<td>102</td><br />
</tr><br />
<br />
<tr><br />
<td>5</td><br />
<td>CFP</td><br />
<td>966</td><br />
<td>16</td><br />
<td>210</td><br />
</tr><br />
<br />
<tr><br />
<td>6</td><br />
<td>CFP</td><br />
<td>966</td><br />
<td>0</td><br />
<td>180</td><br />
</tr><br />
<br />
<tr><br />
<td>7</td><br />
<td>Staph-ORF</td><br />
<td>2450</td><br />
<td>0</td><br />
<td>102</td><br />
</tr><br />
<br />
<tr><br />
<td>8</td><br />
<td>Ricin</td><br />
<td>2392</td><br />
<td>0</td><br />
<td>198</td><br />
</tr><br />
<br />
<tr><br />
<td>9</td><br />
<td>Ebola Matrix Protein</td><br />
<td>1031</td><br />
<td>0</td><br />
<td>0</td><br />
</tr><br />
<br />
<tr><br />
<td>10</td><br />
<td>CFP</td><br />
<td>966</td><br />
<td>16</td><br />
<td>180</td><br />
</tr><br />
<br />
<tr><br />
<td>11</td><br />
<td>Staph-ORF</td><br />
<td>2450</td><br />
<td>0</td><br />
<td>102</td><br />
</tr><br />
<br />
<tr><br />
<td>12</td><br />
<td>CFP</td><br />
<td>966</td><br />
<td>0</td><br />
<td>210</td><br />
</tr><br />
<br />
<tr><br />
<td>13</td><br />
<td>Staph-ORF</td><br />
<td>1869</td><br />
<td>20</td><br />
<td>210</td><br />
</tr><br />
<br />
<tr><br />
<td>14</td><br />
<td>Ricin</td><br />
<td>3139</td><br />
<td>25</td><br />
<td>99</td><br />
</tr><br />
<br />
<tr><br />
<td>15</td><br />
<td>Staph-ORF</td><br />
<td>1869</td><br />
<td>25</td><br />
<td>210</td><br />
</tr><br />
<br />
<tr><br />
<td>16</td><br />
<td>CFP</td><br />
<td>966</td><br />
<td>25</td><br />
<td>210</td><br />
</tr><br />
<br />
<tr><br />
<td>17</td><br />
<td>Ricin</td><br />
<td>3139</td><br />
<td>0</td><br />
<td>99</td><br />
</tr><br />
</table></center><br />
<br><br />
<a href="https://static.igem.org/mediawiki/2013/c/c5/Lethbridge_iGEM_2013_Screening_Sequences.txt"> Click here for sequences used </a><br />
<br />
<br />
<h3><b>Can We Sleep at Night?</b></h3><br />
<br />
<p style="color:black">These sequences were sent to the companies and screened for hazardous elements. One company managed to detect all of the “threats” on the first level of screening. According to them, their next steps would be to do a review of the “threat” sequences using a group of human experts while interviewing the customer to determine their background, shipping and payment information, and the intended use of the synthesized DNA.</p><br />
<br />
<p style="color:black">Another company simply analyzed the sequences to determine if they could actually construct the DNA if we were to order it. All of the sequences containing the pseudoknot elements were flagged as containing high repeats, but sequence 9, the Ebola matrix protein with no pseudoknot elements, was determined to be ready for synthesis. They did not make it clear whether or not there would be another level of screening to determine the origin of the sequences. </p><br />
<br />
<p style="color:black">Based on these results, current industry standard screening protocols appear to be sufficient to detect biosecurity threats, even with codon changes and the distribution of coding sequences amongst many reading frames. What is still a cause for concern is the strictness with which these protocols are applied. There is no legal requirement to execute biosecurity screens on DNA synthesis orders; all of the proposed protocols are currently voluntary guidelines. This could allow companies to relax their security protocols and may increase the potential of a serious bioterrorism threat coming to fruition. </p><br />
<br />
<p style="color:black">In order to make sure that the act of releasing the results of our study did not pose a security threat in itself, we consulted with Edward You. Edward is a representative of the FBI’s WMD department. Many of the industry guidelines for screening call for collaboration between the synthesis companies and government agencies responsible for responding to bioterrorism threats.</p><br />
<br />
<a href="https://static.igem.org/mediawiki/2013/c/c5/Lethbridge_iGEM_2013_Screening_Sequences.txt"> Click here for sequences used </a><br />
<br />
<h3><b>References</h3></b><br />
<br />
<p style="color:black">[1] International Gene Synthesis Consortium. Harmonized screening protocol: gene sequence & customer screening to promote biosecurity. http://www.genesynthesisconsortium.org/wp-content/uploads/2012/02/IGSC-Harmonized-Screening-Protocol1.pdf (2009).</p><br />
<p style="color:black">[2] International Association Synthetic Biology. Code of conduct for best practices in gene synthesis. http://www.ia-sb.eu/tasks/sites/synthetic-biology/assets/File/pdf/iasb_code_of_conduct_final.pdf (2009). </p><br />
<p style="color:black">[3] U.S. Department of Health and Human Services. Screening Framework Guidance for Providers of Synthetic DoublesStranded DNA. http://www.phe.gov/Preparedness/legal/guidance/syndna/Documents/syndna-guidance.pdf (2010).</p><br />
<p style="color:black">[4] Maurer S. M., Fischer M., Schwer H., Stähler C., Stähler P., & Bernauer H. S. Working paper: making commercial biology safer: what the gene synthesis industry has learned about screening customers and orders. http://gspp.berkeley.edu/iths/Maurer_IASB_Screening.pdf (2009). </p><br />
<p style="color:black">[5] Minshull J. & Wagner, R. Nat. Biotechnol. 27, 800-801 (2009).</p><br />
<p style="color:black">[6] Fischer M. & Maurer S. M. Nat. Biotechnol. 28, 20-22 (2010).</p><br />
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</html></div>Dustinhttp://2013.igem.org/Team:Lethbridge/notebookTeam:Lethbridge/notebook2013-10-29T01:32:26Z<p>Dustin: </p>
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<h1>Notebook</h1><br />
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<h2>July</h2><br />
<p>Experimenters: Graeme, Suneet, Zak</p><br />
<p>Objective: Assembly of PK401 Test Construct</p><br />
<p><a href="https://static.igem.org/mediawiki/2013/4/45/Lethbridge_iGEM_Collegiate_2013_Notebook_%28July%29.pdf">Read Entries from July by clicking here...</a></p> <br />
<br />
<h2>August</h2><br />
<p>Experimenters: Graeme, Suneet, Zak, Harland</p><br />
<p>Objective: Assembly of Enhanced Lumazine Construct and Characterization of PK401 Test Construct</p><br />
<p><a href="https://static.igem.org/mediawiki/2013/f/f9/Lethbridge_iGEM_Collegiate_2013_Notebook_%28August%29.pdf">Read Entries from August by clicking here...</a></p> <br />
<br />
<h2>September</h2><br />
<p>Experimenters: Graeme, Suneet, Zak, Harland, Jenna, Dustin</p><br />
<p>Objective: Re-assembly of PK401 Test Construct and Further Characterization. Error-prone PCR of Pseudoknot for Mutant Generation</p><br />
<p><a href="https://static.igem.org/mediawiki/2013/b/b8/Lethbridge_iGEM_Collegiate_2013_Notebook_September.pdf">Read Entries from September by clicking here...</a></p> <br />
</body><br />
</html></div>Dustinhttp://2013.igem.org/File:Lethbridge_iGEM_Collegiate_2013_Notebook_September.pdfFile:Lethbridge iGEM Collegiate 2013 Notebook September.pdf2013-10-29T01:30:57Z<p>Dustin: </p>
<hr />
<div></div>Dustinhttp://2013.igem.org/Team:Lethbridge/notebookTeam:Lethbridge/notebook2013-10-29T01:30:15Z<p>Dustin: </p>
<hr />
<div>{{TeamLethbridgeHead}}<br />
<BLOCKQUOTE><br />
<br />
<html><br />
<center><image src="https://static.igem.org/mediawiki/2013/6/6a/UofL_iGEM_2013.png" width="750px" height="450px"/></center><br><br />
<br />
<h1>Notebook</h1><br />
<br />
<body><br />
<h2>July</h2><br />
<p>Experimenters: Graeme, Suneet, Zak</p><br />
<p>Objective: Assembly of PK401 Test Construct</p><br />
<p><a href="https://static.igem.org/mediawiki/2013/4/45/Lethbridge_iGEM_Collegiate_2013_Notebook_%28July%29.pdf">Read More Entries from July by clicking here...</a></p> <br />
<br />
<h2>August</h2><br />
<p>Experimenters: Graeme, Suneet, Zak, Harland</p><br />
<p>Objective: Assembly of Enhanced Lumazine Construct and Characterization of PK401 Test Construct</p><br />
<p><a href="https://static.igem.org/mediawiki/2013/f/f9/Lethbridge_iGEM_Collegiate_2013_Notebook_%28August%29.pdf">Read More Entries from August by clicking here...</a></p> <br />
<br />
<h2>September</h2><br />
<p>Experimenters: Graeme, Suneet, Zak, Harland, Jenna, Dustin</p><br />
<p>Objective: Re-assembly of PK401 Test Construct and Further Characterization. Error-prone PCR of Pseudoknot for Mutant Generation</p><br />
<p><a href="">Read More Entries from September by clicking here...</a></p> <br />
</body><br />
</html></div>Dustinhttp://2013.igem.org/Team:Lethbridge/teamTeam:Lethbridge/team2013-10-29T01:21:23Z<p>Dustin: </p>
<hr />
<div>{{TeamLethbridgeHead}}<br />
<div style="background-color:#FFFFFF; color:white"><br />
<br />
<BLOCKQUOTE><br />
<html><br />
<style><br />
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</html><br />
<br />
<br><br />
<html><br />
<font color="black"><br />
<h1>Team</h1><br />
<br />
<h2>Suneet Kharey </h2><br />
<table><br />
<tr><td><image src="https://static.igem.org/mediawiki/2013/8/81/UofL_Suneet.png"; position="left"; width="125"; height="150"; /><br />
</td><br />
<td><p position="right">Hello, my name is Suneet (sun-eat). This is my first year in iGEM, I've always been intrigued by synthetic biology so I'm grateful for this opportunity. In my free time (which is currently non-existant) I enjoy learning new languages (currently at 3), painting, buying more Doc Martens (just like my girl Miley), and pointing out obscure constellations in the night sky. Currently I am learning French and teaching myself how to play the Harmonica. </p></td></tr><br />
</table><br />
<br style="clear:both" /><br />
<br />
<h2>Graeme Glaister</h2><br />
<table><br />
<tr><td><image src="https://static.igem.org/mediawiki/2013/6/68/Crackers_igem_2013_uofL.png"; position="left"; width="125"; height="150"; /><br />
</td><br />
<td><p position="right">I am a fourth year B.Sc. Neuroscience student, planning on pursuing a Master’s degree. This is my first year in iGEM. I grew up in Fort McMurray but moved to Lethbridge in Jr. High. The reason I chose to participate in iGEM is so that I could have the unique experience (for an undergrad) of participating in the planning process for an experiment as well as the necessary lab work. I eagerly look forward to continuing on with iGEM in 2014.</p></td></tr><br />
</table><br />
<br style="clear:both" /><br />
<br />
<h2>Zak Stinson</h2><br />
<table><br />
<tr><td><image src="https://static.igem.org/mediawiki/2013/5/53/UofL_Zak.png"; position="left"; width="125"; height="150"; /><br />
</td><br />
<td><p position="right">Hey, I'm Zak, and I am an iGEMmer. I thought I was almost free of my fascination with genetic technologies when I transferred to the neuroscience program at the U of L in 2011 from biology at the U of R, but I lapsed right back when I learned that iGEM existed. I thought I had refocused when I began doing behavioural experiments with rats, but immediately searched out and executed a synthetic biology project designing a system for reporting gene activity in the brains of live animals. After completing my B.Sc. in neuroscience this summer I will likely continue to design synthetic biology projects in the M.Sc. neuroscience program at the U of L as I am dangerously hooked. So I guess I'll talk to you all next year!</p></td></tr><br />
</table><br />
<br style="clear:both" /><br />
<br />
<h2>Dustin Smith</h2><br />
<table><br />
<tr><td><image src="https://static.igem.org/mediawiki/2013/3/37/UofL_Dustin.png"; position="left"; width="125"; height="150"; /><br />
</td><br />
<td><p position="right">Hello, my name is Dustin Smith. I was born and raised in Lethbridge, and I am now a Master’s student majoring in Biochemistry. I’m happy to be back with the University of Lethbridge iGEM team for a third year, and as always it has been a great experience. Outside of school my favorite activities are ice hockey, snowboarding, and lifting.</p></td></tr><br />
</table><br />
<br style="clear:both" /><br />
<br />
<h2>Jenna Friedt</h2><br />
<table><br />
<tr><td><image src="https://static.igem.org/mediawiki/2013/7/7d/UofL_Jenna.png"; position="left"; width="125"; height="150"; /><br />
</td><br />
<td><p position="right">Hello, my name is Jenna and I am one of the graduate student advisors for the 2013 Lethbridge iGEM team. Since I’m convinced I was born in the wrong era, I’m currently looking for assistance in inventing a time machine to send me back to the ‘50s, where I would happily fit in with the bubblegum rockers and poodle skirt-wearing youth of the day. Until that happens, I’ll settle for nights on the patio listening to the Beatles, reading a good book, and watching Audrey Hepburn movies.</p></td></tr><br />
</table><br />
<br style="clear:both" /><br />
<br />
<h2>Harland Brandon</h2><br />
<table><br />
<tr><td><image src="https://static.igem.org/mediawiki/2013/0/0a/UofL_Harland.png"; position="left"; width="125"; height="150"; /></td><br />
<td><p position="right">Harland is an avid biochemist pursuing his Masters degree. In his spare time he enjoys building and creating new things, as such it was only natural that he continued with his interest in iGEM. Having been around the competition for four years now he has decided to take a backseat role in guiding and mentoring the schools iGEM team. When you don't find him in lab or at the university he can most likely be found at his computer playing or enjoying a fine strong Scottish beverage.</p></td></tr><br />
</table><br />
<br style="clear:both" /><br />
<br />
<h2>Supervisor: Hans-Joachim Wieden</h2><br />
<table><br />
<tr><td><image src="https://static.igem.org/mediawiki/2013/e/e4/UofL_HJ_2013.png"; position="left"; width="125"; height="150"; /></td><br />
<td><p position="right">Originally from Germany I moved to Canada in 2005 to start a research group on the structure and function of the bacterial protein synthesis machinery, a cellular process targeted by over 50% of the known antibiotics. I am intrigued by the molecular design and function of this essential bio-nanomachine. I try to unravel the underlying design principles in order to enable the rational design and engineering of novel bio-nanomachines. I am essentially asking the question if such novel bio-machines can be constructed from simple and fundamental principles or are these assemblies just too complex. Well and that’s why it was extremely easy to rope me into doing iGEM.</p></td></tr><br />
</table><br />
<br style="clear:both" /><br />
<br />
</html></div>Dustinhttp://2013.igem.org/Team:Lethbridge/teamTeam:Lethbridge/team2013-10-29T01:11:06Z<p>Dustin: </p>
<hr />
<div>{{TeamLethbridgeHead}}<br />
<div style="background-color:#FFFFFF; color:white"><br />
<br />
<BLOCKQUOTE><br />
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</html><br />
<br />
<br><br />
<html><br />
<font color="black"><br />
<h1>Team</h1><br />
<br />
<h2>Suneet Kharey </h2><br />
<table><br />
<tr><td><image src="https://static.igem.org/mediawiki/2013/8/81/UofL_Suneet.png"; position="left"; width="125"; height="150"; /><br />
</td><br />
<td><p position="right">Hello, my name is Suneet (sun-eat). This is my first year in iGEM, I've always been intrigued by synthetic biology so I'm grateful for this opportunity. In my free time (which is currently non-existant) I enjoy learning new languages (currently at 3), painting, buying more Doc Martens, and pointing out obscure constellations in the night sky. Currently I am learning French and teaching myself how to play the Harmonica. </p></td></tr><br />
</table><br />
<br style="clear:both" /><br />
<br />
<h2>Graeme Glaister</h2><br />
<table><br />
<tr><td><image src="https://static.igem.org/mediawiki/2013/6/68/Crackers_igem_2013_uofL.png"; position="left"; width="125"; height="150"; /><br />
</td><br />
<td><p position="right">I am a fourth year B.Sc. Neuroscience student, planning on pursuing a Master’s degree. This is my first year in iGEM. I grew up in Fort McMurray but moved to Lethbridge in Jr. High. The reason I chose to participate in iGEM is so that I could have the unique experience (for an undergrad) of participating in the planning process for an experiment as well as the necessary lab work. I eagerly look forward to continuing on with iGEM in 2014.</p></td></tr><br />
</table><br />
<br style="clear:both" /><br />
<br />
<h2>Zak Stinson</h2><br />
<table><br />
<tr><td><image src="https://static.igem.org/mediawiki/2013/5/53/UofL_Zak.png"; position="left"; width="125"; height="150"; /><br />
</td><br />
<td><p position="right">Hey, I'm Zak, and I am an iGEMmer. I thought I was almost free of my fascination with genetic technologies when I transferred to the neuroscience program at the U of L in 2011 from biology at the U of R, but I lapsed right back when I learned that iGEM existed. I thought I had refocused when I began doing behavioural experiments with rats, but immediately searched out and executed a synthetic biology project designing a system for reporting gene activity in the brains of live animals. After completing my B.Sc. in neuroscience this summer I will likely continue to design synthetic biology projects in the M.Sc. neuroscience program at the U of L as I am dangerously hooked. So I guess I'll talk to you all next year!</p></td></tr><br />
</table><br />
<br style="clear:both" /><br />
<br />
<h2>Dustin Smith</h2><br />
<table><br />
<tr><td><image src="https://static.igem.org/mediawiki/2013/3/37/UofL_Dustin.png"; position="left"; width="125"; height="150"; /><br />
</td><br />
<td><p position="right">Hello, my name is Dustin Smith. I was born and raised in Lethbridge, and I am now a Master’s student majoring in Biochemistry. I’m happy to be back with the University of Lethbridge iGEM team for a third year, and as always it has been a great experience. Outside of school my favorite activities are ice hockey, snowboarding, and lifting.</p></td></tr><br />
</table><br />
<br style="clear:both" /><br />
<br />
<h2>Jenna Friedt</h2><br />
<table><br />
<tr><td><image src="https://static.igem.org/mediawiki/2013/7/7d/UofL_Jenna.png"; position="left"; width="125"; height="150"; /><br />
</td><br />
<td><p position="right">Hello, my name is Jenna and I am one of the graduate student advisors for the 2013 Lethbridge iGEM team. Since I’m convinced I was born in the wrong era, I’m currently looking for assistance in inventing a time machine to send me back to the ‘50s, where I would happily fit in with the bubblegum rockers and poodle skirt-wearing youth of the day. Until that happens, I’ll settle for nights on the patio listening to the Beatles, reading a good book, and watching Audrey Hepburn movies.</p></td></tr><br />
</table><br />
<br style="clear:both" /><br />
<br />
<h2>Harland Brandon</h2><br />
<table><br />
<tr><td><image src="https://static.igem.org/mediawiki/2013/0/0a/UofL_Harland.png"; position="left"; width="125"; height="150"; /></td><br />
<td><p position="right">Harland is an avid biochemist pursuing his Masters degree. In his spare time he enjoys building and creating new things, as such it was only natural that he continued with his interest in iGEM. Having been around the competition for four years now he has decided to take a backseat role in guiding and mentoring the schools iGEM team. When you don't find him in lab or at the university he can most likely be found at his computer playing or enjoying a fine strong Scottish beverage.</p></td></tr><br />
</table><br />
<br style="clear:both" /><br />
<br />
<h2>Supervisor: Hans-Joachim Wieden</h2><br />
<table><br />
<tr><td><image src="https://static.igem.org/mediawiki/2013/e/e4/UofL_HJ_2013.png"; position="left"; width="125"; height="150"; /></td><br />
<td><p position="right">Originally from Germany I moved to Canada in 2005 to start a research group on the structure and function of the bacterial protein synthesis machinery, a cellular process targeted by over 50% of the known antibiotics. I am intrigued by the molecular design and function of this essential bio-nanomachine. I try to unravel the underlying design principles in order to enable the rational design and engineering of novel bio-nanomachines. I am essentially asking the question if such novel bio-machines can be constructed from simple and fundamental principles or are these assemblies just too complex. Well and that’s why it was extremely easy to rope me into doing iGEM.</p></td></tr><br />
</table><br />
<br style="clear:both" /><br />
<br />
</html></div>Dustinhttp://2013.igem.org/Team:Lethbridge/attributionsTeam:Lethbridge/attributions2013-10-29T01:07:03Z<p>Dustin: /* Attributions */</p>
<hr />
<div>{{TeamLethbridgeHead}}<br />
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<br />
</html><br />
<BLOCKQUOTE><br />
<br />
=<font color="black">Attributions=<br />
All planning and wet lab work was performed by the undergraduate members of our iGEM team.<br />
<br />
<h2>Contributions</h2><br />
<p><b>Dr. Gaymon Bennett</b> (Fred Hutchinson Cancer Research Center)</p><br />
<p><b>Dr. Karmella Haynes</b> (Arizona State University)</p> <br />
<p><b>Edward You</b> (FBI, Weapons of Mass Destruction Directorate)</p> <br />
<p><b>Dallas Thomas</b> (University of Lethbridge, Lethbridge Agriculture and Agri-Food Canada)</p> <br />
<p><b>Sutherland Dubé, Dipankar Goyal, [http://seventhousandplus.weebly.com/index.html Kelsey Kristensen], Richard McLean, Fan Mo, Justin Vigar,</b> and <b>Anthony Vuong</b> (University of Lethbridge)</p><br />
<p>The Department of Chemistry and Biochemistry, the Alberta RNA Research and Training Institute, the Kothe lab, Selinger lab, and Wieden lab at the University of Lethbridge for providing us with equipment and laboratory space.<br />
</p><br />
<br />
<h2>Sponsors</h2><br />
<p>We would also like to acknowledge our sponsors for the following support of the 2013 Lethbridge iGEM team.</p><br><br />
<br />
<html><br />
<p><center><image src="https://static.igem.org/mediawiki/2013/0/00/ULeth2013_Sponsors_-_Platinum.png"; width="400px"; height="200px" /></center></p><br><br />
<br />
<p><center><image src="https://static.igem.org/mediawiki/2013/1/1e/ULeth2013_Sponsors_-_Silver.png"; width="400px"; height="200px" /></center></p><br><br />
<br />
<p><center><image src="https://static.igem.org/mediawiki/2013/4/4c/ULeth2013_Sponsors_-_Bronze.png"; width="600px"; height="200px" /></center></p><br />
</html><br />
<br />
<br />
<p><b>1. Platinum - $5000+ or gift in kind</b></p><br />
<p>Logo on front of team shirts, large logo on scientific poster, large logo on team wiki and verbal recognition during team project presentations/media interviews.</p><br />
<p><b>2. Gold - $2000-$4999 or gift in kind</b></p><br />
<p>Logo on team shirts, medium logo on scientific poster, medium logo on team wiki and written recognition at end of team project presentations.</p><br />
<p><b>3. Silver - $1000-$1999 or gift in kind</b></p><br />
<p>Medium logo on scientific poster, medium logo on team wiki and written recognition at end of team project presentations.</p><br />
<p><b>4. Bronze - <$999 or gift in kind</b></p><br />
<p>Small logo on scientific poster, small logo on team wiki and written recognition at end of team project presentations.</p></div>Dustinhttp://2013.igem.org/Team:Lethbridge/attributionsTeam:Lethbridge/attributions2013-10-29T01:06:09Z<p>Dustin: /* Attributions */</p>
<hr />
<div>{{TeamLethbridgeHead}}<br />
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text-align:justify;<br />
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}<br />
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<center><image src="https://static.igem.org/mediawiki/2013/4/41/Attributions_uofl_2013.png" width="600px" height="300px"/></center><br />
<br />
</html><br />
<BLOCKQUOTE><br />
<br />
=<font color="black">Attributions=<br />
All planning and wet lab work was performed by the undergraduate members of our iGEM team.<br />
<br />
<h2>Contributions</h2><br />
<p><b>Dr. Gaymon Bennett</b> (Fred Hutchinson Cancer Research Center</p><br />
<p><b>Dr. Karmella Haynes</b> (Arizona State University)</p> <br />
<p><b>Edward You</b> (FBI, Weapons of Mass Destruction Directorate)</p> <br />
<p><b>Dallas Thomas</b> (University of Lethbridge, Lethbridge Agriculture and Agri-Food Canada)</p> <br />
<p><b>Sutherland Dubé, Dipankar Goyal, [http://seventhousandplus.weebly.com/index.html Kelsey Kristensen], Richard McLean, Fan Mo, Justin Vigar,</b> and <b>Anthony Vuong</b> (University of Lethbridge)</p><br />
<p>The Department of Chemistry and Biochemistry, the Alberta RNA Research and Training Institute, the Kothe lab, Selinger lab, and Wieden lab at the University of Lethbridge for providing us with equipment and laboratory space.<br />
</p><br />
<br />
<h2>Sponsors</h2><br />
<p>We would also like to acknowledge our sponsors for the following support of the 2013 Lethbridge iGEM team.</p><br><br />
<br />
<html><br />
<p><center><image src="https://static.igem.org/mediawiki/2013/0/00/ULeth2013_Sponsors_-_Platinum.png"; width="400px"; height="200px" /></center></p><br><br />
<br />
<p><center><image src="https://static.igem.org/mediawiki/2013/1/1e/ULeth2013_Sponsors_-_Silver.png"; width="400px"; height="200px" /></center></p><br><br />
<br />
<p><center><image src="https://static.igem.org/mediawiki/2013/4/4c/ULeth2013_Sponsors_-_Bronze.png"; width="600px"; height="200px" /></center></p><br />
</html><br />
<br />
<br />
<p><b>1. Platinum - $5000+ or gift in kind</b></p><br />
<p>Logo on front of team shirts, large logo on scientific poster, large logo on team wiki and verbal recognition during team project presentations/media interviews.</p><br />
<p><b>2. Gold - $2000-$4999 or gift in kind</b></p><br />
<p>Logo on team shirts, medium logo on scientific poster, medium logo on team wiki and written recognition at end of team project presentations.</p><br />
<p><b>3. Silver - $1000-$1999 or gift in kind</b></p><br />
<p>Medium logo on scientific poster, medium logo on team wiki and written recognition at end of team project presentations.</p><br />
<p><b>4. Bronze - <$999 or gift in kind</b></p><br />
<p>Small logo on scientific poster, small logo on team wiki and written recognition at end of team project presentations.</p></div>Dustinhttp://2013.igem.org/Team:Lethbridge/resultsTeam:Lethbridge/results2013-10-29T00:46:08Z<p>Dustin: /* Results */</p>
<hr />
<div>{{TeamLethbridgeHead}}<br />
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<center><image src="https://static.igem.org/mediawiki/2013/4/43/ULeth2013_Construct1.jpg" height="150px" width="600px" /></center><br />
<br><br><br />
</body><br />
</html><br />
<br />
<BLOCKQUOTE><br />
=<font color="black">Results=<br />
<br />
<br />
<br />
<h2>Team Parts Sandbox</h2><br />
<center><br />
<table border="0" style="background-color:#FFF" width="100%" cellpadding="0" cellspacing="5"><br />
<tr><br />
<td><strong>Name</strong></td><br />
<td><strong>Type</strong></td><br />
<td><strong>Description</strong></td><br />
<td><strong>Designer</strong></td><br />
<td><strong>Length</strong></td><br />
<td><strong>Favorite Part</strong></td><br />
</tr><br />
<tr><br />
<td>[http://parts.igem.org/Part:BBa_K1210000 BBa_K1210000]</td><br />
<td>Device</td><br />
<td>LacI-CFP-PK401-YFP</td><br />
<td>Zak Stinson</td><br />
<td>1955</td><br />
<td><center>Yes</center></td><br />
</tr><br />
<tr><br />
<td>[http://parts.igem.org/Part:BBa_K1210001 BBa_K1210001]</td><br />
<td>RNA Part</td><br />
<td>PK401 Pseudoknot</td><br />
<td>Zak Stinson</td><br />
<td>93</td><br />
<td> </td><br />
</tr><br />
<tr><br />
<td>[http://parts.igem.org/Part:BBa_K1210002 BBa_K1210002]</td><br />
<td>Device</td><br />
<td>Enhanced Lumazine Synthase (ELS) Expression Construct</td><br />
<td>Harland Brandon</td><br />
<td>828</td><br />
<td> </td><br />
</tr><br />
<tr><br />
<td>[http://parts.igem.org/Part:BBa_K1210003 BBa_K1210003]</td><br />
<td>Device</td><br />
<td>Tagged dual coding test construct</td><br />
<td>Harland Brandon</td><br />
<td>1850</td><br />
<td> </td><br />
</tr><br />
</table><br />
<br />
</center><br />
<br />
<br><br />
<br />
<h2>Expression and Characterization of PK401 (BBa_K1210000)</h2><br />
<br />
<p><b>PK401 Overexpression</b></p><br />
<p>The PK401 construct was overexpressed to test and characterize the construct for frameshifting ability. To do this, cultures of E. coli DH5α containing either the PK401 plasmid or an empty control plasmid were grown from glycerol stocks overnight at 37°C in 50 mL LB media containing Kanamycin (100 µg/mL). These cultures were used the next day to inoculate 500 mL of LB media to a starting optical density at 600 nm (OD600) near 0.05. The OD600 was monitored and induced with 1 mM IPTG once reaching an OD600 of 0.6 (Fig. 1). Growth was monitored hourly, and equivalent amounts of cells were taken as samples for SDS-PAGE analysis up to 5 h after induction. The cultures were harvested by centrifuging at 5000 x g and shock frozen to store at -80°C.</p><br />
<br />
<center>[[File:ULeth2013_PK401-overexpression.png|600px]]</center><br />
<br />
<p><b>Figure 1. Growth curve of PK401 construct.</b> E. coli DH5α cells containing the PK401 plasmid or a control plasmid were grown at 37°C in LB media. The OD600 was monitored and the cultures were induced with 1 mM IPTG when the OD600 had reached 0.6. The cultures were grown for an additional 5 h, and a sample of 1 OD600 equivalent of cells were taken at each hour after induction.</p><br><br />
<br />
<p>The samples taken for SDS-PAGE analysis were pelleted and resuspended in 80 µL 0.1 M Tris-HCl pH 8.5 containing 5 M urea and 20 µL SDS-PAGE gel-loading buffer. The samples were then analyzed on a 12% SDS-PAGE and stained with Coomassie blue to confirm overexpression of the non-frameshifted and -1 frameshifted protein products from the PK401 construct (Fig. 2). The non-frameshifted product (CFP) has an expected size of 29 kD, and the -1 frameshifted product (a fusion protein of CFP-PK401-YFP) has an expected size of 60 kD. Bands of increasing intensity after induction with IPTG were seen at approximately 30 kD and 60 kD, corresponding to both the non-frameshifted and -1 frameshifted product. These same bands were seen in the uninduced samples, however this could be due to the expression being controlled by the pLacI promoter, which is known to give leaky expression.</p><br />
<br />
<p>To estimate the frameshifting frequency of PK401, the relative band intensities of the 60 kD protein were compared to that of the 29 kD protein. This resulted in a calculated frameshift efficiency of 21 ± 5%, indicating that 21% of the translated product was frameshifted into the -1 frame to produce the CFP-PK401-YFP fusion protein. This result correlates well with the previously reported frameshifting efficiency of 14 ± 2% for this pseudoknot (Tholstrup et al., 2011).</p><br />
<br />
<br><br />
<center>[[Image:ULeth2013_PK401-overexpressiongel.png|400px]]</center><br />
<br><br />
<br />
<p><b>Figure 2. Over-expression of non-frameshifted and -1 frameshifted protein products from the PK401 construct.</b> Equivalent amounts of cells at 0, 1, 3, and 5 h after induction with IPTG (PK401 +IPTG) were analyzed by 12% SDS-PAGE. The same time samples from the uninduced culture were also analyzed (PK401 –IPTG). Black boxes indicate bands of increasing intensity that migrated with an approximate molecular weight of 60 kD and 30 kD, corresponding to the -1 frameshifted CFP-PK401-YFP fusion product and the non-frameshifted CFP product, respectively.</p><br><br />
<br />
<p><b>CFP and YFP Fluorescence from PK401 Overexpression</b></p><br />
<p>To confirm that the protein products seen in the overexpression of the PK401 construct corresponded to the expected fluorescent proteins, fluorescence spectra of the cell lysates from overexpression were measured. The cells from the overexpression were lysed using 1 mg/mL lysozyme and 12.5 mg/g sodium deoxycholate. DNase was added to degrade the DNA followed by centrifugation at 3000 x g for 30 min. The supernatant was collected and centrifuged further at 30 000 x g for 45 min. This supernatant was then used for fluorescence measurements.</p><br />
<br />
<p>The supernatants were diluted 100-fold in the buffer used for cell opening. Excitation and emission scans were measured for all samples in the CFP and YFP range in order to confirm the presence of both fluorescent proteins. CFP was excited at 430 nm, and emission was monitored from 445-650 nm (Fig. 3). YFP was excited at 510 nm, and emission was monitored from 525-650 nm (Fig. 4). These spectra were measured for the cell opening buffer and for the induced and uninduced samples of the cells that contained the PK401 construct, as well as the cells containing the control plasmid.</p><br />
<br />
<br><br />
<center>[[Image:ULeth2013_PK401-CFPexcitation.png|600px]]</center><br />
<br />
<p><b>Figure 3. Emission spectra after excitation at 430 nm.</b> Cell lysates from the cultures used for overexpression were excited near the excitation maximum of CFP (at 430 nm) and the emission was monitored from 445-650 nm. The spectra shown include the uninduced PK401 construct (blue), induced PK401 construct (yellow), uninduced control (dark green), induced control (lightgreen), and cell opening buffer (grey).</p><br><br />
<br />
<p>The cell lysates from the PK401 overexpression show the characteristic emission spectrum of CFP upon excitation at 430 nm. There is an additional shoulder in the spectrum near 527 nm, which is the emission maximum of YFP. When YFP was excited directly in the PK401 overexpression samples, the characteristic emission spectrum of YFP was seen, indicating that both CFP and YFP were expressed in these samples. The fluorescence from YFP could only result from a frameshift during expression of the PK401 transcript, indicating that the pseudoknot worked as expected to induce a -1 frameshift during translation. </p><br />
<br />
<br><br />
<center>[[Image:ULeth2013_PK401-YFPexcitation.png|600px]]</center><br />
<br />
<p><b>Figure 4. Emission spectra after excitation at 510 nm.</b> Cell lysates from the cultures used for overexpression were excited near the excitation maximum of YFP (at 510 nm) and the emission was monitored from 525-650 nm. The spectra shown include the uninduced PK401 construct (blue), induced PK401 construct (yellow), uninduced control (dark green), induced control (lightgreen), and cell opening buffer (grey). The characteristic emission maximum of YFP is at 527 nm, which was observed in the spectra from the PK401 samples.</p><br><br />
<br />
<p>Upon excitation of the cell lysates from the control plasmid cultures at 430 nm, a fluorescence signal of half the intensity of the PK401 cell lysates was observed with a plateau from approximately 500-520 nm. This did not correspond to the emission maximum of CFP or YFP, so the signal was likely due to other cellular components. Additionally, when the sample was excited at 510 nm, there was no detectable emission signal, indicating that YFP was not present in these samples. </p><br />
<br />
<br><br />
<center>[[Image:ULeth2013_PK401-FRET.png|600px]]</center><br />
<br />
<p><b>Figure 5. Maximum fluorescence intensity of overexpression cell lysates.</b> Intensities of cell lysates after excitation at 430 nm and monitoring at 475 nm (CFP fluorescence, blue bars) and excitation at 510 nm and emission at 527 nm (YFP fluorescence, yellow bars).</p><br><br />
<br />
<p>When the peak intensities from each sample were analyzed individually (Fig. 5), more prominent fluorescence measurements were observed for the PK401 cell lysates than for the control cell lysates. Excitation at 430 nm resulted in a fluorescence emission at 475 nm, which is a characteristic emission maximum of CFP, for the PK401 samples, but not for the control samples. Additionally, direct excitation and emission from YFP was seen for the PK401 cell lysates, but no fluorescence emission was seen for the control samples after excitation at 510 nm. This further indicates that the PK401 pseudoknot induced a -1 frameshift during translation of the PK401 transcript to produce both CFP and YFP.</p><br><br />
<br />
<h2>Generating a Pseudoknot Library</h2><br />
<p><b>Generating a Library of Pseudoknots with Variable Frameshifting Frequencies</b></p><br />
<br />
<p>To make better use of the frameshifting capability of the pseudoknot, we are also generating a randomized library of pseudoknot sequences that can be characterized for frameshifting frequency. It is our goal to create a library of pseudoknots that have a range of frameshifting frequencies to better facilitate broad application of this part. To do this, the PK401 sequence will be mutagenized using error-prone PCR to generate a library of primers that will be used to amplify the plasmid containing the original sequence. The newly generated plasmids contained mutated PK401 sequences will be transformed into E. coli cells, sequenced, and characterized for their specific frameshifting efficiency using a construct such as BBa_K1210000.</p><br><br />
<br />
<p><b>Optimizing Conditions for the Error-Prone PCR</b></p><br />
<br />
<p>To generate a library with high variability of sequence, conditions for the error-prone PCR had to first be optimized. We wanted to disrupt the PCR experiment enough to produce a large variety of primer sequences, but not too much so that the PCR was no longer successful. To do this, we used a temperature gradient to determine the annealing temperature that would best facilitate the amplification of the expected product. Figure 6 shows the resulting PCR products from the temperature gradient reactions, using temperatures from 41.9-62.1°C. The expected size of the PCR product is 93 bp, which was seen in the first three reaction samples. Various unspecific PCR products were seen in the remaining reactions samples. For this reason, the temperature chosen for the subsequent error-prone PCRs was 43.6°C, the temperature used to produce the PCR product in lane 3.</p><br />
<br />
<br><br />
<center>[[Image:ULeth2013iGEM tempgradient.png|400px]]</center><br><br />
<br />
<p><b>Figure 6. Determining the ideal temperature for error-prone PCR reactions.</b> PCR reactions were performed using various annealing temperatures from 41.9-62.1°C (lanes 1-12). The primers used in the reaction flank the PK401 pseudoknot, making the expected product size 93 bp. The temperature from the reaction in lane 3 (43.6°C) was selected for subsequent error-prone PCRs.</p><br><br />
<br />
<p>Next, we modulated a few of the typical PCR conditions to induce errors during the reaction. First, we used Taq polymerase, which is known to have higher error rates than higher fidelity polymerases commonly used in PCRs. Second, we added MnCl2 to the reaction to further hinder the accuracy of the polymerase. Finally, different ratios of deoxynucleotides were used in the reactions, either by increasing the dGTP concentration or by using a higher concentration of both dTTP and dCTP. A number of these conditions worked to produce the expected PCR product at 93 bp (Fig. 7, lanes 1-4, 6). The conditions tested in lanes 1-4 included MnCl2 at concentrations of 0-0.45 mM, and lane 6 tested the effect of increasing dGTP concentrations to 1 mM while the other dNTPs were at a concentration of 0.2 mM.</p><br />
<br />
<br><br />
<center>[[Image:ULeth2013iGEM_errorpronepcr.png|400px]]</center><br><br />
<br />
<p><b>Figure 7. Error-prone PCR of PK401 sequence.</b> PCR conditions were adjusted to induce errors in the PK401 sequence. Reactions were performed with the following modified condtions: lanes 1-6 used MnCl2 concentrations from 0-0.65 mM, lanes 4-6 used dGTP concentrations that were increased to 0.7-1.2 mM, lane 7 used increased concentrations of dTTP and dCTP, lane 8 used increased concentrations of dGTP only, and lane 9 used 0.65 mM MnCl2 only.</p><br><br />
<br />
<p>Now that we have determined the conditions for the error-prone PCR, we will perform a large-scale amplification so that we can purify the resulting PK401 primers by extracting it from an agarose gel. The primers will then be used in a high fidelity PCR reaction to amplify the entire plasmid, which will create the library of PK401 sequences. The final steps will be to transform the final PCR products into E. coli cells, screen them for frameshifting frequency using a similar construct as BBa_K1210000, and sequence the pseudoknot before submitting the parts to the registry to complete the library of frameshifting elements.</p><br />
<br />
<br><h2>References</h2><br />
<p>Tholstrup, J., Oddershede, L. B., & Sørensen, M. A. (2011). mRNA pseudoknot structures can act as ribosomal roadblocks. Nucleic acids research, 40(1), 303-313.</p></div>Dustinhttp://2013.igem.org/Team:Lethbridge/human_practicesTeam:Lethbridge/human practices2013-10-29T00:41:28Z<p>Dustin: </p>
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<h1>Human Practices</h1><br />
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<h2>Biosecurity and DNA Synthesis</h2><br />
<br />
<h3><b>Current Sequence Screening Methods</b></h3><br />
<br />
<p style="color:black"> In the last 5 years, there has been increased recognition of the powers of gene synthesis. It is now easy and affordable to look up genetic sequences for nearly any organism, design an expression construct, and order that gene from a synthesis company. This allows for the creative projects we see each year at the iGEM jamborees, but it also allows those with malevolent intentions and adequate knowledge to easily order genes that may pose a hazard to others. </p><br />
<p style="color:black"> The recognition of this potential has led members of governments and large synthesis companies to try and establish a framework for screening these synthesis orders to ensure that potentially hazardous sequences stay in the hands of those who would use them for legitimate research purposes. This effort to regulate the gene synthesis industry has largely come from within. In the late 2000's, both Europe's International Association of Synthetic Biology (IASP) and North America's International Gene Synthesis Consortium (IGSC) put forth reports on the state of synthesis order screening as well as a set of best practices to follow [1-2]. These bodies are made up of individuals from the major gene synthesis companies in each region as well as experts from major universities.</p><br />
<p style="color:black">Both groups outline a very similar approach to screening these orders for legitimacy. This entails a two part approach that first compares the ordered sequence to sequences on a list of known bio-hazardous agents and second, verifies the legitimacy of the customer and their intended use of the final product. In both reports the sequence screening utilizes existing pathogen databases, such as the US Select Agents and Toxins List or the Australia Group List as well as internal pathogen databases, and BLASTs the submitted sequence against these regulated ones. This first step in screening is conducted automatically. If there is a similarity between the submitted sequence and one of the sequences on these lists that exceeds the specified threshold, human investigation is used to further characterize the sequence [2].</p><br />
<p style="color:black">Customer screening is arguably the most important aspect of the current gene synthesis security strategies. It is possible that ordering sequences that could be considered hazardous is necessary for research applications and adequate customer screening could determine if this sequence was going to someone at a research facility for legitimate use. European and North American groups recommend collection of the name, mailing address, and institutional affiliation of the customer to ensure that they are individuals working in verifiable positions within companies or academic institutions [1-2]. This information is then independently verified and checked against a number of national and international lists of individuals of concern, such as the US Specially Designated Nationals list.</p><br />
<p style="color:black">While these protocols are put forth by consortium members in both Europe and North America, as well as there being a set of guidelines published by the US Department of Health and Human Services, all of these measures are voluntary [3]. There are no penalties to synthesis companies that do not screen the sequences or customers they deal with, outside of restrictions on international shipment of dual-use goods. This lack of legal regulation has the potential to allow dangerous sequences into the hands of malevolent individuals if any company decides to loosen their security criteria in order to save time or money in processing an order. </p><br />
<br />
<h3><b>Potential Weaknesses of Current Screening Procedures</b></h3><br />
<br />
<p style="color:black">Although companies included in the IASB and IGSC adhere to the regulations of the Code of Conduct for Best Practices in Gene Synthesis or the Harmonized Screening Protocol, respectively [1-2], these protocols have a few potential weaknesses. Both of these protocols require that all synthesis orders are at minimum screened against a regulated pathogen database. However, these lists are by no means complete and there is a chance that potentially hazardous sequences can be ordered and synthesized without any efforts made to investigate the source of the order. This is currently one of the major weaknesses of screening protocols, and efforts are being made to compile a list of data from organisms on the Select Agents list, the Australia Group List, and other national lists of regulated pathogens. Once complete, this list will provide a more comprehensive database of potential pathogenic and toxic organism sequences as a step toward higher biosecurity.</p><br />
<p style="color:black">The following are a few other weaknesses associated with current screening protocols. First, the IASB requires its member companies to screen orders of a minimum 200 base pairs in length, but there is also the potential of larger sequences being ordered as a series of short oligonucleotide sequences, from one company or multiple companies, that could bypass the screening process entirely. Though it can be more difficult to get direct database hits for shorter sequences, including these types of orders in the screening procedure is still feasible and may only require extra processing time for human investigation for these database matches. Second, though a legitimate customer can be approved for ordering hazardous sequences, the synthesis company cannot be sure of the final end user. There is no way to ensure that the customer does not ship the product to a third-party user that has not been investigated. Finally, and almost the most concerning weakness of current screening protocol, is the accountability of DNA synthesis companies. While most of the larger synthesis companies are members of the IASB or IGSC, complying to the standards mandated by these groups is still only a voluntary practice. There are no regulations in place that require a synthesis company to screen their orders for hazardous sequences or to follow-up with customer investigations of suspicious orders [4]. Even for orders that do not give a direct match to a hazardous sequence, any additional steps to associate function with the sequence is at the discretion of the company. Minshull and Wagner (representing DNA2.0 and GENEART) suggest that synthesis companies should be subject to routine “tests” of their screening protocols by their respective government bodies to ensure that they are complying to screening protocols and using the most up-to-date screening databases [5].</p><br />
<br />
<h3><b>How elements of our project were used to examine synthesis screening procedures</b></h3><br />
<br />
<p style="color:black">Our project involves the characterization of pseudoknot RNA secondary structural motifs. These motifs can be used to express dual-coding gene sequences to give protein products whose expression can be regulated by the pseudoknot’s ability to induce ribosomal frameshifting. This method of coding can allow for the expression of a protein which may be encoded by fragments in alternating reading frames. This technology adds another level of complexity in terms of screening for controlled sequences, in that the protein produced from a synthesized construct may not be the product of translating a gene in one continuous reading frame.</p><br />
<p style="color:black">It was our goal to investigate the ability of DNA synthesis companies to identify hazardous sequences in their screening procedures in the presence of frameshifting elements. A series of hazardous sequences containing intervening pseudoknots were designed and tested by two of the leading synthesis companies in North America in their standard screening procedures. These constructs contained all the necessary components to form a dangerous protein product, with DNA segments allocated into different reading frames and successively frameshifted using pseudoknots. The results from this screening test indicate that the current screening methods are successful at identifying hazardous sequences that had been “hidden” in multiple reading frames. The companies expressed their support of our efforts to investigate loopholes and problems in current screening procedures with regards to this new type of technology.</p><br />
<br />
<h3><b>Possible Methods for Bypassing Screening</h3></b><br />
<br />
<p style="color:black"><b>Codon redundancy</p></b><br />
<p style="color:black">Codon redundancy in the genetic code refers to having multiple codons that code for a single amino acid. This redundancy allows for the DNA sequence of a protein to be changed without altering the resulting amino acid sequence. By utilizing codon redundancy, bioterrorists could drastically change the known DNA sequence of a harmful virus or protein. Fortunately, synthesis companies scan both the DNA and protein sequence of sequences submitted for synthesis, and in this way would still be able to identify a harmful sequence that had been changed using codon redundancy. However, this method in conjunction with others, such as frameshifting elements or those others listed below, could potentially be used to bypass the DNA and amino acid sequence screening performed by synthesis companies.</p><br />
<br />
<p style="color:black"><b>Utilizing conservative and non-conservative regions of proteins</p></b><br />
<p style="color:black">Homologous proteins are those that are derived from the same ancestor; however, the two proteins do not have to share 100% amino acid identity. Multiple sequence alignments of amino acid sequences of homologous proteins from different organisms can be used to identify functionally important residues in a protein by indicating which residues are absolutely conserved, semi-conserved, and non-conserved. This would allow an individual to alter a controlled protein sequence by changing all or some of the conserved and semi-conserved residues to residues with similar physiochemical properties. In addition, all or some of the non-conserved residues could be substituted with essentially any other amino acid without risking loss of the protein’s function. This method, in combination with utilizing codon redundancy, would allow for more drastic alterations to be made to both the DNA and protein sequence from a pathogenic organism that could bypass screening procedures.</p><br />
<br />
<p style="color:black"><b>Using “custom” tRNAs</p></b><br />
<p style="color:black">A more complicated means for bypassing screening procedures by decoupling protein sequence from function would be to use a highly engineered system with non-canonical tRNAs. An organism could be designed that uses engineered amino acyl-tRNA synthetases that recognize non-cognate tRNAs and therefore aminoacylate the tRNA with the incorrect amino acid. By using this alternative genetic code in the engineered organism, the DNA sequence from a pathogenic organism could be altered in an almost indistinguishable way while still producing the protein of interest.</p><br />
<br />
<p style="color:black"><b>Do-it-yourself synthesis</p></b><br />
<p style="color:black">As time progresses, the cost of a DNA synthesizer is getting more affordable to research labs and independent users. Initially this may seem like a good thing, but there are tremendous dangers that are associated with this development. Directly bypassing screening procedures by not requiring the services of synthesis companies allows the owner of the DNA synthesizer unrestricted access to synthesize whatever sequence they choose. This would make any techniques to bypass the screening methods of synthesis companies obsolete. As a result, there may need to be regulations put in place to limit or restrict the access of DNA synthesizers. This can be done for example by requiring the owner to upload any sequences they synthesize to a governing body that will scan them for harmful sequences, or by installing software that will screen sequences prior to allowing them to be synthesized. A combination of these two methods as well as additional advances in screening procedures is crucial to ensure the safety of the general public.</p><br />
<br />
<h3><b>Changes recommended for screening protocols</h3></b><br />
<br />
<p style="color:black">Though commendable biosecurity efforts have been put forward by major international synthesis companies, these groups are aware that standard protocols may not be enough to mitigate the risk of the synthesis and delivery of hazardous sequences. In the IASB Code of Conduct for Best Practices in Gene Synthesis, all member companies are mandated to take part in ongoing efforts to refine and improve the current screening technologies by establishing a review committee to update and expand the Code of Conduct as new or changing threats emerge, maintain open communication with member companies through the exchange of research and literature searches, and regularly collaborating on best practices and new screening ideas [2]. While these practices are important for synthesis companies to implement, DNA synthesis is becoming less expensive and more accessible by non-professionals. According to Minshull and Wagner “[a]nyone who is sufficiently motivated could synthesize the gene for a toxin or even an entire viral genome using readily available reagents and without ever going near a specialized synthesizer” [5]. With molecular biology equipment becoming available through avenues such as E-Bay and other online dealers, individuals with limited molecular biology experience could soon realistically synthesize their own DNA sequences in the next few years [4]. Screening protocols could thereafter become obsolete. Until then, further steps are required to assure the public, government, and research community that biosecurity is being upheld to the highest standards possible. This may involve expanding the use of online forums, such as VIREP (Virulence Factor Information Repository), to allow researchers to deposit and access information about genes and organisms. Additionally, government regulations may need to be implemented that require all synthesis companies to adhere to standard practices and implement human investigation of suspicious orders [6]. This may best be achieved through the integration of both the IASB and IGSC protocols into an industry-wide Code of Conduct.</p><br />
<br />
<h2>Testing the System</h2><br />
<h3><b>Learning to Be Bad</b></h3><br />
<p style="color:black">This year, we focused on the implications our frameshifting project might have on biosecurity. In thinking about the ways our pseudoknots could be used to do new, exciting things in synthetic biology, we came up with a use that is more frightening than exciting. Bioterrorism.</p><br />
<br />
<p style="color:black">The idea is this: There are guidelines put forward by a number of industry groups on how DNA synthesis orders should be screened to ensure no biohazardous sequences get into the hands of the wrong people. The standard protocol for screening sequences involves taking the submitted DNA sequences and translating all six reading frames, then using BLAST to compare the DNA and amino acid sequences to those of organisms on a list of controlled agents.</p><br />
<br />
<p style="color:black">Our pseudoknot enables the ribosome to switch frames mid-translation, essentially splitting the entire protein amongst as many reading frames as there are pseudoknots. If someone were to split a protein from the Ebola virus into small fragments distributed across the reading frames, could they bypass this initial automatic screening step? </p><br />
<br />
<h3><b>Putting our White Hats On</b></h3><br />
<br />
<p style="color:black">To investigate this potential for abuse of our project, we worked together with major North American synthesis companies to see if we could try and fool their screening methods using our frameshifting elements. <br />
We designed and submitted sequences with vary coding changes and coding fragment sizes between the sequences for our PK401 pseudoknot to the synthesis companies we had partnered with. There is a full description of the sequences and a link to the raw data files below.</p><br />
<br />
<center><table width="800px"; border="1px"; border-color="black"><br />
<tr><br />
<td><b>Sequence ID Number</td><br />
<td><b>Sequence Origin</td><br />
<td><b>Total Length (bp)</td><br />
<td><b>Codon Changes (%)</td><br />
<td><b>Length between PK (bp)</td><br />
</tr><br />
<br />
<tr><br />
<td>1</td><br />
<td>CFP</td><br />
<td>966</td><br />
<td>25</td><br />
<td>180</td><br />
</tr><br />
<br />
<tr><br />
<td>2</td><br />
<td>Staph-ORF</td><br />
<td>1869</td><br />
<td>0</td><br />
<td>210</td><br />
</tr><br />
<br />
<tr><br />
<td>3</td><br />
<td>Ricin</td><br />
<td>2392</td><br />
<td>25</td><br />
<td>198</td><br />
</tr><br />
<br />
<tr><br />
<td>4</td><br />
<td>Staph-ORF</td><br />
<td>2450</td><br />
<td>25</td><br />
<td>102</td><br />
</tr><br />
<br />
<tr><br />
<td>5</td><br />
<td>CFP</td><br />
<td>966</td><br />
<td>16</td><br />
<td>210</td><br />
</tr><br />
<br />
<tr><br />
<td>6</td><br />
<td>CFP</td><br />
<td>966</td><br />
<td>0</td><br />
<td>180</td><br />
</tr><br />
<br />
<tr><br />
<td>7</td><br />
<td>Staph-ORF</td><br />
<td>2450</td><br />
<td>0</td><br />
<td>102</td><br />
</tr><br />
<br />
<tr><br />
<td>8</td><br />
<td>Ricin</td><br />
<td>2392</td><br />
<td>0</td><br />
<td>198</td><br />
</tr><br />
<br />
<tr><br />
<td>9</td><br />
<td>Ebola Matrix Protein</td><br />
<td>1031</td><br />
<td>0</td><br />
<td>0</td><br />
</tr><br />
<br />
<tr><br />
<td>10</td><br />
<td>CFP</td><br />
<td>966</td><br />
<td>16</td><br />
<td>180</td><br />
</tr><br />
<br />
<tr><br />
<td>11</td><br />
<td>Staph-ORF</td><br />
<td>2450</td><br />
<td>0</td><br />
<td>102</td><br />
</tr><br />
<br />
<tr><br />
<td>12</td><br />
<td>CFP</td><br />
<td>966</td><br />
<td>0</td><br />
<td>210</td><br />
</tr><br />
<br />
<tr><br />
<td>13</td><br />
<td>Staph-ORF</td><br />
<td>1869</td><br />
<td>20</td><br />
<td>210</td><br />
</tr><br />
<br />
<tr><br />
<td>14</td><br />
<td>Ricin</td><br />
<td>3139</td><br />
<td>25</td><br />
<td>99</td><br />
</tr><br />
<br />
<tr><br />
<td>15</td><br />
<td>Staph-ORF</td><br />
<td>1869</td><br />
<td>25</td><br />
<td>210</td><br />
</tr><br />
<br />
<tr><br />
<td>16</td><br />
<td>CFP</td><br />
<td>966</td><br />
<td>25</td><br />
<td>210</td><br />
</tr><br />
<br />
<tr><br />
<td>17</td><br />
<td>Ricin</td><br />
<td>3139</td><br />
<td>0</td><br />
<td>99</td><br />
</tr><br />
</table></center><br />
<br><br />
<a href="https://static.igem.org/mediawiki/2013/c/c5/Lethbridge_iGEM_2013_Screening_Sequences.txt"> Click here for sequences used </a><br />
<br />
<h3><b>Can We Sleep at Night?</b></h3><br />
<br />
<p style="color:black">These sequences were sent to the companies and screened for hazardous elements. One company managed to detect all of the “threats” on the first level of screening. According to them, their next steps would be to do a review of the “threat” sequences using a group of human experts while interviewing the customer to determine their background, shipping and payment information, and the intended use of the synthesized DNA.</p><br />
<br />
<p style="color:black">Another company simply analyzed the sequences to determine if they could actually construct the DNA if we were to order it. All of the sequences containing the pseudoknot elements were flagged as containing high repeats, but sequence 9, the Ebola matrix protein with no pseudoknot elements, was determined to be ready for synthesis. They did not make it clear whether or not there would be another level of screening to determine the origin of the sequences. </p><br />
<br />
<p style="color:black">Based on these results, current industry standard screening protocols appear to be sufficient to detect biosecurity threats, even with codon changes and the distribution of coding sequences amongst many reading frames. What is still a cause for concern is the strictness with which these protocols are applied. There is no legal requirement to execute biosecurity screens on DNA synthesis orders; all of the proposed protocols are currently voluntary guidelines. This could allow companies to relax their security protocols and may increase the potential of a serious bioterrorism threat coming to fruition. </p><br />
<br />
<p style="color:black">In order to make sure that the act of releasing the results of our study did not pose a security threat in itself, we consulted with Edward You. Edward is a representative of the FBI’s WMD department. Many of the industry guidelines for screening call for collaboration between the synthesis companies and government agencies responsible for responding to bioterrorism threats.</p><br />
<br />
<h3><b>References</h3></b><br />
<br />
<p style="color:black">[1] International Gene Synthesis Consortium. Harmonized screening protocol: gene sequence & customer screening to promote biosecurity. http://www.genesynthesisconsortium.org/wp-content/uploads/2012/02/IGSC-Harmonized-Screening-Protocol1.pdf (2009).</p><br />
<p style="color:black">[2] International Association Synthetic Biology. Code of conduct for best practices in gene synthesis. http://www.ia-sb.eu/tasks/sites/synthetic-biology/assets/File/pdf/iasb_code_of_conduct_final.pdf (2009). </p><br />
<p style="color:black">[3] U.S. Department of Health and Human Services. Screening Framework Guidance for Providers of Synthetic DoublesStranded DNA. http://www.phe.gov/Preparedness/legal/guidance/syndna/Documents/syndna-guidance.pdf (2010).</p><br />
<p style="color:black">[4] Maurer S. M., Fischer M., Schwer H., Stähler C., Stähler P., & Bernauer H. S. Working paper: making commercial biology safer: what the gene synthesis industry has learned about screening customers and orders. http://gspp.berkeley.edu/iths/Maurer_IASB_Screening.pdf (2009). </p><br />
<p style="color:black">[5] Minshull J. & Wagner, R. Nat. Biotechnol. 27, 800-801 (2009).</p><br />
<p style="color:black">[6] Fischer M. & Maurer S. M. Nat. Biotechnol. 28, 20-22 (2010).</p><br />
<br />
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</html></div>Dustinhttp://2013.igem.org/Team:Lethbridge/human_practicesTeam:Lethbridge/human practices2013-09-28T04:00:40Z<p>Dustin: </p>
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<h1>Human Practices</h1><br />
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<h2>Biosecurity and DNA Synthesis</h2><br />
<br />
<h3><b>Current Sequence Screening Methods</b></h3><br />
<br />
<p style="color:black"> In the last 5 years, there has been increased recognition of the powers of gene synthesis. It is now easy and affordable to look up genetic sequences for nearly any organism, design an expression construct, and order that gene from a synthesis company. This allows for the creative projects we see each year at the iGEM jamborees, but it also allows those with malevolent intentions and adequate knowledge to easily order genes that may pose a hazard to others. </p><br />
<p style="color:black"> The recognition of this potential has led members of governments and large synthesis companies to try and establish a framework for screening these synthesis orders to ensure that potentially hazardous sequences stay in the hands of those who would use them for legitimate research purposes. This effort to regulate the gene synthesis industry has largely come from within. In the late 2000's, both Europe's International Association of Synthetic Biology (IASP) and North America's International Gene Synthesis Consortium (IGSC) put forth reports on the state of synthesis order screening as well as a set of best practices to follow [1-2]. These bodies are made up of individuals from the major gene synthesis companies in each region as well as experts from major universities.</p><br />
<p style="color:black">Both groups outline a very similar approach to screening these orders for legitimacy. This entails a two part approach that first compares the ordered sequence to sequences on a list of known bio-hazardous agents and second, verifies the legitimacy of the customer and their intended use of the final product. In both reports the sequence screening utilizes existing pathogen databases, such as the US Select Agents and Toxins List or the Australia Group List as well as internal pathogen databases, and BLASTs the submitted sequence against these regulated ones. This first step in screening is conducted automatically. If there is a similarity between the submitted sequence and one of the sequences on these lists that exceeds the specified threshold, human investigation is used to further characterize the sequence [2].</p><br />
<p style="color:black">Customer screening is arguably the most important aspect of the current gene synthesis security strategies. It is possible that ordering sequences that could be considered hazardous is necessary for research applications and adequate customer screening could determine if this sequence was going to someone at a research facility for legitimate use. European and North American groups recommend collection of the name, mailing address, and institutional affiliation of the customer to ensure that they are individuals working in verifiable positions within companies or academic institutions [1-2]. This information is then independently verified and checked against a number of national and international lists of individuals of concern, such as the US Specially Designated Nationals list.</p><br />
<p style="color:black">While these protocols are put forth by consortium members in both Europe and North America, as well as there being a set of guidelines published by the US Department of Health and Human Services, all of these measures are voluntary [3]. There are no penalties to synthesis companies that do not screen the sequences or customers they deal with, outside of restrictions on international shipment of dual-use goods. This lack of legal regulation has the potential to allow dangerous sequences into the hands of malevolent individuals if any company decides to loosen their security criteria in order to save time or money in processing an order. </p><br />
<br />
<h3><b>Potential Weaknesses of Current Screening Procedures</b></h3><br />
<br />
<p style="color:black">Although companies included in the IASB and IGSC adhere to the regulations of the Code of Conduct for Best Practices in Gene Synthesis or the Harmonized Screening Protocol, respectively [1-2], these protocols have a few potential weaknesses. Both of these protocols require that all synthesis orders are at minimum screened against a regulated pathogen database. However, these lists are by no means complete and there is a chance that potentially hazardous sequences can be ordered and synthesized without any efforts made to investigate the source of the order. This is currently one of the major weaknesses of screening protocols, and efforts are being made to compile a list of data from organisms on the Select Agents list, the Australia Group List, and other national lists of regulated pathogens. Once complete, this list will provide a more comprehensive database of potential pathogenic and toxic organism sequences as a step toward higher biosecurity.</p><br />
<p style="color:black">The following are a few other weaknesses associated with current screening protocols. First, the IASB requires its member companies to screen orders of a minimum 200 base pairs in length, but there is also the potential of larger sequences being ordered as a series of short oligonucleotide sequences, from one company or multiple companies, that could bypass the screening process entirely. Though it can be more difficult to get direct database hits for shorter sequences, including these types of orders in the screening procedure is still feasible and may only require extra processing time for human investigation for these database matches. Second, though a legitimate customer can be approved for ordering hazardous sequences, the synthesis company cannot be sure of the final end user. There is no way to ensure that the customer does not ship the product to a third-party user that has not been investigated. Finally, and almost the most concerning weakness of current screening protocol, is the accountability of DNA synthesis companies. While most of the larger synthesis companies are members of the IASB or IGSC, complying to the standards mandated by these groups is still only a voluntary practice. There are no regulations in place that require a synthesis company to screen their orders for hazardous sequences or to follow-up with customer investigations of suspicious orders [4]. Even for orders that do not give a direct match to a hazardous sequence, any additional steps to associate function with the sequence is at the discretion of the company. Minshull and Wagner (representing DNA2.0 and GENEART) suggest that synthesis companies should be subject to routine “tests” of their screening protocols by their respective government bodies to ensure that they are complying to screening protocols and using the most up-to-date screening databases [5].</p><br />
<br />
<h3><b>How elements of our project were used to examine synthesis screening procedures</b></h3><br />
<br />
<p style="color:black">Our project involves the characterization of pseudoknot RNA secondary structural motifs. These motifs can be used to express dual-coding gene sequences to give protein products whose expression can be regulated by the pseudoknot’s ability to induce ribosomal frameshifting. This method of coding can allow for the expression of a protein which may be encoded by fragments in alternating reading frames. This technology adds another level of complexity in terms of screening for controlled sequences, in that the protein produced from a synthesized construct may not be the product of translating a gene in one continuous reading frame.</p><br />
<p style="color:black">It was our goal to investigate the ability of DNA synthesis companies to identify hazardous sequences in their screening procedures in the presence of frameshifting elements. A series of hazardous sequences containing intervening pseudoknots were designed and tested by two of the leading synthesis companies in North America in their standard screening procedures. These constructs contained all the necessary components to form a dangerous protein product, with DNA segments allocated into different reading frames and successively frameshifted using pseudoknots. The results from this screening test indicate that the current screening methods are successful at identifying hazardous sequences that had been “hidden” in multiple reading frames. The companies expressed their support of our efforts to investigate loopholes and problems in current screening procedures with regards to this new type of technology.</p><br />
<br />
<h3><b>Possible Methods for Bypassing Screening</h3></b><br />
<br />
<p style="color:black"><b>Codon redundancy</p></b><br />
<p style="color:black">Codon redundancy in the genetic code refers to having multiple codons that code for a single amino acid. This redundancy allows for the DNA sequence of a protein to be changed without altering the resulting amino acid sequence. By utilizing codon redundancy, bioterrorists could drastically change the known DNA sequence of a harmful virus or protein. Fortunately, synthesis companies scan both the DNA and protein sequence of sequences submitted for synthesis, and in this way would still be able to identify a harmful sequence that had been changed using codon redundancy. However, this method in conjunction with others, such as frameshifting elements or those others listed below, could potentially be used to bypass the DNA and amino acid sequence screening performed by synthesis companies.</p><br />
<br />
<p style="color:black"><b>Utilizing conservative and non-conservative regions of proteins</p></b><br />
<p style="color:black">Homologous proteins are those that are derived from the same ancestor; however, the two proteins do not have to share 100% amino acid identity. Multiple sequence alignments of amino acid sequences of homologous proteins from different organisms can be used to identify functionally important residues in a protein by indicating which residues are absolutely conserved, semi-conserved, and non-conserved. This would allow an individual to alter a controlled protein sequence by changing all or some of the conserved and semi-conserved residues to residues with similar physiochemical properties. In addition, all or some of the non-conserved residues could be substituted with essentially any other amino acid without risking loss of the protein’s function. This method, in combination with utilizing codon redundancy, would allow for more drastic alterations to be made to both the DNA and protein sequence from a pathogenic organism that could bypass screening procedures.</p><br />
<br />
<p style="color:black"><b>Using “custom” tRNAs</p></b><br />
<p style="color:black">A more complicated means for bypassing screening procedures by decoupling protein sequence from function would be to use a highly engineered system with non-canonical tRNAs. An organism could be designed that uses engineered amino acyl-tRNA synthetases that recognize non-cognate tRNAs and therefore aminoacylate the tRNA with the incorrect amino acid. By using this alternative genetic code in the engineered organism, the DNA sequence from a pathogenic organism could be altered in an almost indistinguishable way while still producing the protein of interest.</p><br />
<br />
<p style="color:black"><b>Do-it-yourself synthesis</p></b><br />
<p style="color:black">As time progresses, the cost of a DNA synthesizer is getting more affordable to research labs and independent users. Initially this may seem like a good thing, but there are tremendous dangers that are associated with this development. Directly bypassing screening procedures by not requiring the services of synthesis companies allows the owner of the DNA synthesizer unrestricted access to synthesize whatever sequence they choose. This would make any techniques to bypass the screening methods of synthesis companies obsolete. As a result, there may need to be regulations put in place to limit or restrict the access of DNA synthesizers. This can be done for example by requiring the owner to upload any sequences they synthesize to a governing body that will scan them for harmful sequences, or by installing software that will screen sequences prior to allowing them to be synthesized. A combination of these two methods as well as additional advances in screening procedures is crucial to ensure the safety of the general public.</p><br />
<br />
<h3><b>Changes recommended for screening protocols</h3></b><br />
<br />
<p style="color:black">Though commendable biosecurity efforts have been put forward by major international synthesis companies, these groups are aware that standard protocols may not be enough to mitigate the risk of the synthesis and delivery of hazardous sequences. In the IASB Code of Conduct for Best Practices in Gene Synthesis, all member companies are mandated to take part in ongoing efforts to refine and improve the current screening technologies by establishing a review committee to update and expand the Code of Conduct as new or changing threats emerge, maintain open communication with member companies through the exchange of research and literature searches, and regularly collaborating on best practices and new screening ideas [2]. While these practices are important for synthesis companies to implement, DNA synthesis is becoming less expensive and more accessible by non-professionals. According to Minshull and Wagner “[a]nyone who is sufficiently motivated could synthesize the gene for a toxin or even an entire viral genome using readily available reagents and without ever going near a specialized synthesizer” [5]. With molecular biology equipment becoming available through avenues such as E-Bay and other online dealers, individuals with limited molecular biology experience could soon realistically synthesize their own DNA sequences in the next few years [4]. Screening protocols could thereafter become obsolete. Until then, further steps are required to assure the public, government, and research community that biosecurity is being upheld to the highest standards possible. This may involve expanding the use of online forums, such as VIREP (Virulence Factor Information Repository), to allow researchers to deposit and access information about genes and organisms. Additionally, government regulations may need to be implemented that require all synthesis companies to adhere to standard practices and implement human investigation of suspicious orders [6]. This may best be achieved through the integration of both the IASB and IGSC protocols into an industry-wide Code of Conduct.</p><br />
<br />
<h2>Testing the System</h2><br />
<h3><b>Learning to Be Bad</b></h3><br />
<p style="color:black">This year, we focused on the implications our frameshifting project might have on biosecurity. In thinking about the ways our pseudoknots could be used to do new, exciting things in synthetic biology, we came up with a use that is more frightening than exciting. Bioterrorism.</p><br />
<br />
<p style="color:black">The idea is this: There are guidelines put forward by a number of industry groups on how DNA synthesis orders should be screened to ensure no biohazardous sequences get into the hands of the wrong people. The standard protocol for screening sequences involves taking the submitted DNA sequences and translating all six reading frames, then using BLAST to compare the DNA and amino acid sequences to those of organisms on a list of controlled agents.</p><br />
<br />
<p style="color:black">Our pseudoknot enables the ribosome to switch frames mid-translation, essentially splitting the entire protein amongst as many reading frames as there are pseudoknots. If someone were to split a protein from the Ebola virus into small fragments distributed across the reading frames, could they bypass this initial automatic screening step? </p><br />
<br />
<h3><b>Putting our White Hats On</b></h3><br />
<br />
<p style="color:black">To investigate this potential for abuse of our project, we worked together with major North American synthesis companies to see if we could try and fool their screening methods using our frameshifting elements. <br />
We designed and submitted sequences with vary coding changes and coding fragment sizes between the sequences for our PK401 pseudoknot to the synthesis companies we had partnered with. There is a full description of the sequences and a link to the raw data files below.</p><br />
<br />
<center><table width="800px"; border="1px"; border-color="black"><br />
<tr><br />
<td><b>Sequence ID Number</td><br />
<td><b>Sequence Origin</td><br />
<td><b>Total Length (bp)</td><br />
<td><b>Codon Changes (%)</td><br />
<td><b>Length between PK (bp)</td><br />
</tr><br />
<br />
<tr><br />
<td>1</td><br />
<td>CFP</td><br />
<td>966</td><br />
<td>25</td><br />
<td>180</td><br />
</tr><br />
<br />
<tr><br />
<td>2</td><br />
<td>SARS-CoV</td><br />
<td>1869</td><br />
<td>0</td><br />
<td>210</td><br />
</tr><br />
<br />
<tr><br />
<td>3</td><br />
<td>Ricin</td><br />
<td>2392</td><br />
<td>25</td><br />
<td>198</td><br />
</tr><br />
<br />
<tr><br />
<td>4</td><br />
<td>SARS-CoV</td><br />
<td>2450</td><br />
<td>25</td><br />
<td>102</td><br />
</tr><br />
<br />
<tr><br />
<td>5</td><br />
<td>CFP</td><br />
<td>966</td><br />
<td>16</td><br />
<td>210</td><br />
</tr><br />
<br />
<tr><br />
<td>6</td><br />
<td>CFP</td><br />
<td>966</td><br />
<td>0</td><br />
<td>180</td><br />
</tr><br />
<br />
<tr><br />
<td>7</td><br />
<td>SARS-CoV</td><br />
<td>2450</td><br />
<td>0</td><br />
<td>102</td><br />
</tr><br />
<br />
<tr><br />
<td>8</td><br />
<td>Ricin</td><br />
<td>2392</td><br />
<td>0</td><br />
<td>198</td><br />
</tr><br />
<br />
<tr><br />
<td>9</td><br />
<td>Ebola Matrix Protein</td><br />
<td>1031</td><br />
<td>0</td><br />
<td>0</td><br />
</tr><br />
<br />
<tr><br />
<td>10</td><br />
<td>CFP</td><br />
<td>966</td><br />
<td>16</td><br />
<td>180</td><br />
</tr><br />
<br />
<tr><br />
<td>11</td><br />
<td>SARS-CoV</td><br />
<td>2450</td><br />
<td>0</td><br />
<td>102</td><br />
</tr><br />
<br />
<tr><br />
<td>12</td><br />
<td>CFP</td><br />
<td>966</td><br />
<td>0</td><br />
<td>210</td><br />
</tr><br />
<br />
<tr><br />
<td>13</td><br />
<td>SARS-CoV</td><br />
<td>1869</td><br />
<td>20</td><br />
<td>210</td><br />
</tr><br />
<br />
<tr><br />
<td>14</td><br />
<td>Ricin</td><br />
<td>3139</td><br />
<td>25</td><br />
<td>99</td><br />
</tr><br />
<br />
<tr><br />
<td>15</td><br />
<td>SARS-CoV</td><br />
<td>1869</td><br />
<td>25</td><br />
<td>210</td><br />
</tr><br />
<br />
<tr><br />
<td>16</td><br />
<td>CFP</td><br />
<td>966</td><br />
<td>25</td><br />
<td>210</td><br />
</tr><br />
<br />
<tr><br />
<td>17</td><br />
<td>Ricin</td><br />
<td>3139</td><br />
<td>0</td><br />
<td>99</td><br />
</tr><br />
</table></center><br />
<br><br />
<a href="https://static.igem.org/mediawiki/2013/c/c5/Lethbridge_iGEM_2013_Screening_Sequences.txt"> Click here for sequences used </a><br />
<br />
<h3><b>Can We Sleep at Night?</b></h3><br />
<br />
<p style="color:black">These sequences were sent to the companies and screened for hazardous elements. One company managed to detect all of the “threats” on the first level of screening. According to them, their next steps would be to do a review of the “threat” sequences using a group of human experts while interviewing the customer to determine their background, shipping and payment information, and the intended use of the synthesized DNA.</p><br />
<br />
<p style="color:black">Another company simply analyzed the sequences to determine if they could actually construct the DNA if we were to order it. All of the sequences containing the pseudoknot elements were flagged as containing high repeats, but sequence 9, the Ebola matrix protein with no pseudoknot elements, was determined to be ready for synthesis. They did not make it clear whether or not there would be another level of screening to determine the origin of the sequences. </p><br />
<br />
<p style="color:black">Based on these results, current industry standard screening protocols appear to be sufficient to detect biosecurity threats, even with codon changes and the distribution of coding sequences amongst many reading frames. What is still a cause for concern is the strictness with which these protocols are applied. There is no legal requirement to execute biosecurity screens on DNA synthesis orders; all of the proposed protocols are currently voluntary guidelines. This could allow companies to relax their security protocols and may increase the potential of a serious bioterrorism threat coming to fruition. </p><br />
<br />
<p style="color:black">In order to make sure that the act of releasing the results of our study did not pose a security threat in itself, we consulted with Edward You. Edward is a representative of the FBI’s WMD department. Many of the industry guidelines for screening call for collaboration between the synthesis companies and government agencies responsible for responding to bioterrorism threats.</p><br />
<br />
<h3><b>References</h3></b><br />
<br />
<p style="color:black">[1] International Gene Synthesis Consortium. Harmonized screening protocol: gene sequence & customer screening to promote biosecurity. http://www.genesynthesisconsortium.org/wp-content/uploads/2012/02/IGSC-Harmonized-Screening-Protocol1.pdf (2009).</p><br />
<p style="color:black">[2] International Association Synthetic Biology. Code of conduct for best practices in gene synthesis. http://www.ia-sb.eu/tasks/sites/synthetic-biology/assets/File/pdf/iasb_code_of_conduct_final.pdf (2009). </p><br />
<p style="color:black">[3] U.S. Department of Health and Human Services. Screening Framework Guidance for Providers of Synthetic DoublesStranded DNA. http://www.phe.gov/Preparedness/legal/guidance/syndna/Documents/syndna-guidance.pdf (2010).</p><br />
<p style="color:black">[4] Maurer S. M., Fischer M., Schwer H., Stähler C., Stähler P., & Bernauer H. S. Working paper: making commercial biology safer: what the gene synthesis industry has learned about screening customers and orders. http://gspp.berkeley.edu/iths/Maurer_IASB_Screening.pdf (2009). </p><br />
<p style="color:black">[5] Minshull J. & Wagner, R. Nat. Biotechnol. 27, 800-801 (2009).</p><br />
<p style="color:black">[6] Fischer M. & Maurer S. M. Nat. Biotechnol. 28, 20-22 (2010).</p><br />
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</html></div>Dustinhttp://2013.igem.org/Team:Lethbridge/resultsTeam:Lethbridge/results2013-09-28T03:57:26Z<p>Dustin: </p>
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=<font color="black">Results=<br />
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<br />
<h2>Team Parts Sandbox</h2><br />
<center><br />
<table border="0" style="background-color:#FFF" width="100%" cellpadding="0" cellspacing="5"><br />
<tr><br />
<td><strong>Name</strong></td><br />
<td><strong>Type</strong></td><br />
<td><strong>Description</strong></td><br />
<td><strong>Designer</strong></td><br />
<td><strong>Length</strong></td><br />
<td><strong>Favorite Part</strong></td><br />
</tr><br />
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<td>[http://parts.igem.org/Part:BBa_K1210000 BBa_K1210000]</td><br />
<td>Device</td><br />
<td>LacI-CFP-PK401-YFP</td><br />
<td>Zak Stinson</td><br />
<td>1955</td><br />
<td><center>Yes</center></td><br />
</tr><br />
<tr><br />
<td>[http://parts.igem.org/Part:BBa_K1210001 BBa_K1210001]</td><br />
<td>RNA Part</td><br />
<td>PK401 Pseudoknot</td><br />
<td>Zak Stinson</td><br />
<td>93</td><br />
<td> </td><br />
</tr><br />
<tr><br />
<td>[http://parts.igem.org/Part:BBa_K1210002 BBa_K1210002]</td><br />
<td>Device</td><br />
<td>Enhanced Lumazine Synthase (ELS) Expression Construct</td><br />
<td>Harland Brandon</td><br />
<td>828</td><br />
<td> </td><br />
</tr><br />
</table><br />
<br />
</center><br />
<br />
<br><br />
<br />
<h2>Expression and Characterization of PK401 (BBa_K1210000)</h2><br />
<br />
<p><b>PK401 Overexpression</b></p><br />
<p>The PK401 construct was overexpressed to test and characterize the construct for frameshifting ability. To do this, cultures of E. coli DH5α containing either the PK401 plasmid or an empty control plasmid were grown from glycerol stocks overnight at 37°C in 50 mL LB media containing Kanamycin (100 µg/mL). These cultures were used the next day to inoculate 500 mL of LB media to a starting optical density at 600 nm (OD600) near 0.05. The OD600 was monitored and induced with 1 mM IPTG once reaching an OD600 of 0.6 (Fig. 1). Growth was monitored hourly, and equivalent amounts of cells were taken as samples for SDS-PAGE analysis up to 5 h after induction. The cultures were harvested by centrifuging at 5000 x g and shock frozen to store at -80°C.</p><br />
<br />
<center>[[File:ULeth2013_PK401-overexpression.png|600px]]</center><br />
<br />
<p><b>Figure 1. Growth curve of PK401 construct.</b> E. coli DH5α cells containing the PK401 plasmid or a control plasmid were grown at 37°C in LB media. The OD600 was monitored and the cultures were induced with 1 mM IPTG when the OD600 had reached 0.6. The cultures were grown for an additional 5 h, and a sample of 1 OD600 equivalent of cells were taken at each hour after induction.</p><br><br />
<br />
<p>The samples taken for SDS-PAGE analysis were pelleted and resuspended in 80 µL 0.1 M Tris-HCl pH 8.5 containing 5 M urea and 20 µL SDS-PAGE gel-loading buffer. The samples were then analyzed on a 12% SDS-PAGE and stained with Coomassie blue to confirm overexpression of the non-frameshifted and -1 frameshifted protein products from the PK401 construct (Fig. 2). The non-frameshifted product (CFP) has an expected size of 29 kD, and the -1 frameshifted product (a fusion protein of CFP-PK401-YFP) has an expected size of 60 kD. Bands of increasing intensity after induction with IPTG were seen at approximately 30 kD and 60 kD, corresponding to both the non-frameshifted and -1 frameshifted product. These same bands were seen in the uninduced samples, however this could be due to the expression being controlled by the pLacI promoter, which is known to give leaky expression.</p><br />
<br />
<p>To estimate the frameshifting frequency of PK401, the relative band intensities of the 60 kD protein were compared to that of the 29 kD protein. This resulted in a calculated frameshift efficiency of 21 ± 5%, indicating that 21% of the translated product was frameshifted into the -1 frame to produce the CFP-PK401-YFP fusion protein. This result correlates well with the previously reported frameshifting efficiency of 14 ± 2% for this pseudoknot (Tholstrup et al., 2011).</p><br />
<br />
<br><br />
<center>[[Image:ULeth2013_PK401-overexpressiongel.png|400px]]</center><br />
<br><br />
<br />
<p><b>Figure 2. Over-expression of non-frameshifted and -1 frameshifted protein products from the PK401 construct.</b> Equivalent amounts of cells at 0, 1, 3, and 5 h after induction with IPTG (PK401 +IPTG) were analyzed by 12% SDS-PAGE. The same time samples from the uninduced culture were also analyzed (PK401 –IPTG). Black boxes indicate bands of increasing intensity that migrated with an approximate molecular weight of 60 kD and 30 kD, corresponding to the -1 frameshifted CFP-PK401-YFP fusion product and the non-frameshifted CFP product, respectively.</p><br><br />
<br />
<p><b>CFP and YFP Fluorescence from PK401 Overexpression</b></p><br />
<p>To confirm that the protein products seen in the overexpression of the PK401 construct corresponded to the expected fluorescent proteins, fluorescence spectra of the cell lysates from overexpression were measured. The cells from the overexpression were lysed using 1 mg/mL lysozyme and 12.5 mg/g sodium deoxycholate. DNase was added to degrade the DNA followed by centrifugation at 3000 x g for 30 min. The supernatant was collected and centrifuged further at 30 000 x g for 45 min. This supernatant was then used for fluorescence measurements.</p><br />
<br />
<p>The supernatants were diluted 100-fold in the buffer used for cell opening. Excitation and emission scans were measured for all samples in the CFP and YFP range in order to confirm the presence of both fluorescent proteins. CFP was excited at 430 nm, and emission was monitored from 445-650 nm (Fig. 3). YFP was excited at 510 nm, and emission was monitored from 525-650 nm (Fig. 4). These spectra were measured for the cell opening buffer and for the induced and uninduced samples of the cells that contained the PK401 construct, as well as the cells containing the control plasmid.</p><br />
<br />
<br><br />
<center>[[Image:ULeth2013_PK401-CFPexcitation.png|600px]]</center><br />
<br />
<p><b>Figure 3. Emission spectra after excitation at 430 nm.</b> Cell lysates from the cultures used for overexpression were excited near the excitation maximum of CFP (at 430 nm) and the emission was monitored from 445-650 nm. The spectra shown include the uninduced PK401 construct (blue), induced PK401 construct (yellow), uninduced control (dark green), induced control (lightgreen), and cell opening buffer (grey).</p><br><br />
<br />
<p>The cell lysates from the PK401 overexpression show the characteristic emission spectrum of CFP upon excitation at 430 nm. There is an additional shoulder in the spectrum near 527 nm, which is the emission maximum of YFP. When YFP was excited directly in the PK401 overexpression samples, the characteristic emission spectrum of YFP was seen, indicating that both CFP and YFP were expressed in these samples. The fluorescence from YFP could only result from a frameshift during expression of the PK401 transcript, indicating that the pseudoknot worked as expected to induce a -1 frameshift during translation. </p><br />
<br />
<br><br />
<center>[[Image:ULeth2013_PK401-YFPexcitation.png|600px]]</center><br />
<br />
<p><b>Figure 4. Emission spectra after excitation at 510 nm.</b> Cell lysates from the cultures used for overexpression were excited near the excitation maximum of YFP (at 510 nm) and the emission was monitored from 525-650 nm. The spectra shown include the uninduced PK401 construct (blue), induced PK401 construct (yellow), uninduced control (dark green), induced control (lightgreen), and cell opening buffer (grey). The characteristic emission maximum of YFP is at 527 nm, which was observed in the spectra from the PK401 samples.</p><br><br />
<br />
<p>Upon excitation of the cell lysates from the control plasmid cultures at 430 nm, a fluorescence signal of half the intensity of the PK401 cell lysates was observed with a plateau from approximately 500-520 nm. This did not correspond to the emission maximum of CFP or YFP, so the signal was likely due to other cellular components. Additionally, when the sample was excited at 510 nm, there was no detectable emission signal, indicating that YFP was not present in these samples. </p><br />
<br />
<br><br />
<center>[[Image:ULeth2013_PK401-FRET.png|600px]]</center><br />
<br />
<p><b>Figure 5. Maximum fluorescence intensity of overexpression cell lysates.</b> Intensities of cell lysates after excitation at 430 nm and monitoring at 475 nm (CFP fluorescence, blue bars) and excitation at 510 nm and emission at 527 nm (YFP fluorescence, yellow bars).</p><br><br />
<br />
<p>When the peak intensities from each sample were analyzed individually (Fig. 5), more prominent fluorescence measurements were observed for the PK401 cell lysates than for the control cell lysates. Excitation at 430 nm resulted in a fluorescence emission at 475 nm, which is a characteristic emission maximum of CFP, for the PK401 samples, but not for the control samples. Additionally, direct excitation and emission from YFP was seen for the PK401 cell lysates, but no fluorescence emission was seen for the control samples after excitation at 510 nm. This further indicates that the PK401 pseudoknot induced a -1 frameshift during translation of the PK401 transcript to produce both CFP and YFP.</p><br><br />
<br />
<h2>Generating a Pseudoknot Library</h2><br />
<p><b>Generating a Library of Pseudoknots with Variable Frameshifting Frequencies</b></p><br />
<br />
<p>To make better use of the frameshifting capability of the pseudoknot, we are also generating a randomized library of pseudoknot sequences that can be characterized for frameshifting frequency. It is our goal to create a library of pseudoknots that have a range of frameshifting frequencies to better facilitate broad application of this part. To do this, the PK401 sequence will be mutagenized using error-prone PCR to generate a library of primers that will be used to amplify the plasmid containing the original sequence. The newly generated plasmids contained mutated PK401 sequences will be transformed into E. coli cells, sequenced, and characterized for their specific frameshifting efficiency using a construct such as BBa_K1210000.</p><br><br />
<br />
<p><b>Optimizing Conditions for the Error-Prone PCR</b></p><br />
<br />
<p>To generate a library with high variability of sequence, conditions for the error-prone PCR had to first be optimized. We wanted to disrupt the PCR experiment enough to produce a large variety of primer sequences, but not too much so that the PCR was no longer successful. To do this, we used a temperature gradient to determine the annealing temperature that would best facilitate the amplification of the expected product. Figure 6 shows the resulting PCR products from the temperature gradient reactions, using temperatures from 41.9-62.1°C. The expected size of the PCR product is 93 bp, which was seen in the first three reaction samples. Various unspecific PCR products were seen in the remaining reactions samples. For this reason, the temperature chosen for the subsequent error-prone PCRs was 43.6°C, the temperature used to produce the PCR product in lane 3.</p><br />
<br />
<br><br />
<center>[[Image:ULeth2013iGEM tempgradient.png|400px]]</center><br><br />
<br />
<p><b>Figure 6. Determining the ideal temperature for error-prone PCR reactions.</b> PCR reactions were performed using various annealing temperatures from 41.9-62.1°C (lanes 1-12). The primers used in the reaction flank the PK401 pseudoknot, making the expected product size 93 bp. The temperature from the reaction in lane 3 (43.6°C) was selected for subsequent error-prone PCRs.</p><br><br />
<br />
<p>Next, we modulated a few of the typical PCR conditions to induce errors during the reaction. First, we used Taq polymerase, which is known to have higher error rates than higher fidelity polymerases commonly used in PCRs. Second, we added MnCl2 to the reaction to further hinder the accuracy of the polymerase. Finally, different ratios of deoxynucleotides were used in the reactions, either by increasing the dGTP concentration or by using a higher concentration of both dTTP and dCTP. A number of these conditions worked to produce the expected PCR product at 93 bp (Fig. 7, lanes 1-4, 6). The conditions tested in lanes 1-4 included MnCl2 at concentrations of 0-0.45 mM, and lane 6 tested the effect of increasing dGTP concentrations to 1 mM while the other dNTPs were at a concentration of 0.2 mM.</p><br />
<br />
<br><br />
<center>[[Image:ULeth2013iGEM_errorpronepcr.png|400px]]</center><br><br />
<br />
<p><b>Figure 7. Error-prone PCR of PK401 sequence.</b> PCR conditions were adjusted to induce errors in the PK401 sequence. Reactions were performed with the following modified condtions: lanes 1-6 used MnCl2 concentrations from 0-0.65 mM, lanes 4-6 used dGTP concentrations that were increased to 0.7-1.2 mM, lane 7 used increased concentrations of dTTP and dCTP, lane 8 used increased concentrations of dGTP only, and lane 9 used 0.65 mM MnCl2 only.</p><br><br />
<br />
<p>Now that we have determined the conditions for the error-prone PCR, we will perform a large-scale amplification so that we can purify the resulting PK401 primers by extracting it from an agarose gel. The primers will then be used in a high fidelity PCR reaction to amplify the entire plasmid, which will create the library of PK401 sequences. The final steps will be to transform the final PCR products into E. coli cells, screen them for frameshifting frequency using a similar construct as BBa_K1210000, and sequence the pseudoknot before submitting the parts to the registry to complete the library of frameshifting elements.</p><br />
<br />
<br><h2>References</h2><br />
<p>Tholstrup, J., Oddershede, L. B., & Sørensen, M. A. (2011). mRNA pseudoknot structures can act as ribosomal roadblocks. Nucleic acids research, 40(1), 303-313.</p></div>Dustinhttp://2013.igem.org/Team:Lethbridge/notebookTeam:Lethbridge/notebook2013-09-28T03:54:21Z<p>Dustin: </p>
<hr />
<div>{{TeamLethbridgeHead}}<br />
<BLOCKQUOTE><br />
<br />
<html><br />
<center><image src="https://static.igem.org/mediawiki/2013/6/6a/UofL_iGEM_2013.png" width="750px" height="450px"/></center><br><br />
<br />
<h1>Notebook</h1><br />
<br />
<body><br />
<h2>July</h2><br />
<p>Experimenters: Graeme, Suneet, Zak</p><br />
<p>Objective: Assembly of PK401 Test Construct</p><br />
<p><a href="https://static.igem.org/mediawiki/2013/4/45/Lethbridge_iGEM_Collegiate_2013_Notebook_%28July%29.pdf">Read More Entries from July by clicking here...</a></p> <br />
<br />
<h2>August</h2><br />
<p>Experimenters: Graeme, Suneet, Zak, Harland</p><br />
<p>Objective: Assembly of Enhanced Lumazine Construct and Characterization of PK401 Test Construct</p><br />
<p><a href="https://static.igem.org/mediawiki/2013/f/f9/Lethbridge_iGEM_Collegiate_2013_Notebook_%28August%29.pdf">Read More Entries from August by clicking here...</a></p> <br />
<br />
<h2>September</h2><br />
<p>Experimenters: Graeme, Suneet, Zak, Harland, Jenna, Dustin</p><br />
<p>Objective: Re-assembly of PK401 Test Construct and Further Characterization. Error-prone PCR of Pseudoknot for Mutant Generation</p><br />
<p><a href="https://static.igem.org/mediawiki/2013/5/5e/Lethbridge_iGEM_Collegiate_2013_Notebook_%28September%29.pdf">Read More Entries from September by clicking here...</a></p> <br />
</body><br />
</html></div>Dustinhttp://2013.igem.org/Team:Lethbridge/resultsTeam:Lethbridge/results2013-09-28T03:53:50Z<p>Dustin: /* Results */</p>
<hr />
<div>{{TeamLethbridgeHead}}<br />
<div style="background-color:#FFFFFF; color:black"><br />
<html><br />
<style><br />
div<br />
{<br />
text-align:justify;<br />
text-justify:inter-word;<br />
}<br />
</style><br />
<br><br><br />
<center><image src="https://static.igem.org/mediawiki/2013/4/43/ULeth2013_Construct1.jpg" height="150px" width="600px" /></center><br />
<br><br><br />
</body><br />
</html><br />
<br />
<BLOCKQUOTE><br />
=<font color="black">Results=<br />
<br />
<br />
<br />
<h2>Team Parts Sandbox</h2><br />
<center><br />
<table border="0" style="background-color:#FFF" width="100%" cellpadding="0" cellspacing="5"><br />
<tr><br />
<td><strong>Name</strong></td><br />
<td><strong>Type</strong></td><br />
<td><strong>Description</strong></td><br />
<td><strong>Designer</strong></td><br />
<td><strong>Length</strong></td><br />
<td><strong>Favorite Part</strong></td><br />
</tr><br />
<tr><br />
<td>[http://parts.igem.org/Part:BBa_K1210000 BBa_K1210000]</td><br />
<td>Device</td><br />
<td>LacI-CFP-PK401-YFP</td><br />
<td>Zak Stinson</td><br />
<td>1955</td><br />
<td><center>Yes</center></td><br />
</tr><br />
<tr><br />
<td>[http://parts.igem.org/Part:BBa_K1210001 BBa_K1210001]</td><br />
<td>RNA Part</td><br />
<td>PK401 Pseudoknot</td><br />
<td>Zak Stinson</td><br />
<td>93</td><br />
<td> </td><br />
</tr><br />
<tr><br />
<td>[http://parts.igem.org/Part:BBa_K1210002 BBa_K1210002]</td><br />
<td>Device</td><br />
<td>Enhanced Lumazine Synthase (ELS) Expression Construct</td><br />
<td>Harland Brandon</td><br />
<td>828</td><br />
<td> </td><br />
</tr><br />
</table><br />
<br />
</center><br />
<br />
<br><br />
<br />
<h2>Expression and Characterization of PK401 (BBa_K1210000)</h2><br />
<br />
<p><b>PK401 Overexpression</b></p><br />
<p>The PK401 construct was overexpressed to test and characterize the construct for frameshifting ability. To do this, cultures of E. coli DH5α containing either the PK401 plasmid or an empty control plasmid were grown from glycerol stocks overnight at 37°C in 50 mL LB media containing Kanamycin (100 µg/mL). These cultures were used the next day to inoculate 500 mL of LB media to a starting optical density at 600 nm (OD600) near 0.05. The OD600 was monitored and induced with 1 mM IPTG once reaching an OD600 of 0.6 (Fig. 1). Growth was monitored hourly, and equivalent amounts of cells were taken as samples for SDS-PAGE analysis up to 5 h after induction. The cultures were harvested by centrifuging at 5000 x g and shock frozen to store at -80°C.</p><br />
<br />
<center>[[File:ULeth2013_PK401-overexpression.png|600px]]</center><br />
<br />
<p><b>Figure 1. Growth curve of PK401 construct.</b> E. coli DH5α cells containing the PK401 plasmid or a control plasmid were grown at 37°C in LB media. The OD600 was monitored and the cultures were induced with 1 mM IPTG when the OD600 had reached 0.6. The cultures were grown for an additional 5 h, and a sample of 1 OD600 equivalent of cells were taken at each hour after induction.</p><br><br />
<br />
<p>The samples taken for SDS-PAGE analysis were pelleted and resuspended in 80 µL 0.1 M Tris-HCl pH 8.5 containing 5 M urea and 20 µL SDS-PAGE gel-loading buffer. The samples were then analyzed on a 12% SDS-PAGE and stained with Coomassie blue to confirm overexpression of the non-frameshifted and -1 frameshifted protein products from the PK401 construct (Fig. 2). The non-frameshifted product (CFP) has an expected size of 29 kD, and the -1 frameshifted product (a fusion protein of CFP-PK401-YFP) has an expected size of 60 kD. Bands of increasing intensity after induction with IPTG were seen at approximately 30 kD and 60 kD, corresponding to both the non-frameshifted and -1 frameshifted product. These same bands were seen in the uninduced samples, however this could be due to the expression being controlled by the pLacI promoter, which is known to give leaky expression.</p><br />
<br />
<p>To estimate the frameshifting frequency of PK401, the relative band intensities of the 60 kD protein were compared to that of the 29 kD protein. This resulted in a calculated frameshift efficiency of 21 ± 5%, indicating that 21% of the translated product was frameshifted into the -1 frame to produce the CFP-PK401-YFP fusion protein. This result correlates well with the previously reported frameshifting efficiency of 14 ± 2% for this pseudoknot (Tholstrup et al., 2011).</p><br />
<br />
<br><br />
<center>[[Image:ULeth2013_PK401-overexpressiongel.png|400px]]</center><br />
<br><br />
<br />
<p><b>Figure 2. Over-expression of non-frameshifted and -1 frameshifted protein products from the PK401 construct.</b> Equivalent amounts of cells at 0, 1, 3, and 5 h after induction with IPTG (PK401 +IPTG) were analyzed by 12% SDS-PAGE. The same time samples from the uninduced culture were also analyzed (PK401 –IPTG). Black boxes indicate bands of increasing intensity that migrated with an approximate molecular weight of 60 kD and 30 kD, corresponding to the -1 frameshifted CFP-PK401-YFP fusion product and the non-frameshifted CFP product, respectively.</p><br><br />
<br />
<p><b>CFP and YFP Fluorescence from PK401 Overexpression</b></p><br />
<p>To confirm that the protein products seen in the overexpression of the PK401 construct corresponded to the expected fluorescent proteins, fluorescence spectra of the cell lysates from overexpression were measured. The cells from the overexpression were lysed using 1 mg/mL lysozyme and 12.5 mg/g sodium deoxycholate. DNase was added to degrade the DNA followed by centrifugation at 3000 x g for 30 min. The supernatant was collected and centrifuged further at 30 000 x g for 45 min. This supernatant was then used for fluorescence measurements.</p><br />
<br />
<p>The supernatants were diluted 100-fold in the buffer used for cell opening. Excitation and emission scans were measured for all samples in the CFP and YFP range in order to confirm the presence of both fluorescent proteins. CFP was excited at 430 nm, and emission was monitored from 445-650 nm (Fig. 3). YFP was excited at 510 nm, and emission was monitored from 525-650 nm (Fig. 4). These spectra were measured for the cell opening buffer and for the induced and uninduced samples of the cells that contained the PK401 construct, as well as the cells containing the control plasmid.</p><br />
<br />
<br><br />
<center>[[Image:ULeth2013_PK401-CFPexcitation.png|600px]]</center><br />
<br />
<p><b>Figure 3. Emission spectra after excitation at 430 nm.</b> Cell lysates from the cultures used for overexpression were excited near the excitation maximum of CFP (at 430 nm) and the emission was monitored from 445-650 nm. The spectra shown include the uninduced PK401 construct (blue), induced PK401 construct (yellow), uninduced control (dark green), induced control (lightgreen), and cell opening buffer (grey).</p><br><br />
<br />
<p>The cell lysates from the PK401 overexpression show the characteristic emission spectrum of CFP upon excitation at 430 nm. There is an additional shoulder in the spectrum near 527 nm, which is the emission maximum of YFP. When YFP was excited directly in the PK401 overexpression samples, the characteristic emission spectrum of YFP was seen, indicating that both CFP and YFP were expressed in these samples. The fluorescence from YFP could only result from a frameshift during expression of the PK401 transcript, indicating that the pseudoknot worked as expected to induce a -1 frameshift during translation. </p><br />
<br />
<br><br />
<center>[[Image:ULeth2013_PK401-YFPexcitation.png|600px]]</center><br />
<br />
<p><b>Figure 4. Emission spectra after excitation at 510 nm.</b> Cell lysates from the cultures used for overexpression were excited near the excitation maximum of YFP (at 510 nm) and the emission was monitored from 525-650 nm. The spectra shown include the uninduced PK401 construct (blue), induced PK401 construct (yellow), uninduced control (dark green), induced control (lightgreen), and cell opening buffer (grey). The characteristic emission maximum of YFP is at 527 nm, which was observed in the spectra from the PK401 samples.</p><br><br />
<br />
<p>Upon excitation of the cell lysates from the control plasmid cultures at 430 nm, a fluorescence signal of half the intensity of the PK401 cell lysates was observed with a plateau from approximately 500-520 nm. This did not correspond to the emission maximum of CFP or YFP, so the signal was likely due to other cellular components. Additionally, when the sample was excited at 510 nm, there was no detectable emission signal, indicating that YFP was not present in these samples. </p><br />
<br />
<br><br />
<center>[[Image:ULeth2013_PK401-FRET.png|600px]]</center><br />
<br />
<p><b>Figure 5. Maximum fluorescence intensity of overexpression cell lysates.</b> Intensities of cell lysates after excitation at 430 nm and monitoring at 475 nm (CFP fluorescence, blue bars) and excitation at 510 nm and emission at 527 nm (YFP fluorescence, yellow bars).</p><br><br />
<br />
<p>When the peak intensities from each sample were analyzed individually (Fig. 5), more prominent fluorescence measurements were observed for the PK401 cell lysates than for the control cell lysates. Excitation at 430 nm resulted in a fluorescence emission at 475 nm, which is a characteristic emission maximum of CFP, for the PK401 samples, but not for the control samples. Additionally, direct excitation and emission from YFP was seen for the PK401 cell lysates, but no fluorescence emission was seen for the control samples after excitation at 510 nm. This further indicates that the PK401 pseudoknot induced a -1 frameshift during translation of the PK401 transcript to produce both CFP and YFP.</p><br><br />
<br />
<h2>Generating a Pseudoknot Library</h2><br />
<p><b>Generating a Library of Pseudoknots with Variable Frameshifting Frequencies</b></p><br />
<br />
<p>To make better use of the frameshifting capability of the pseudoknot, we are also generating a randomized library of pseudoknot sequences that can be characterized for frameshifting frequency. It is our goal to create a library of pseudoknots that have a range of frameshifting frequencies to better facilitate broad application of this part. To do this, the PK401 sequence will be mutagenized using error-prone PCR to generate a library of primers that will be used to amplify the plasmid containing the original sequence. The newly generated plasmids contained mutated PK401 sequences will be transformed into E. coli cells, sequenced, and characterized for their specific frameshifting efficiency using a construct such as BBa_K1210000.</p><br><br />
<br />
<p><b>Optimizing Conditions for the Error-Prone PCR</b></p><br />
<br />
<p>To generate a library with high variability of sequence, conditions for the error-prone PCR had to first be optimized. We wanted to disrupt the PCR experiment enough to produce a large variety of primer sequences, but not too much so that the PCR was no longer successful. To do this, we used a temperature gradient to determine the annealing temperature that would best facilitate the amplification of the expected product. Figure 6 shows the resulting PCR products from the temperature gradient reactions, using temperatures from 41.9-62.1°C. The expected size of the PCR product is 93 bp, which was seen in the first three reaction samples. Various unspecific PCR products were seen in the remaining reactions samples. For this reason, the temperature chosen for the subsequent error-prone PCRs was 43.6°C, the temperature used to produce the PCR product in lane 3.</p><br />
<br />
<br><br />
<center>[[Image:ULeth2013iGEM tempgradient.png|400px]]</center><br><br />
<br />
<p><b>Figure 6. Determining the ideal temperature for error-prone PCR reactions.</b> PCR reactions were performed using various annealing temperatures from 41.9-62.1°C (lanes 1-12). The primers used in the reaction flank the PK401 pseudoknot, making the expected product size 93 bp. The temperature from the reaction in lane 3 (43.6°C) was selected for subsequent error-prone PCRs.</p><br><br />
<br />
<p>Next, we modulated a few of the typical PCR conditions to induce errors during the reaction. First, we used Taq polymerase, which is known to have higher error rates than higher fidelity polymerases commonly used in PCRs. Second, we added MnCl2 to the reaction to further hinder the accuracy of the polymerase. Finally, different ratios of deoxynucleotides were used in the reactions, either by increasing the dGTP concentration or by using a higher concentration of both dTTP and dCTP. A number of these conditions worked to produce the expected PCR product at 93 bp (Fig. 7, lanes 1-4, 6). The conditions tested in lanes 1-4 included MnCl2 at concentrations of 0-0.45 mM, and lane 6 tested the effect of increasing dGTP concentrations to 1 mM while the other dNTPs were at a concentration of 0.2 mM.</p><br />
<br />
<br><br />
<center>[[Image:ULeth2013iGEM_errorpronepcr.png|400px]]</center><br><br />
<br />
<p><b>Figure 7. Error-prone PCR of PK401 sequence.</b> PCR conditions were adjusted to induce errors in the PK401 sequence. Reactions were performed with the following modified condtions: lanes 1-6 used MnCl2 concentrations from 0-0.65 mM, lanes 4-6 used dGTP concentrations that were increased to 0.7-1.2 mM, lane 7 used increased concentrations of dTTP and dCTP, lane 8 used increased concentrations of dGTP only, and lane 9 used 0.65 mM MnCl2 only.</p><br><br />
<br />
<p>Now that we have determined the conditions for the error-prone PCR, we will perform a large-scale amplification so that we can purify the resulting PK401 primers by extracting it from an agarose gel. The primers will then be used in a high fidelity PCR reaction to amplify the entire plasmid, which will create the library of PK401 sequences. The final steps will be to transform the final PCR products into E. coli cells, screen them for frameshifting frequency using a similar construct as BBa_K1210000, and sequence the pseudoknot before submitting the parts to the registry to complete the library of frameshifting elements.</p><br />
<br />
<br><b>References</b><br />
<p>Tholstrup, J., Oddershede, L. B., & Sørensen, M. A. (2011). mRNA pseudoknot structures can act as ribosomal roadblocks. Nucleic acids research, 40(1), 303-313.</p></div>Dustinhttp://2013.igem.org/Team:Lethbridge/notebookTeam:Lethbridge/notebook2013-09-28T03:51:03Z<p>Dustin: </p>
<hr />
<div>{{TeamLethbridgeHead}}<br />
<BLOCKQUOTE><br />
<br />
<html><br />
<center><image src="https://static.igem.org/mediawiki/2013/6/6a/UofL_iGEM_2013.png" width="750px" height="450px"/></center><br><br />
<br />
<h1>Notebook</h1><br />
<br />
<body><br />
<h2>July</h2><br />
<p>Experimenters: Graeme, Suneet, Zak</p><br />
<p>Objective: Assembly of PK401 Test Construct</p><br />
<p><a href="https://static.igem.org/mediawiki/2013/4/45/Lethbridge_iGEM_Collegiate_2013_Notebook_%28July%29.pdf">Read More Entries from July by clicking here...</a></p> <br />
<br />
<h2>August</h2><br />
<p>Experimenters: Graeme, Suneet, Zak, Harland</p><br />
<p>Objective: Assembly of Enhanced Lumazine Construct and Characterization of PK401 Test Construct</p><br />
<p><a href="https://static.igem.org/mediawiki/2013/f/f9/Lethbridge_iGEM_Collegiate_2013_Notebook_%28August%29.pdf">Read More Entries from August by clicking here...</a></p> <br />
<br />
<h2>September</h2><br />
<p>Experimenters: Graeme, Suneet, Zak, Harland, Jenna, Dustin</p><br />
<p>Objective: Re-assembly of PK401 Test Construct and Further Characterization. Error-prone PCR of Pseudoknot for Mutant Generaion</p><br />
<p><a href="https://static.igem.org/mediawiki/2013/5/5e/Lethbridge_iGEM_Collegiate_2013_Notebook_%28September%29.pdf">Read More Entries from September by clicking here...</a></p> <br />
</body><br />
</html></div>Dustinhttp://2013.igem.org/File:Lethbridge_iGEM_Collegiate_2013_Notebook_(September).pdfFile:Lethbridge iGEM Collegiate 2013 Notebook (September).pdf2013-09-28T03:49:58Z<p>Dustin: </p>
<hr />
<div></div>Dustinhttp://2013.igem.org/Team:Lethbridge/resultsTeam:Lethbridge/results2013-09-28T03:49:16Z<p>Dustin: </p>
<hr />
<div>{{TeamLethbridgeHead}}<br />
<div style="background-color:#FFFFFF; color:black"><br />
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<center><image src="https://static.igem.org/mediawiki/2013/4/43/ULeth2013_Construct1.jpg" height="150px" width="600px" /></center><br />
<br><br><br />
</body><br />
</html><br />
<br />
<BLOCKQUOTE><br />
=<font color="black">Results=<br />
<br />
<br />
<br />
<h2>Team Parts Sandbox</h2><br />
<center><br />
<table border="0" style="background-color:#FFF" width="100%" cellpadding="0" cellspacing="5"><br />
<tr><br />
<td><strong>Name</strong></td><br />
<td><strong>Type</strong></td><br />
<td><strong>Description</strong></td><br />
<td><strong>Designer</strong></td><br />
<td><strong>Length</strong></td><br />
<td><strong>Favorite Part</strong></td><br />
</tr><br />
<tr><br />
<td>[http://parts.igem.org/Part:BBa_K1210000 BBa_K1210000]</td><br />
<td>Device</td><br />
<td>LacI-CFP-PK401-YFP</td><br />
<td>Zak Stinson</td><br />
<td>1955</td><br />
<td><center>Yes</center></td><br />
</tr><br />
<tr><br />
<td>[http://parts.igem.org/Part:BBa_K1210001 BBa_K1210001]</td><br />
<td>RNA Part</td><br />
<td>PK401 Pseudoknot</td><br />
<td>Zak Stinson</td><br />
<td>93</td><br />
<td> </td><br />
</tr><br />
<tr><br />
<td>[http://parts.igem.org/Part:BBa_K1210002 BBa_K1210002]</td><br />
<td>Device</td><br />
<td>Enhanced Lumazine Synthase (ELS) Expression Construct</td><br />
<td>Harland Brandon</td><br />
<td>828</td><br />
<td> </td><br />
</tr><br />
</table><br />
<br />
</center><br />
<br />
<br><br />
<br />
<h2>Expression and Characterization of PK401 (BBa_K1210000)</h2><br />
<br />
<p><b>PK401 Overexpression</b></p><br />
<p>The PK401 construct was overexpressed to test and characterize the construct for frameshifting ability. To do this, cultures of E. coli DH5α containing either the PK401 plasmid or an empty control plasmid were grown from glycerol stocks overnight at 37°C in 50 mL LB media containing Kanamycin (100 µg/mL). These cultures were used the next day to inoculate 500 mL of LB media to a starting optical density at 600 nm (OD600) near 0.05. The OD600 was monitored and induced with 1 mM IPTG once reaching an OD600 of 0.6 (Fig. 1). Growth was monitored hourly, and equivalent amounts of cells were taken as samples for SDS-PAGE analysis up to 5 h after induction. The cultures were harvested by centrifuging at 5000 x g and shock frozen to store at -80°C.</p><br />
<br />
<center>[[File:ULeth2013_PK401-overexpression.png|600px]]</center><br />
<br />
<p><b>Figure 1. Growth curve of PK401 construct.</b> E. coli DH5α cells containing the PK401 plasmid or a control plasmid were grown at 37°C in LB media. The OD600 was monitored and the cultures were induced with 1 mM IPTG when the OD600 had reached 0.6. The cultures were grown for an additional 5 h, and a sample of 1 OD600 equivalent of cells were taken at each hour after induction.</p><br><br />
<br />
<p>The samples taken for SDS-PAGE analysis were pelleted and resuspended in 80 µL 0.1 M Tris-HCl pH 8.5 containing 5 M urea and 20 µL SDS-PAGE gel-loading buffer. The samples were then analyzed on a 12% SDS-PAGE and stained with Coomassie blue to confirm overexpression of the non-frameshifted and -1 frameshifted protein products from the PK401 construct (Fig. 2). The non-frameshifted product (CFP) has an expected size of 29 kD, and the -1 frameshifted product (a fusion protein of CFP-PK401-YFP) has an expected size of 60 kD. Bands of increasing intensity after induction with IPTG were seen at approximately 30 kD and 60 kD, corresponding to both the non-frameshifted and -1 frameshifted product. These same bands were seen in the uninduced samples, however this could be due to the expression being controlled by the pLacI promoter, which is known to give leaky expression.</p><br />
<br />
<p>To estimate the frameshifting frequency of PK401, the relative band intensities of the 60 kD protein were compared to that of the 29 kD protein. This resulted in a calculated frameshift efficiency of 21 ± 5%, indicating that 21% of the translated product was frameshifted into the -1 frame to produce the CFP-PK401-YFP fusion protein. This result correlates well with the previously reported frameshifting efficiency of 14 ± 2% for this pseudoknot (Tholstrup et al., 2011).</p><br />
<br />
<br><br />
<center>[[Image:ULeth2013_PK401-overexpressiongel.png|400px]]</center><br />
<br><br />
<br />
<p><b>Figure 2. Over-expression of non-frameshifted and -1 frameshifted protein products from the PK401 construct.</b> Equivalent amounts of cells at 0, 1, 3, and 5 h after induction with IPTG (PK401 +IPTG) were analyzed by 12% SDS-PAGE. The same time samples from the uninduced culture were also analyzed (PK401 –IPTG). Black boxes indicate bands of increasing intensity that migrated with an approximate molecular weight of 60 kD and 30 kD, corresponding to the -1 frameshifted CFP-PK401-YFP fusion product and the non-frameshifted CFP product, respectively.</p><br><br />
<br />
<p><b>CFP and YFP Fluorescence from PK401 Overexpression</b></p><br />
<p>To confirm that the protein products seen in the overexpression of the PK401 construct corresponded to the expected fluorescent proteins, fluorescence spectra of the cell lysates from overexpression were measured. The cells from the overexpression were lysed using 1 mg/mL lysozyme and 12.5 mg/g sodium deoxycholate. DNase was added to degrade the DNA followed by centrifugation at 3000 x g for 30 min. The supernatant was collected and centrifuged further at 30 000 x g for 45 min. This supernatant was then used for fluorescence measurements.</p><br />
<br />
<p>The supernatants were diluted 100-fold in the buffer used for cell opening. Excitation and emission scans were measured for all samples in the CFP and YFP range in order to confirm the presence of both fluorescent proteins. CFP was excited at 430 nm, and emission was monitored from 445-650 nm (Fig. 3). YFP was excited at 510 nm, and emission was monitored from 525-650 nm (Fig. 4). These spectra were measured for the cell opening buffer and for the induced and uninduced samples of the cells that contained the PK401 construct, as well as the cells containing the control plasmid.</p><br />
<br />
<br><br />
<center>[[Image:ULeth2013_PK401-CFPexcitation.png|600px]]</center><br />
<br />
<p><b>Figure 3. Emission spectra after excitation at 430 nm.</b> Cell lysates from the cultures used for overexpression were excited near the excitation maximum of CFP (at 430 nm) and the emission was monitored from 445-650 nm. The spectra shown include the uninduced PK401 construct (blue), induced PK401 construct (yellow), uninduced control (dark green), induced control (lightgreen), and cell opening buffer (grey).</p><br><br />
<br />
<p>The cell lysates from the PK401 overexpression show the characteristic emission spectrum of CFP upon excitation at 430 nm. There is an additional shoulder in the spectrum near 527 nm, which is the emission maximum of YFP. When YFP was excited directly in the PK401 overexpression samples, the characteristic emission spectrum of YFP was seen, indicating that both CFP and YFP were expressed in these samples. The fluorescence from YFP could only result from a frameshift during expression of the PK401 transcript, indicating that the pseudoknot worked as expected to induce a -1 frameshift during translation. </p><br />
<br />
<br><br />
<center>[[Image:ULeth2013_PK401-YFPexcitation.png|600px]]</center><br />
<br />
<p><b>Figure 4. Emission spectra after excitation at 510 nm.</b> Cell lysates from the cultures used for overexpression were excited near the excitation maximum of YFP (at 510 nm) and the emission was monitored from 525-650 nm. The spectra shown include the uninduced PK401 construct (blue), induced PK401 construct (yellow), uninduced control (dark green), induced control (lightgreen), and cell opening buffer (grey). The characteristic emission maximum of YFP is at 527 nm, which was observed in the spectra from the PK401 samples.</p><br><br />
<br />
<p>Upon excitation of the cell lysates from the control plasmid cultures at 430 nm, a fluorescence signal of half the intensity of the PK401 cell lysates was observed with a plateau from approximately 500-520 nm. This did not correspond to the emission maximum of CFP or YFP, so the signal was likely due to other cellular components. Additionally, when the sample was excited at 510 nm, there was no detectable emission signal, indicating that YFP was not present in these samples. </p><br />
<br />
<br><br />
<center>[[Image:ULeth2013_PK401-FRET.png|600px]]</center><br />
<br />
<p><b>Figure 5. Maximum fluorescence intensity of overexpression cell lysates.</b> Intensities of cell lysates after excitation at 430 nm and monitoring at 475 nm (CFP fluorescence, blue bars) and excitation at 510 nm and emission at 527 nm (YFP fluorescence, yellow bars).</p><br><br />
<br />
<p>When the peak intensities from each sample were analyzed individually (Fig. 5), more prominent fluorescence measurements were observed for the PK401 cell lysates than for the control cell lysates. Excitation at 430 nm resulted in a fluorescence emission at 475 nm, which is a characteristic emission maximum of CFP, for the PK401 samples, but not for the control samples. Additionally, direct excitation and emission from YFP was seen for the PK401 cell lysates, but no fluorescence emission was seen for the control samples after excitation at 510 nm. This further indicates that the PK401 pseudoknot induced a -1 frameshift during translation of the PK401 transcript to produce both CFP and YFP.</p><br><br />
<br />
<h2>Generating a Pseudoknot Library</h2><br />
<p><b>Generating a Library of Pseudoknots with Variable Frameshifting Frequencies</b></p><br />
<br />
<p>To make better use of the frameshifting capability of the pseudoknot, we are also generating a randomized library of pseudoknot sequences that can be characterized for frameshifting frequency. It is our goal to create a library of pseudoknots that have a range of frameshifting frequencies to better facilitate broad application of this part. To do this, the PK401 sequence will be mutagenized using error-prone PCR to generate a library of primers that will be used to amplify the plasmid containing the original sequence. The newly generated plasmids contained mutated PK401 sequences will be transformed into E. coli cells, sequenced, and characterized for their specific frameshifting efficiency using a construct such as BBa_K1210000.</p><br><br />
<br />
<p><b>Optimizing Conditions for the Error-Prone PCR</b></p><br />
<br />
<p>To generate a library with high variability of sequence, conditions for the error-prone PCR had to first be optimized. We wanted to disrupt the PCR experiment enough to produce a large variety of primer sequences, but not too much so that the PCR was no longer successful. To do this, we used a temperature gradient to determine the annealing temperature that would best facilitate the amplification of the expected product. Figure 6 shows the resulting PCR products from the temperature gradient reactions, using temperatures from 41.9-62.1°C. The expected size of the PCR product is 93 bp, which was seen in the first three reaction samples. Various unspecific PCR products were seen in the remaining reactions samples. For this reason, the temperature chosen for the subsequent error-prone PCRs was 43.6°C, the temperature used to produce the PCR product in lane 3.</p><br />
<br />
<br><br />
<center>[[Image:ULeth2013iGEM tempgradient.png|400px]]</center><br><br />
<br />
<p><b>Figure 6. Determining the ideal temperature for error-prone PCR reactions.</b> PCR reactions were performed using various annealing temperatures from 41.9-62.1°C (lanes 1-12). The primers used in the reaction flank the PK401 pseudoknot, making the expected product size 93 bp. The temperature from the reaction in lane 3 (43.6°C) was selected for subsequent error-prone PCRs.</p><br><br />
<br />
<p>Next, we modulated a few of the typical PCR conditions to induce errors during the reaction. First, we used Taq polymerase, which is known to have higher error rates than higher fidelity polymerases commonly used in PCRs. Second, we added MnCl2 to the reaction to further hinder the accuracy of the polymerase. Finally, different ratios of deoxynucleotides were used in the reactions, either by increasing the dGTP concentration or by using a higher concentration of both dTTP and dCTP. A number of these conditions worked to produce the expected PCR product at 93 bp (Fig. 7, lanes 1-4, 6). The conditions tested in lanes 1-4 included MnCl2 at concentrations of 0-0.45 mM, and lane 6 tested the effect of increasing dGTP concentrations to 1 mM while the other dNTPs were at a concentration of 0.2 mM.</p><br />
<br />
<br><br />
<center>[[Image:ULeth2013iGEM_errorpronepcr.png|400px]]</center><br><br />
<br />
<p><b>Figure 7. Error-prone PCR of PK401 sequence.</b> PCR conditions were adjusted to induce errors in the PK401 sequence. Reactions were performed with the following modified condtions: lanes 1-6 used MnCl2 concentrations from 0-0.65 mM, lanes 4-6 used dGTP concentrations that were increased to 0.7-1.2 mM, lane 7 used increased concentrations of dTTP and dCTP, lane 8 used increased concentrations of dGTP only, and lane 9 used 0.65 mM MnCl2 only.</p><br><br />
<br />
<p>Now that we have determined the conditions for the error-prone PCR, we will perform a large-scale amplification so that we can purify the resulting PK401 primers by extracting it from an agarose gel. The primers will then be used in a high fidelity PCR reaction to amplify the entire plasmid, which will create the library of PK401 sequences. The final steps will be to transform the final PCR products into E. coli cells, screen them for frameshifting frequency using a similar construct as BBa_K1210000, and sequence the pseudoknot before submitting the parts to the registry to complete the library of frameshifting elements.</p></div>Dustinhttp://2013.igem.org/Team:Lethbridge/notebookTeam:Lethbridge/notebook2013-09-28T03:45:24Z<p>Dustin: </p>
<hr />
<div>{{TeamLethbridgeHead}}<br />
<BLOCKQUOTE><br />
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<center><image src="https://static.igem.org/mediawiki/2013/6/6a/UofL_iGEM_2013.png" width="750px" height="450px"/></center><br><br />
<br />
<h1>Notebook</h1><br />
<br />
<body><br />
<h2>July</h2><br />
<p>Experimenters: Graeme, Suneet, Zak</p><br />
<p>Objective: Assembly of PK401 Test Construct</p><br />
<p><a href="https://static.igem.org/mediawiki/2013/4/45/Lethbridge_iGEM_Collegiate_2013_Notebook_%28July%29.pdf">Read More Entries from July by clicking here...</a></p> <br />
<br />
<h2>August</h2><br />
<p>Experimenters: Graeme, Suneet, Zak, Harland</p><br />
<p>Objective: Assembly of Enhanced Lumazine Construct and Characterization of PK401 Test Construct</p><br />
<p><a href="https://static.igem.org/mediawiki/2013/f/f9/Lethbridge_iGEM_Collegiate_2013_Notebook_%28August%29.pdf">Read More Entries from August by clicking here...</a></p> <br />
</body><br />
</html></div>Dustinhttp://2013.igem.org/Team:Lethbridge/notebookTeam:Lethbridge/notebook2013-09-28T03:44:41Z<p>Dustin: </p>
<hr />
<div>{{TeamLethbridgeHead}}<br />
<BLOCKQUOTE><br />
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<center><image src="https://static.igem.org/mediawiki/2013/6/6a/UofL_iGEM_2013.png" width="750px" height="450px"/></center><br><br />
<br />
<h1>Notebook</h1><br />
<br />
<body><br />
<h2>July</h2><br />
<p>Experimenters: Graeme, Suneet, Zak</p><br />
<p>Objective: Assembly of PK401 Test Construct</p><br />
<p><a href="https://static.igem.org/mediawiki/2013/4/45/Lethbridge_iGEM_Collegiate_2013_Notebook_%28July%29.pdf">Read More Entries from July by clicking here...</a></p> <br />
<br />
<h2>August</h2><br />
<p>Experimenters: Graeme, Suneet, Zak, Harland</p><br />
<p>Objective: Assembly of Enhanced Lumazine Construct and Characterization of PK401 Test Construct</p><br />
<p><a href="https://static.igem.org/mediawiki/2013/f/f9/Lethbridge_iGEM_Collegiate_2013_Notebook_%28August%29.pdf"</a></p> <br />
</body><br />
</html></div>Dustinhttp://2013.igem.org/File:Lethbridge_iGEM_Collegiate_2013_Notebook_(August).pdfFile:Lethbridge iGEM Collegiate 2013 Notebook (August).pdf2013-09-28T03:44:15Z<p>Dustin: uploaded a new version of &quot;File:Lethbridge iGEM Collegiate 2013 Notebook (August).pdf&quot;</p>
<hr />
<div></div>Dustinhttp://2013.igem.org/Team:Lethbridge/notebookTeam:Lethbridge/notebook2013-09-28T03:42:53Z<p>Dustin: </p>
<hr />
<div>{{TeamLethbridgeHead}}<br />
<BLOCKQUOTE><br />
<br />
<html><br />
<center><image src="https://static.igem.org/mediawiki/2013/6/6a/UofL_iGEM_2013.png" width="750px" height="450px"/></center><br><br />
<br />
<h1>Notebook</h1><br />
<br />
<body><br />
<h2>July</h2><br />
<p>Experimenters: Graeme, Suneet, Zak</p><br />
<p>Objective: Assembly of PK401 Test Construct</p><br />
<p><a href="https://static.igem.org/mediawiki/2013/4/45/Lethbridge_iGEM_Collegiate_2013_Notebook_%28July%29.pdf">Read More Entries from July by clicking here...</a></p> <br />
<br />
<h2>August</h2><br />
<p>Experimenters: Graeme, Suneet, Zak, Harland</p><br />
<p>Objective: Assembly of Enhanced Lumazine Construct and Characterization of PK401 Test Construct</p><br />
<p><a href="https://static.igem.org/mediawiki/2013/4/45/Lethbridge_iGEM_Collegiate_2013_Notebook_%28July%29.pdf">Read More Entries from July by clicking here...</a></p> <br />
</body><br />
</html></div>Dustinhttp://2013.igem.org/File:Lethbridge_iGEM_Collegiate_2013_Notebook_(August).pdfFile:Lethbridge iGEM Collegiate 2013 Notebook (August).pdf2013-09-28T03:42:26Z<p>Dustin: </p>
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<div></div>Dustinhttp://2013.igem.org/Team:Lethbridge/notebookTeam:Lethbridge/notebook2013-09-28T03:39:04Z<p>Dustin: </p>
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<div>{{TeamLethbridgeHead}}<br />
<BLOCKQUOTE><br />
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<html><br />
<center><image src="https://static.igem.org/mediawiki/2013/6/6a/UofL_iGEM_2013.png" width="750px" height="450px"/></center><br><br />
<br />
<h1>Notebook</h1><br />
<br />
<body><br />
<h2>July 2013</h2><br />
<p>Experimenters: Graeme, Suneet, Zak</p><br />
<p>Objective: Assembly of PK401 Test Construct</p><br />
<p><a href="https://static.igem.org/mediawiki/2013/4/45/Lethbridge_iGEM_Collegiate_2013_Notebook_%28July%29.pdf">Read More Entries from July by clicking here...</a></p> <br />
</body><br />
</html></div>Dustinhttp://2013.igem.org/File:Lethbridge_iGEM_Collegiate_2013_Notebook_(July).pdfFile:Lethbridge iGEM Collegiate 2013 Notebook (July).pdf2013-09-28T03:36:42Z<p>Dustin: </p>
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<h1>Notebook</h1><br />
<br />
<body><br />
<h2>July 2013</h2><br />
<p>Experimenters: Graeme, Suneet, Zak</p><br />
</body><br />
</html></div>Dustinhttp://2013.igem.org/Team:Lethbridge/notebookTeam:Lethbridge/notebook2013-09-28T03:28:53Z<p>Dustin: </p>
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<div>{{TeamLethbridgeHead}}<br />
<BLOCKQUOTE><br />
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<h1>Notebook</h1><br />
<br />
<body><br />
<h2>July 2013</h2><br />
</body><br />
</html></div>Dustinhttp://2013.igem.org/Team:Lethbridge/notebookTeam:Lethbridge/notebook2013-09-28T03:26:28Z<p>Dustin: </p>
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=<font color="black">Notebook=<br />
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</html></div>Dustinhttp://2013.igem.org/Team:Lethbridge/notebookTeam:Lethbridge/notebook2013-09-28T03:26:00Z<p>Dustin: </p>
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<div>{{TeamLethbridgeHead}}<br />
<BLOCKQUOTE><br />
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<center><image src="https://static.igem.org/mediawiki/2013/6/6a/UofL_iGEM_2013.png" width="850px" height="550px"/></center><br />
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=<font color="black">Notebook=<br />
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<html><br />
<body><br />
</body><br />
</html></div>Dustinhttp://2013.igem.org/Team:Lethbridge/resultsTeam:Lethbridge/results2013-09-28T03:23:25Z<p>Dustin: </p>
<hr />
<div>{{TeamLethbridgeHead}}<br />
<div style="background-color:#FFFFFF; color:black"><br />
<html><br />
<style><br />
div<br />
{<br />
text-align:justify;<br />
text-justify:inter-word;<br />
}<br />
</style><br />
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<center><image src="https://static.igem.org/mediawiki/2013/4/43/ULeth2013_Construct1.jpg" height="150px" width="600px" /></center><br />
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</body><br />
</html><br />
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<BLOCKQUOTE><br />
=<font color="black">Results=<br />
<br />
<br />
<br />
<h2>Team Parts Sandbox</h2><br />
<center><br />
<table border="0" style="background-color:#FFF" width="100%" cellpadding="0" cellspacing="5"><br />
<tr><br />
<td><strong>Name</strong></td><br />
<td><strong>Type</strong></td><br />
<td><strong>Description</strong></td><br />
<td><strong>Designer</strong></td><br />
<td><strong>Length</strong></td><br />
<td><strong>Favorite Part</strong></td><br />
</tr><br />
<tr><br />
<td>[http://parts.igem.org/Part:BBa_K1210000 BBa_K1210000]</td><br />
<td>Device</td><br />
<td>LacI-CFP-PK401-YFP</td><br />
<td>Zak Stinson</td><br />
<td>1955</td><br />
<td><center>Yes</center></td><br />
</tr><br />
<tr><br />
<td>[http://parts.igem.org/Part:BBa_K1210001 BBa_K1210001]</td><br />
<td>RNA Part</td><br />
<td>PK401 Pseudoknot</td><br />
<td>Zak Stinson</td><br />
<td>93</td><br />
<td> </td><br />
</tr><br />
<tr><br />
<td>[http://parts.igem.org/Part:BBa_K1210002 BBa_K1210002]</td><br />
<td>Device</td><br />
<td>Enhanced Lumazine Synthase (ELS) Expression Construct</td><br />
<td>Harland Brandon</td><br />
<td>828</td><br />
<td> </td><br />
</tr><br />
</table><br />
<br />
</center><br />
<br />
<br><br />
<br />
<h2>Expression and Characterization of PK401 (BBa_K1210000)</h2><br />
<br />
<p><b>PK401 Overexpression</b></p><br />
<p>The PK401 construct was overexpressed to test and characterize the construct for frameshifting ability. To do this, cultures of E. coli DH5α containing either the PK401 plasmid or an empty control plasmid were grown from glycerol stocks overnight at 37°C in 50 mL LB media containing Kanamycin (100 µg/mL). These cultures were used the next day to inoculate 500 mL of LB media to a starting optical density at 600 nm (OD600) near 0.05. The OD600 was monitored and induced with 1 mM IPTG once reaching an OD600 of 0.6 (Fig. 1). Growth was monitored hourly, and equivalent amounts of cells were taken as samples for SDS-PAGE analysis up to 5 h after induction. The cultures were harvested by centrifuging at 5000 x g and shock frozen to store at -80°C.</p><br />
<br />
<center>[[File:ULeth2013_PK401-overexpression.png|600px]]</center><br />
<br />
<p><b>Figure 1. Growth curve of PK401 construct.</b> E. coli DH5α cells containing the PK401 plasmid or a control plasmid were grown at 37°C in LB media. The OD600 was monitored and the cultures were induced with 1 mM IPTG when the OD600 had reached 0.6. The cultures were grown for an additional 5 h, and a sample of 1 OD600 equivalent of cells were taken at each hour after induction.</p><br><br />
<br />
<p>The samples taken for SDS-PAGE analysis were pelleted and resuspended in 80 µL 0.1 M Tris-HCl pH 8.5 containing 5 M urea and 20 µL SDS-PAGE gel-loading buffer. The samples were then analyzed on a 12% SDS-PAGE and stained with Coomassie blue to confirm overexpression of the non-frameshifted and -1 frameshifted protein products from the PK401 construct (Fig. 2). The non-frameshifted product (CFP) has an expected size of 29 kD, and the -1 frameshifted product (a fusion protein of CFP-PK401-YFP) has an expected size of 60 kD. Bands of increasing intensity after induction with IPTG were seen at approximately 30 kD and 60 kD, corresponding to both the non-frameshifted and -1 frameshifted product. These same bands were seen in the uninduced samples, however this could be due to the expression being controlled by the pLacI promoter, which is known to give leaky expression.</p><br />
<br />
<p>To estimate the frameshifting frequency of PK401, the relative band intensities of the 60 kD protein were compared to that of the 29 kD protein. This resulted in a calculated frameshift efficiency of 42 ± 5%, indicating that 42% of the translated product was frameshifted into the -1 frame to produce the CFP-PK401-YFP fusion protein.</p><br />
<br />
<br><br />
<center>[[Image:ULeth2013_PK401-overexpressiongel.png|400px]]</center><br />
<br><br />
<br />
<p><b>Figure 2. Over-expression of non-frameshifted and -1 frameshifted protein products from the PK401 construct.</b> Equivalent amounts of cells at 0, 1, 3, and 5 h after induction with IPTG (PK401 +IPTG) were analyzed by 12% SDS-PAGE. The same time samples from the uninduced culture were also analyzed (PK401 –IPTG). Black boxes indicate bands of increasing intensity that migrated with an approximate molecular weight of 60 kD and 30 kD, corresponding to the -1 frameshifted CFP-PK401-YFP fusion product and the non-frameshifted CFP product, respectively.</p><br><br />
<br />
<p><b>CFP and YFP Fluorescence from PK401 Overexpression</b></p><br />
<p>To confirm that the protein products seen in the overexpression of the PK401 construct corresponded to the expected fluorescent proteins, fluorescence spectra of the cell lysates from overexpression were measured. The cells from the overexpression were lysed using 1 mg/mL lysozyme and 12.5 mg/g sodium deoxycholate. DNase was added to degrade the DNA followed by centrifugation at 3000 x g for 30 min. The supernatant was collected and centrifuged further at 30 000 x g for 45 min. This supernatant was then used for fluorescence measurements.</p><br />
<br />
<p>The supernatants were diluted 100-fold in the buffer used for cell opening. Excitation and emission scans were measured for all samples in the CFP and YFP range in order to confirm the presence of both fluorescent proteins. CFP was excited at 430 nm, and emission was monitored from 445-650 nm (Fig. 3). YFP was excited at 510 nm, and emission was monitored from 525-650 nm (Fig. 4). These spectra were measured for the cell opening buffer and for the induced and uninduced samples of the cells that contained the PK401 construct, as well as the cells containing the control plasmid.</p><br />
<br />
<br><br />
<center>[[Image:ULeth2013_PK401-CFPexcitation.png|600px]]</center><br />
<br />
<p><b>Figure 3. Emission spectra after excitation at 430 nm.</b> Cell lysates from the cultures used for overexpression were excited near the excitation maximum of CFP (at 430 nm) and the emission was monitored from 445-650 nm. The spectra shown include the uninduced PK401 construct (blue), induced PK401 construct (yellow), uninduced control (dark green), induced control (lightgreen), and cell opening buffer (grey).</p><br><br />
<br />
<p>The cell lysates from the PK401 overexpression show the characteristic emission spectrum of CFP upon excitation at 430 nm. There is an additional shoulder in the spectrum near 527 nm, which is the emission maximum of YFP. When YFP was excited directly in the PK401 overexpression samples, the characteristic emission spectrum of YFP was seen, indicating that both CFP and YFP were expressed in these samples. The fluorescence from YFP could only result from a frameshift during expression of the PK401 transcript, indicating that the pseudoknot worked as expected to induce a -1 frameshift during translation. </p><br />
<br />
<br><br />
<center>[[Image:ULeth2013_PK401-YFPexcitation.png|600px]]</center><br />
<br />
<p><b>Figure 4. Emission spectra after excitation at 510 nm.</b> Cell lysates from the cultures used for overexpression were excited near the excitation maximum of YFP (at 510 nm) and the emission was monitored from 525-650 nm. The spectra shown include the uninduced PK401 construct (blue), induced PK401 construct (yellow), uninduced control (dark green), induced control (lightgreen), and cell opening buffer (grey). The characteristic emission maximum of YFP is at 527 nm, which was observed in the spectra from the PK401 samples.</p><br><br />
<br />
<p>Upon excitation of the cell lysates from the control plasmid cultures at 430 nm, a fluorescence signal of half the intensity of the PK401 cell lysates was observed with a plateau from approximately 500-520 nm. This did not correspond to the emission maximum of CFP or YFP, so the signal was likely due to other cellular components. Additionally, when the sample was excited at 510 nm, there was no detectable emission signal, indicating that YFP was not present in these samples. </p><br />
<br />
<br><br />
<center>[[Image:ULeth2013_PK401-FRET.png|600px]]</center><br />
<br />
<p><b>Figure 5. Maximum fluorescence intensity of overexpression cell lysates.</b> Intensities of cell lysates after excitation at 430 nm and monitoring at 475 nm (CFP fluorescence, blue bars) and excitation at 510 nm and emission at 527 nm (YFP fluorescence, yellow bars).</p><br><br />
<br />
<p>When the peak intensities from each sample were analyzed individually (Fig. 5), more prominent fluorescence measurements were observed for the PK401 cell lysates than for the control cell lysates. Excitation at 430 nm resulted in a fluorescence emission at 475 nm, which is a characteristic emission maximum of CFP, for the PK401 samples, but not for the control samples. Additionally, direct excitation and emission from YFP was seen for the PK401 cell lysates, but no fluorescence emission was seen for the control samples after excitation at 510 nm. This further indicates that the PK401 pseudoknot induced a -1 frameshift during translation of the PK401 transcript to produce both CFP and YFP.</p><br><br />
<br />
<h2>Generating a Pseudoknot Library</h2><br />
<p><b>Generating a Library of Pseudoknots with Variable Frameshifting Frequencies</b></p><br />
<br />
<p>To make better use of the frameshifting capability of the pseudoknot, we are also generating a randomized library of pseudoknot sequences that can be characterized for frameshifting frequency. It is our goal to create a library of pseudoknots that have a range of frameshifting frequencies to better facilitate broad application of this part. To do this, the PK401 sequence will be mutagenized using error-prone PCR to generate a library of primers that will be used to amplify the plasmid containing the original sequence. The newly generated plasmids contained mutated PK401 sequences will be transformed into E. coli cells, sequenced, and characterized for their specific frameshifting efficiency using a construct such as BBa_K1210000.</p><br><br />
<br />
<p><b>Optimizing Conditions for the Error-Prone PCR</b></p><br />
<br />
<p>To generate a library with high variability of sequence, conditions for the error-prone PCR had to first be optimized. We wanted to disrupt the PCR experiment enough to produce a large variety of primer sequences, but not too much so that the PCR was no longer successful. To do this, we used a temperature gradient to determine the annealing temperature that would best facilitate the amplification of the expected product. Figure 6 shows the resulting PCR products from the temperature gradient reactions, using temperatures from 41.9-62.1°C. The expected size of the PCR product is 93 bp, which was seen in the first three reaction samples. Various unspecific PCR products were seen in the remaining reactions samples. For this reason, the temperature chosen for the subsequent error-prone PCRs was 43.6°C, the temperature used to produce the PCR product in lane 3.</p><br />
<br />
<br><br />
<center>[[Image:ULeth2013iGEM tempgradient.png|400px]]</center><br><br />
<br />
<p><b>Figure 6. Determining the ideal temperature for error-prone PCR reactions.</b> PCR reactions were performed using various annealing temperatures from 41.9-62.1°C (lanes 1-12). The primers used in the reaction flank the PK401 pseudoknot, making the expected product size 93 bp. The temperature from the reaction in lane 3 (43.6°C) was selected for subsequent error-prone PCRs.</p><br><br />
<br />
<p>Next, we modulated a few of the typical PCR conditions to induce errors during the reaction. First, we used Taq polymerase, which is known to have higher error rates than higher fidelity polymerases commonly used in PCRs. Second, we added MnCl2 to the reaction to further hinder the accuracy of the polymerase. Finally, different ratios of deoxynucleotides were used in the reactions, either by increasing the dGTP concentration or by using a higher concentration of both dTTP and dCTP. A number of these conditions worked to produce the expected PCR product at 93 bp (Fig. 7, lanes 1-4, 6). The conditions tested in lanes 1-4 included MnCl2 at concentrations of 0-0.45 mM, and lane 6 tested the effect of increasing dGTP concentrations to 1 mM while the other dNTPs were at a concentration of 0.2 mM.</p><br />
<br />
<br><br />
<center>[[Image:ULeth2013iGEM_errorpronepcr.png|400px]]</center><br><br />
<br />
<p><b>Figure 7. Error-prone PCR of PK401 sequence.</b> PCR conditions were adjusted to induce errors in the PK401 sequence. Reactions were performed with the following modified condtions: lanes 1-6 used MnCl2 concentrations from 0-0.65 mM, lanes 4-6 used dGTP concentrations that were increased to 0.7-1.2 mM, lane 7 used increased concentrations of dTTP and dCTP, lane 8 used increased concentrations of dGTP only, and lane 9 used 0.65 mM MnCl2 only.</p><br><br />
<br />
<p>Now that we have determined the conditions for the error-prone PCR, we will perform a large-scale amplification so that we can purify the resulting PK401 primers by extracting it from an agarose gel. The primers will then be used in a high fidelity PCR reaction to amplify the entire plasmid, which will create the library of PK401 sequences. The final steps will be to transform the final PCR products into E. coli cells, screen them for frameshifting frequency using a similar construct as BBa_K1210000, and sequence the pseudoknot before submitting the parts to the registry to complete the library of frameshifting elements.</p></div>Dustinhttp://2013.igem.org/Team:Lethbridge/resultsTeam:Lethbridge/results2013-09-28T03:23:05Z<p>Dustin: </p>
<hr />
<div>{{TeamLethbridgeHead}}<br />
<div style="background-color:#FFFFFF; color:black"><br />
<html><br />
<style><br />
div<br />
{<br />
text-align:justify;<br />
text-justify:inter-word;<br />
}<br />
</style><br />
<br><br><br />
<center><image src="https://static.igem.org/mediawiki/2013/4/43/ULeth2013_Construct1.jpg" height="150px" width="600px" /></center><br />
<br><br><br />
</body><br />
</html><br />
<br />
<BLOCKQUOTE><br />
=<font color="black">Results=<br />
<br />
<br />
<br />
<h2>Team Parts Sandbox</h2><br />
<center><br />
<table border="0" style="background-color:#FFF" width="100%" cellpadding="0" cellspacing="5"><br />
<tr><br />
<td><strong>Name</strong></td><br />
<td><strong>Type</strong></td><br />
<td><strong>Description</strong></td><br />
<td><strong>Designer</strong></td><br />
<td><strong>Length</strong></td><br />
<td><strong>Favorite Part</strong></td><br />
</tr><br />
<tr><br />
<td>[http://parts.igem.org/Part:BBa_K1210000 BBa_K1210000]</td><br />
<td>Device</td><br />
<td>LacI-CFP-PK401-YFP</td><br />
<td>Zak Stinson</td><br />
<td>1955</td><br />
<td><center>Yes</center></td><br />
</tr><br />
<tr><br />
<td>[http://parts.igem.org/Part:BBa_K1210001 BBa_K1210001]</td><br />
<td>RNA Part</td><br />
<td>PK401 Pseudoknot</td><br />
<td>Zak Stinson</td><br />
<td>93</td><br />
<td> </td><br />
</tr><br />
<tr><br />
<td>[http://parts.igem.org/Part:BBa_K1210002 BBa_K1210002]</td><br />
<td>Device</td><br />
<td>Enhanced Lumazine Synthase (ELS) Expression Construct</td><br />
<td>Harland Brandon</td><br />
<td>828</td><br />
<td> </td><br />
</tr><br />
</table><br />
<br />
</center><br />
<br />
<br><br />
<br />
<br />
<br />
<h2>Expression and Characterization of PK401 (BBa_K1210000)</h2><br />
<br />
<p><b>PK401 Overexpression</b></p><br />
<p>The PK401 construct was overexpressed to test and characterize the construct for frameshifting ability. To do this, cultures of E. coli DH5α containing either the PK401 plasmid or an empty control plasmid were grown from glycerol stocks overnight at 37°C in 50 mL LB media containing Kanamycin (100 µg/mL). These cultures were used the next day to inoculate 500 mL of LB media to a starting optical density at 600 nm (OD600) near 0.05. The OD600 was monitored and induced with 1 mM IPTG once reaching an OD600 of 0.6 (Fig. 1). Growth was monitored hourly, and equivalent amounts of cells were taken as samples for SDS-PAGE analysis up to 5 h after induction. The cultures were harvested by centrifuging at 5000 x g and shock frozen to store at -80°C.</p><br />
<br />
<center>[[File:ULeth2013_PK401-overexpression.png|600px]]</center><br />
<br />
<p><b>Figure 1. Growth curve of PK401 construct.</b> E. coli DH5α cells containing the PK401 plasmid or a control plasmid were grown at 37°C in LB media. The OD600 was monitored and the cultures were induced with 1 mM IPTG when the OD600 had reached 0.6. The cultures were grown for an additional 5 h, and a sample of 1 OD600 equivalent of cells were taken at each hour after induction.</p><br><br />
<br />
<p>The samples taken for SDS-PAGE analysis were pelleted and resuspended in 80 µL 0.1 M Tris-HCl pH 8.5 containing 5 M urea and 20 µL SDS-PAGE gel-loading buffer. The samples were then analyzed on a 12% SDS-PAGE and stained with Coomassie blue to confirm overexpression of the non-frameshifted and -1 frameshifted protein products from the PK401 construct (Fig. 2). The non-frameshifted product (CFP) has an expected size of 29 kD, and the -1 frameshifted product (a fusion protein of CFP-PK401-YFP) has an expected size of 60 kD. Bands of increasing intensity after induction with IPTG were seen at approximately 30 kD and 60 kD, corresponding to both the non-frameshifted and -1 frameshifted product. These same bands were seen in the uninduced samples, however this could be due to the expression being controlled by the pLacI promoter, which is known to give leaky expression.</p><br />
<br />
<p>To estimate the frameshifting frequency of PK401, the relative band intensities of the 60 kD protein were compared to that of the 29 kD protein. This resulted in a calculated frameshift efficiency of 42 ± 5%, indicating that 42% of the translated product was frameshifted into the -1 frame to produce the CFP-PK401-YFP fusion protein.</p><br />
<br />
<br><br />
<center>[[Image:ULeth2013_PK401-overexpressiongel.png|400px]]</center><br />
<br><br />
<br />
<p><b>Figure 2. Over-expression of non-frameshifted and -1 frameshifted protein products from the PK401 construct.</b> Equivalent amounts of cells at 0, 1, 3, and 5 h after induction with IPTG (PK401 +IPTG) were analyzed by 12% SDS-PAGE. The same time samples from the uninduced culture were also analyzed (PK401 –IPTG). Black boxes indicate bands of increasing intensity that migrated with an approximate molecular weight of 60 kD and 30 kD, corresponding to the -1 frameshifted CFP-PK401-YFP fusion product and the non-frameshifted CFP product, respectively.</p><br><br />
<br />
<p><b>CFP and YFP Fluorescence from PK401 Overexpression</b></p><br />
<p>To confirm that the protein products seen in the overexpression of the PK401 construct corresponded to the expected fluorescent proteins, fluorescence spectra of the cell lysates from overexpression were measured. The cells from the overexpression were lysed using 1 mg/mL lysozyme and 12.5 mg/g sodium deoxycholate. DNase was added to degrade the DNA followed by centrifugation at 3000 x g for 30 min. The supernatant was collected and centrifuged further at 30 000 x g for 45 min. This supernatant was then used for fluorescence measurements.</p><br />
<br />
<p>The supernatants were diluted 100-fold in the buffer used for cell opening. Excitation and emission scans were measured for all samples in the CFP and YFP range in order to confirm the presence of both fluorescent proteins. CFP was excited at 430 nm, and emission was monitored from 445-650 nm (Fig. 3). YFP was excited at 510 nm, and emission was monitored from 525-650 nm (Fig. 4). These spectra were measured for the cell opening buffer and for the induced and uninduced samples of the cells that contained the PK401 construct, as well as the cells containing the control plasmid.</p><br />
<br />
<br><br />
<center>[[Image:ULeth2013_PK401-CFPexcitation.png|600px]]</center><br />
<br />
<p><b>Figure 3. Emission spectra after excitation at 430 nm.</b> Cell lysates from the cultures used for overexpression were excited near the excitation maximum of CFP (at 430 nm) and the emission was monitored from 445-650 nm. The spectra shown include the uninduced PK401 construct (blue), induced PK401 construct (yellow), uninduced control (dark green), induced control (lightgreen), and cell opening buffer (grey).</p><br><br />
<br />
<p>The cell lysates from the PK401 overexpression show the characteristic emission spectrum of CFP upon excitation at 430 nm. There is an additional shoulder in the spectrum near 527 nm, which is the emission maximum of YFP. When YFP was excited directly in the PK401 overexpression samples, the characteristic emission spectrum of YFP was seen, indicating that both CFP and YFP were expressed in these samples. The fluorescence from YFP could only result from a frameshift during expression of the PK401 transcript, indicating that the pseudoknot worked as expected to induce a -1 frameshift during translation. </p><br />
<br />
<br><br />
<center>[[Image:ULeth2013_PK401-YFPexcitation.png|600px]]</center><br />
<br />
<p><b>Figure 4. Emission spectra after excitation at 510 nm.</b> Cell lysates from the cultures used for overexpression were excited near the excitation maximum of YFP (at 510 nm) and the emission was monitored from 525-650 nm. The spectra shown include the uninduced PK401 construct (blue), induced PK401 construct (yellow), uninduced control (dark green), induced control (lightgreen), and cell opening buffer (grey). The characteristic emission maximum of YFP is at 527 nm, which was observed in the spectra from the PK401 samples.</p><br><br />
<br />
<p>Upon excitation of the cell lysates from the control plasmid cultures at 430 nm, a fluorescence signal of half the intensity of the PK401 cell lysates was observed with a plateau from approximately 500-520 nm. This did not correspond to the emission maximum of CFP or YFP, so the signal was likely due to other cellular components. Additionally, when the sample was excited at 510 nm, there was no detectable emission signal, indicating that YFP was not present in these samples. </p><br />
<br />
<br><br />
<center>[[Image:ULeth2013_PK401-FRET.png|600px]]</center><br />
<br />
<p><b>Figure 5. Maximum fluorescence intensity of overexpression cell lysates.</b> Intensities of cell lysates after excitation at 430 nm and monitoring at 475 nm (CFP fluorescence, blue bars) and excitation at 510 nm and emission at 527 nm (YFP fluorescence, yellow bars).</p><br><br />
<br />
<p>When the peak intensities from each sample were analyzed individually (Fig. 5), more prominent fluorescence measurements were observed for the PK401 cell lysates than for the control cell lysates. Excitation at 430 nm resulted in a fluorescence emission at 475 nm, which is a characteristic emission maximum of CFP, for the PK401 samples, but not for the control samples. Additionally, direct excitation and emission from YFP was seen for the PK401 cell lysates, but no fluorescence emission was seen for the control samples after excitation at 510 nm. This further indicates that the PK401 pseudoknot induced a -1 frameshift during translation of the PK401 transcript to produce both CFP and YFP.</p><br><br />
<br />
<h2>Generating a Pseudoknot Library</h2><br />
<p><b>Generating a Library of Pseudoknots with Variable Frameshifting Frequencies</b></p><br />
<br />
<p>To make better use of the frameshifting capability of the pseudoknot, we are also generating a randomized library of pseudoknot sequences that can be characterized for frameshifting frequency. It is our goal to create a library of pseudoknots that have a range of frameshifting frequencies to better facilitate broad application of this part. To do this, the PK401 sequence will be mutagenized using error-prone PCR to generate a library of primers that will be used to amplify the plasmid containing the original sequence. The newly generated plasmids contained mutated PK401 sequences will be transformed into E. coli cells, sequenced, and characterized for their specific frameshifting efficiency using a construct such as BBa_K1210000.</p><br><br />
<br />
<p><b>Optimizing Conditions for the Error-Prone PCR</b></p><br />
<br />
<p>To generate a library with high variability of sequence, conditions for the error-prone PCR had to first be optimized. We wanted to disrupt the PCR experiment enough to produce a large variety of primer sequences, but not too much so that the PCR was no longer successful. To do this, we used a temperature gradient to determine the annealing temperature that would best facilitate the amplification of the expected product. Figure 6 shows the resulting PCR products from the temperature gradient reactions, using temperatures from 41.9-62.1°C. The expected size of the PCR product is 93 bp, which was seen in the first three reaction samples. Various unspecific PCR products were seen in the remaining reactions samples. For this reason, the temperature chosen for the subsequent error-prone PCRs was 43.6°C, the temperature used to produce the PCR product in lane 3.</p><br />
<br />
<br><br />
<center>[[Image:ULeth2013iGEM tempgradient.png|400px]]</center><br><br />
<br />
<p><b>Figure 6. Determining the ideal temperature for error-prone PCR reactions.</b> PCR reactions were performed using various annealing temperatures from 41.9-62.1°C (lanes 1-12). The primers used in the reaction flank the PK401 pseudoknot, making the expected product size 93 bp. The temperature from the reaction in lane 3 (43.6°C) was selected for subsequent error-prone PCRs.</p><br><br />
<br />
<p>Next, we modulated a few of the typical PCR conditions to induce errors during the reaction. First, we used Taq polymerase, which is known to have higher error rates than higher fidelity polymerases commonly used in PCRs. Second, we added MnCl2 to the reaction to further hinder the accuracy of the polymerase. Finally, different ratios of deoxynucleotides were used in the reactions, either by increasing the dGTP concentration or by using a higher concentration of both dTTP and dCTP. A number of these conditions worked to produce the expected PCR product at 93 bp (Fig. 7, lanes 1-4, 6). The conditions tested in lanes 1-4 included MnCl2 at concentrations of 0-0.45 mM, and lane 6 tested the effect of increasing dGTP concentrations to 1 mM while the other dNTPs were at a concentration of 0.2 mM.</p><br />
<br />
<br><br />
<center>[[Image:ULeth2013iGEM_errorpronepcr.png|400px]]</center><br><br />
<br />
<p><b>Figure 7. Error-prone PCR of PK401 sequence.</b> PCR conditions were adjusted to induce errors in the PK401 sequence. Reactions were performed with the following modified condtions: lanes 1-6 used MnCl2 concentrations from 0-0.65 mM, lanes 4-6 used dGTP concentrations that were increased to 0.7-1.2 mM, lane 7 used increased concentrations of dTTP and dCTP, lane 8 used increased concentrations of dGTP only, and lane 9 used 0.65 mM MnCl2 only.</p><br><br />
<br />
<p>Now that we have determined the conditions for the error-prone PCR, we will perform a large-scale amplification so that we can purify the resulting PK401 primers by extracting it from an agarose gel. The primers will then be used in a high fidelity PCR reaction to amplify the entire plasmid, which will create the library of PK401 sequences. The final steps will be to transform the final PCR products into E. coli cells, screen them for frameshifting frequency using a similar construct as BBa_K1210000, and sequence the pseudoknot before submitting the parts to the registry to complete the library of frameshifting elements.</p></div>Dustinhttp://2013.igem.org/Team:Lethbridge/resultsTeam:Lethbridge/results2013-09-28T03:21:44Z<p>Dustin: </p>
<hr />
<div>{{TeamLethbridgeHead}}<br />
<div style="background-color:#FFFFFF; color:black"><br />
<html><br />
<style><br />
div<br />
{<br />
text-align:justify;<br />
text-justify:inter-word;<br />
}<br />
</style><br />
<br><br><br />
<center><image src="https://static.igem.org/mediawiki/2013/4/43/ULeth2013_Construct1.jpg" height="150px" width="600px" /></center><br />
<br><br><br />
</body><br />
</html><br />
<br />
<BLOCKQUOTE><br />
=<font color="black">Results=<br />
<br />
<br />
<br />
<h2>Team Parts Sandbox</h2><br />
<center><br />
<table border="0" style="background-color:#FFF" width="100%" cellpadding="0" cellspacing="5"><br />
<tr><br />
<td><strong>Name</strong></td><br />
<td><strong>Type</strong></td><br />
<td><strong>Description</strong></td><br />
<td><strong>Designer</strong></td><br />
<td><strong>Length</strong></td><br />
<td><strong>Favorite Part</strong></td><br />
</tr><br />
<tr><br />
<td>[http://parts.igem.org/Part:BBa_K1210000 BBa_K1210000]</td><br />
<td>Device</td><br />
<td>LacI-CFP-PK401-YFP</td><br />
<td>Zak Stinson</td><br />
<td>1955</td><br />
<td><center>Yes</center></td><br />
</tr><br />
<tr><br />
<td>[http://parts.igem.org/Part:BBa_K1210001 BBa_K1210001]</td><br />
<td>RNA Part</td><br />
<td>PK401 Pseudoknot</td><br />
<td>Zak Stinson</td><br />
<td>93</td><br />
<td> </td><br />
</tr><br />
<tr><br />
<td>[http://parts.igem.org/Part:BBa_K1210002 BBa_K1210002]</td><br />
<td>Device</td><br />
<td>Enhanced Lumazine Synthase (ELS) Expression Construct</td><br />
<td>Harland Brandon</td><br />
<td>828</td><br />
<td> </td><br />
</tr><br />
</table><br />
<br />
</center><br />
<br />
<br><br />
<br />
<br />
<br><br />
<h2>Expression and Characterization of PK401 (BBa_K1210000)</h2><br />
<br />
<p><b>PK401 Overexpression</b></p><br />
<p>The PK401 construct was overexpressed to test and characterize the construct for frameshifting ability. To do this, cultures of E. coli DH5α containing either the PK401 plasmid or an empty control plasmid were grown from glycerol stocks overnight at 37°C in 50 mL LB media containing Kanamycin (100 µg/mL). These cultures were used the next day to inoculate 500 mL of LB media to a starting optical density at 600 nm (OD600) near 0.05. The OD600 was monitored and induced with 1 mM IPTG once reaching an OD600 of 0.6 (Fig. 1). Growth was monitored hourly, and equivalent amounts of cells were taken as samples for SDS-PAGE analysis up to 5 h after induction. The cultures were harvested by centrifuging at 5000 x g and shock frozen to store at -80°C.</p><br />
<br />
<center>[[File:ULeth2013_PK401-overexpression.png|600px]]</center><br />
<br />
<p><b>Figure 1. Growth curve of PK401 construct.</b> E. coli DH5α cells containing the PK401 plasmid or a control plasmid were grown at 37°C in LB media. The OD600 was monitored and the cultures were induced with 1 mM IPTG when the OD600 had reached 0.6. The cultures were grown for an additional 5 h, and a sample of 1 OD600 equivalent of cells were taken at each hour after induction.</p><br><br />
<br />
<p>The samples taken for SDS-PAGE analysis were pelleted and resuspended in 80 µL 0.1 M Tris-HCl pH 8.5 containing 5 M urea and 20 µL SDS-PAGE gel-loading buffer. The samples were then analyzed on a 12% SDS-PAGE and stained with Coomassie blue to confirm overexpression of the non-frameshifted and -1 frameshifted protein products from the PK401 construct (Fig. 2). The non-frameshifted product (CFP) has an expected size of 29 kD, and the -1 frameshifted product (a fusion protein of CFP-PK401-YFP) has an expected size of 60 kD. Bands of increasing intensity after induction with IPTG were seen at approximately 30 kD and 60 kD, corresponding to both the non-frameshifted and -1 frameshifted product. These same bands were seen in the uninduced samples, however this could be due to the expression being controlled by the pLacI promoter, which is known to give leaky expression.</p><br />
<br />
<p>To estimate the frameshifting frequency of PK401, the relative band intensities of the 60 kD protein were compared to that of the 29 kD protein. This resulted in a calculated frameshift efficiency of 42 ± 5%, indicating that 42% of the translated product was frameshifted into the -1 frame to produce the CFP-PK401-YFP fusion protein.</p><br />
<br />
<br><br />
<center>[[Image:ULeth2013_PK401-overexpressiongel.png|400px]]</center><br />
<br><br />
<br />
<p><b>Figure 2. Over-expression of non-frameshifted and -1 frameshifted protein products from the PK401 construct.</b> Equivalent amounts of cells at 0, 1, 3, and 5 h after induction with IPTG (PK401 +IPTG) were analyzed by 12% SDS-PAGE. The same time samples from the uninduced culture were also analyzed (PK401 –IPTG). Black boxes indicate bands of increasing intensity that migrated with an approximate molecular weight of 60 kD and 30 kD, corresponding to the -1 frameshifted CFP-PK401-YFP fusion product and the non-frameshifted CFP product, respectively.</p><br><br />
<br />
<p><b>CFP and YFP Fluorescence from PK401 Overexpression</b></p><br />
<p>To confirm that the protein products seen in the overexpression of the PK401 construct corresponded to the expected fluorescent proteins, fluorescence spectra of the cell lysates from overexpression were measured. The cells from the overexpression were lysed using 1 mg/mL lysozyme and 12.5 mg/g sodium deoxycholate. DNase was added to degrade the DNA followed by centrifugation at 3000 x g for 30 min. The supernatant was collected and centrifuged further at 30 000 x g for 45 min. This supernatant was then used for fluorescence measurements.</p><br />
<br />
<p>The supernatants were diluted 100-fold in the buffer used for cell opening. Excitation and emission scans were measured for all samples in the CFP and YFP range in order to confirm the presence of both fluorescent proteins. CFP was excited at 430 nm, and emission was monitored from 445-650 nm (Fig. 3). YFP was excited at 510 nm, and emission was monitored from 525-650 nm (Fig. 4). These spectra were measured for the cell opening buffer and for the induced and uninduced samples of the cells that contained the PK401 construct, as well as the cells containing the control plasmid.</p><br />
<br />
<br><br />
<center>[[Image:ULeth2013_PK401-CFPexcitation.png|600px]]</center><br />
<br />
<p><b>Figure 3. Emission spectra after excitation at 430 nm.</b> Cell lysates from the cultures used for overexpression were excited near the excitation maximum of CFP (at 430 nm) and the emission was monitored from 445-650 nm. The spectra shown include the uninduced PK401 construct (blue), induced PK401 construct (yellow), uninduced control (dark green), induced control (lightgreen), and cell opening buffer (grey).</p><br><br />
<br />
<p>The cell lysates from the PK401 overexpression show the characteristic emission spectrum of CFP upon excitation at 430 nm. There is an additional shoulder in the spectrum near 527 nm, which is the emission maximum of YFP. When YFP was excited directly in the PK401 overexpression samples, the characteristic emission spectrum of YFP was seen, indicating that both CFP and YFP were expressed in these samples. The fluorescence from YFP could only result from a frameshift during expression of the PK401 transcript, indicating that the pseudoknot worked as expected to induce a -1 frameshift during translation. </p><br />
<br />
<br><br />
<center>[[Image:ULeth2013_PK401-YFPexcitation.png|600px]]</center><br />
<br />
<p><b>Figure 4. Emission spectra after excitation at 510 nm.</b> Cell lysates from the cultures used for overexpression were excited near the excitation maximum of YFP (at 510 nm) and the emission was monitored from 525-650 nm. The spectra shown include the uninduced PK401 construct (blue), induced PK401 construct (yellow), uninduced control (dark green), induced control (lightgreen), and cell opening buffer (grey). The characteristic emission maximum of YFP is at 527 nm, which was observed in the spectra from the PK401 samples.</p><br><br />
<br />
<p>Upon excitation of the cell lysates from the control plasmid cultures at 430 nm, a fluorescence signal of half the intensity of the PK401 cell lysates was observed with a plateau from approximately 500-520 nm. This did not correspond to the emission maximum of CFP or YFP, so the signal was likely due to other cellular components. Additionally, when the sample was excited at 510 nm, there was no detectable emission signal, indicating that YFP was not present in these samples. </p><br />
<br />
<br><br />
<center>[[Image:ULeth2013_PK401-FRET.png|600px]]</center><br />
<br />
<p><b>Figure 5. Maximum fluorescence intensity of overexpression cell lysates.</b> Intensities of cell lysates after excitation at 430 nm and monitoring at 475 nm (CFP fluorescence, blue bars) and excitation at 510 nm and emission at 527 nm (YFP fluorescence, yellow bars).</p><br><br />
<br />
<p>When the peak intensities from each sample were analyzed individually (Fig. 5), more prominent fluorescence measurements were observed for the PK401 cell lysates than for the control cell lysates. Excitation at 430 nm resulted in a fluorescence emission at 475 nm, which is a characteristic emission maximum of CFP, for the PK401 samples, but not for the control samples. Additionally, direct excitation and emission from YFP was seen for the PK401 cell lysates, but no fluorescence emission was seen for the control samples after excitation at 510 nm. This further indicates that the PK401 pseudoknot induced a -1 frameshift during translation of the PK401 transcript to produce both CFP and YFP.</p><br><br />
<br />
<h2>Generating a Pseudoknot Library</h2><br />
<p><b>Generating a Library of Pseudoknots with Variable Frameshifting Frequencies</b></p><br />
<br />
<p>To make better use of the frameshifting capability of the pseudoknot, we are also generating a randomized library of pseudoknot sequences that can be characterized for frameshifting frequency. It is our goal to create a library of pseudoknots that have a range of frameshifting frequencies to better facilitate broad application of this part. To do this, the PK401 sequence will be mutagenized using error-prone PCR to generate a library of primers that will be used to amplify the plasmid containing the original sequence. The newly generated plasmids contained mutated PK401 sequences will be transformed into E. coli cells, sequenced, and characterized for their specific frameshifting efficiency using a construct such as BBa_K1210000.</p><br><br />
<br />
<p><b>Optimizing Conditions for the Error-Prone PCR</b></p><br />
<br />
<p>To generate a library with high variability of sequence, conditions for the error-prone PCR had to first be optimized. We wanted to disrupt the PCR experiment enough to produce a large variety of primer sequences, but not too much so that the PCR was no longer successful. To do this, we used a temperature gradient to determine the annealing temperature that would best facilitate the amplification of the expected product. Figure 6 shows the resulting PCR products from the temperature gradient reactions, using temperatures from 41.9-62.1°C. The expected size of the PCR product is 93 bp, which was seen in the first three reaction samples. Various unspecific PCR products were seen in the remaining reactions samples. For this reason, the temperature chosen for the subsequent error-prone PCRs was 43.6°C, the temperature used to produce the PCR product in lane 3.</p><br />
<br />
<br><br />
<center>[[Image:ULeth2013iGEM tempgradient.png|400px]]</center><br><br />
<br />
<p><b>Figure 6. Determining the ideal temperature for error-prone PCR reactions.</b> PCR reactions were performed using various annealing temperatures from 41.9-62.1°C (lanes 1-12). The primers used in the reaction flank the PK401 pseudoknot, making the expected product size 93 bp. The temperature from the reaction in lane 3 (43.6°C) was selected for subsequent error-prone PCRs.</p><br><br />
<br />
<p>Next, we modulated a few of the typical PCR conditions to induce errors during the reaction. First, we used Taq polymerase, which is known to have higher error rates than higher fidelity polymerases commonly used in PCRs. Second, we added MnCl2 to the reaction to further hinder the accuracy of the polymerase. Finally, different ratios of deoxynucleotides were used in the reactions, either by increasing the dGTP concentration or by using a higher concentration of both dTTP and dCTP. A number of these conditions worked to produce the expected PCR product at 93 bp (Fig. 7, lanes 1-4, 6). The conditions tested in lanes 1-4 included MnCl2 at concentrations of 0-0.45 mM, and lane 6 tested the effect of increasing dGTP concentrations to 1 mM while the other dNTPs were at a concentration of 0.2 mM.</p><br />
<br />
<br><br />
<center>[[Image:ULeth2013iGEM_errorpronepcr.png|400px]]</center><br><br />
<br />
<p><b>Figure 7. Error-prone PCR of PK401 sequence.</b> PCR conditions were adjusted to induce errors in the PK401 sequence. Reactions were performed with the following modified condtions: lanes 1-6 used MnCl2 concentrations from 0-0.65 mM, lanes 4-6 used dGTP concentrations that were increased to 0.7-1.2 mM, lane 7 used increased concentrations of dTTP and dCTP, lane 8 used increased concentrations of dGTP only, and lane 9 used 0.65 mM MnCl2 only.</p><br><br />
<br />
<p>Now that we have determined the conditions for the error-prone PCR, we will perform a large-scale amplification so that we can purify the resulting PK401 primers by extracting it from an agarose gel. The primers will then be used in a high fidelity PCR reaction to amplify the entire plasmid, which will create the library of PK401 sequences. The final steps will be to transform the final PCR products into E. coli cells, screen them for frameshifting frequency using a similar construct as BBa_K1210000, and sequence the pseudoknot before submitting the parts to the registry to complete the library of frameshifting elements.</p></div>Dustinhttp://2013.igem.org/Team:Lethbridge/attributionsTeam:Lethbridge/attributions2013-09-28T03:02:50Z<p>Dustin: </p>
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<BLOCKQUOTE><br />
<br />
=<font color="black">Attributions=<br />
All planning and wet lab work was performed by the undergraduate members of our iGEM team.<br />
<br />
<h2>Contributions</h2><br />
<p><b>Dallas Thomas</b> (University of Lethbridge, Lethbridge Agriculture and Agri-Food Canada): For developing the software code to overlap protein coding sequences</p><br />
<p><b>Dipankar Goyal</b> (University of Lethbridge): For assistance with our Wiki design</p><br />
<p><b>Kelsey Kristensen</b> (University of Lethbridge): For taking our team photos, please visit her [http://seventhousandplus.weebly.com/index.html website]</p><br />
<p>The Department of Chemistry and Biochemistry, the Alberta RNA Research and Training Institute, the Wieden Lab, the Kothe Lab, and the Selinger Lab at the University of Lethbridge for providing us with equipment and laboratory space.</p><br />
<br />
<h2>Sponsors</h2><br />
<p>We would also like to acknowledge our sponsors for the following support of the 2013 Lethbridge iGEM team.</p><br><br />
<br />
<html><br />
<p><center><image src="https://static.igem.org/mediawiki/2013/0/00/ULeth2013_Sponsors_-_Platinum.png"; width="400px"; height="200px" /></center></p><br><br />
<br />
<p><center><image src="https://static.igem.org/mediawiki/2013/1/1e/ULeth2013_Sponsors_-_Silver.png"; width="400px"; height="200px" /></center></p><br><br />
<br />
<p><center><image src="https://static.igem.org/mediawiki/2013/4/4c/ULeth2013_Sponsors_-_Bronze.png"; width="600px"; height="200px" /></center></p><br />
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<br />
<br />
<p><b>1. Platinum - $5000+ or gift in kind</b></p><br />
<p>Logo on front of team shirts, large logo on scientific poster, large logo on team wiki and verbal recognition during team project presentations/media interviews.</p><br />
<p><b>2. Gold - $2000-$4999 or gift in kind</b></p><br />
<p>Logo on team shirts, medium logo on scientific poster, medium logo on team wiki and written recognition at end of team project presentations.</p><br />
<p><b>3. Silver - $1000-$1999 or gift in kind</b></p><br />
<p>Medium logo on scientific poster, medium logo on team wiki and written recognition at end of team project presentations.</p><br />
<p><b>4. Bronze - <$999 or gift in kind</b></p><br />
<p>Small logo on scientific poster, small logo on team wiki and written recognition at end of team project presentations.</p></div>Dustinhttp://2013.igem.org/Team:Lethbridge/resultsTeam:Lethbridge/results2013-09-28T03:01:58Z<p>Dustin: </p>
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<br />
<BLOCKQUOTE><br />
=<font color="black">Results=<br />
<br />
<br />
<br />
<h2>Team Parts Sandbox</h2><br />
<center><br />
<table border="0" style="background-color:#FFF" width="100%" cellpadding="0" cellspacing="5"><br />
<tr><br />
<td><strong>Name</strong></td><br />
<td><strong>Type</strong></td><br />
<td><strong>Description</strong></td><br />
<td><strong>Designer</strong></td><br />
<td><strong>Length</strong></td><br />
<td><strong>Favorite Part</strong></td><br />
</tr><br />
<tr><br />
<td>[http://parts.igem.org/Part:BBa_K1210000 BBa_K1210000]</td><br />
<td>Device</td><br />
<td>LacI-CFP-PK401-YFP</td><br />
<td>Zak Stinson</td><br />
<td>1955</td><br />
<td><center>Yes</center></td><br />
</tr><br />
<tr><br />
<td>[http://parts.igem.org/Part:BBa_K1210001 BBa_K1210001]</td><br />
<td>RNA Part</td><br />
<td>PK401 Pseudoknot</td><br />
<td>Zak Stinson</td><br />
<td>93</td><br />
<td> </td><br />
</tr><br />
<tr><br />
<td>[http://parts.igem.org/Part:BBa_K1210002 BBa_K1210002]</td><br />
<td>Device</td><br />
<td>Enhanced Lumazine Synthase (ELS) Expression Construct</td><br />
<td>Harland Brandon</td><br />
<td>828</td><br />
<td> </td><br />
</tr><br />
</table><br />
<br />
</center><br />
<br />
<br><br />
<center>[[File:ULeth2013_Construct1.jpg|800px]]</center><br />
<br />
<br><br />
<h2>Expression and Characterization of PK401 (BBa_K1210000)</h2><br />
<br />
<p><b>PK401 Overexpression</b></p><br />
<p>The PK401 construct was overexpressed to test and characterize the construct for frameshifting ability. To do this, cultures of E. coli DH5α containing either the PK401 plasmid or an empty control plasmid were grown from glycerol stocks overnight at 37°C in 50 mL LB media containing Kanamycin (100 µg/mL). These cultures were used the next day to inoculate 500 mL of LB media to a starting optical density at 600 nm (OD600) near 0.05. The OD600 was monitored and induced with 1 mM IPTG once reaching an OD600 of 0.6 (Fig. 1). Growth was monitored hourly, and equivalent amounts of cells were taken as samples for SDS-PAGE analysis up to 5 h after induction. The cultures were harvested by centrifuging at 5000 x g and shock frozen to store at -80°C.</p><br />
<br />
<center>[[File:ULeth2013_PK401-overexpression.png|600px]]</center><br />
<br />
<p><b>Figure 1. Growth curve of PK401 construct.</b> E. coli DH5α cells containing the PK401 plasmid or a control plasmid were grown at 37°C in LB media. The OD600 was monitored and the cultures were induced with 1 mM IPTG when the OD600 had reached 0.6. The cultures were grown for an additional 5 h, and a sample of 1 OD600 equivalent of cells were taken at each hour after induction.</p><br><br />
<br />
<p>The samples taken for SDS-PAGE analysis were pelleted and resuspended in 80 µL 0.1 M Tris-HCl pH 8.5 containing 5 M urea and 20 µL SDS-PAGE gel-loading buffer. The samples were then analyzed on a 12% SDS-PAGE and stained with Coomassie blue to confirm overexpression of the non-frameshifted and -1 frameshifted protein products from the PK401 construct (Fig. 2). The non-frameshifted product (CFP) has an expected size of 29 kD, and the -1 frameshifted product (a fusion protein of CFP-PK401-YFP) has an expected size of 60 kD. Bands of increasing intensity after induction with IPTG were seen at approximately 30 kD and 60 kD, corresponding to both the non-frameshifted and -1 frameshifted product. These same bands were seen in the uninduced samples, however this could be due to the expression being controlled by the pLacI promoter, which is known to give leaky expression.</p><br />
<br />
<p>To estimate the frameshifting frequency of PK401, the relative band intensities of the 60 kD protein were compared to that of the 29 kD protein. This resulted in a calculated frameshift efficiency of 42 ± 5%, indicating that 42% of the translated product was frameshifted into the -1 frame to produce the CFP-PK401-YFP fusion protein.</p><br />
<br />
<br><br />
<center>[[Image:ULeth2013_PK401-overexpressiongel.png|400px]]</center><br />
<br><br />
<br />
<p><b>Figure 2. Over-expression of non-frameshifted and -1 frameshifted protein products from the PK401 construct.</b> Equivalent amounts of cells at 0, 1, 3, and 5 h after induction with IPTG (PK401 +IPTG) were analyzed by 12% SDS-PAGE. The same time samples from the uninduced culture were also analyzed (PK401 –IPTG). Black boxes indicate bands of increasing intensity that migrated with an approximate molecular weight of 60 kD and 30 kD, corresponding to the -1 frameshifted CFP-PK401-YFP fusion product and the non-frameshifted CFP product, respectively.</p><br><br />
<br />
<p><b>CFP and YFP Fluorescence from PK401 Overexpression</b></p><br />
<p>To confirm that the protein products seen in the overexpression of the PK401 construct corresponded to the expected fluorescent proteins, fluorescence spectra of the cell lysates from overexpression were measured. The cells from the overexpression were lysed using 1 mg/mL lysozyme and 12.5 mg/g sodium deoxycholate. DNase was added to degrade the DNA followed by centrifugation at 3000 x g for 30 min. The supernatant was collected and centrifuged further at 30 000 x g for 45 min. This supernatant was then used for fluorescence measurements.</p><br />
<br />
<p>The supernatants were diluted 100-fold in the buffer used for cell opening. Excitation and emission scans were measured for all samples in the CFP and YFP range in order to confirm the presence of both fluorescent proteins. CFP was excited at 430 nm, and emission was monitored from 445-650 nm (Fig. 3). YFP was excited at 510 nm, and emission was monitored from 525-650 nm (Fig. 4). These spectra were measured for the cell opening buffer and for the induced and uninduced samples of the cells that contained the PK401 construct, as well as the cells containing the control plasmid.</p><br />
<br />
<br><br />
<center>[[Image:ULeth2013_PK401-CFPexcitation.png|600px]]</center><br />
<br />
<p><b>Figure 3. Emission spectra after excitation at 430 nm.</b> Cell lysates from the cultures used for overexpression were excited near the excitation maximum of CFP (at 430 nm) and the emission was monitored from 445-650 nm. The spectra shown include the uninduced PK401 construct (blue), induced PK401 construct (yellow), uninduced control (dark green), induced control (lightgreen), and cell opening buffer (grey).</p><br><br />
<br />
<p>The cell lysates from the PK401 overexpression show the characteristic emission spectrum of CFP upon excitation at 430 nm. There is an additional shoulder in the spectrum near 527 nm, which is the emission maximum of YFP. When YFP was excited directly in the PK401 overexpression samples, the characteristic emission spectrum of YFP was seen, indicating that both CFP and YFP were expressed in these samples. The fluorescence from YFP could only result from a frameshift during expression of the PK401 transcript, indicating that the pseudoknot worked as expected to induce a -1 frameshift during translation. </p><br />
<br />
<br><br />
<center>[[Image:ULeth2013_PK401-YFPexcitation.png|600px]]</center><br />
<br />
<p><b>Figure 4. Emission spectra after excitation at 510 nm.</b> Cell lysates from the cultures used for overexpression were excited near the excitation maximum of YFP (at 510 nm) and the emission was monitored from 525-650 nm. The spectra shown include the uninduced PK401 construct (blue), induced PK401 construct (yellow), uninduced control (dark green), induced control (lightgreen), and cell opening buffer (grey). The characteristic emission maximum of YFP is at 527 nm, which was observed in the spectra from the PK401 samples.</p><br><br />
<br />
<p>Upon excitation of the cell lysates from the control plasmid cultures at 430 nm, a fluorescence signal of half the intensity of the PK401 cell lysates was observed with a plateau from approximately 500-520 nm. This did not correspond to the emission maximum of CFP or YFP, so the signal was likely due to other cellular components. Additionally, when the sample was excited at 510 nm, there was no detectable emission signal, indicating that YFP was not present in these samples. </p><br />
<br />
<br><br />
<center>[[Image:ULeth2013_PK401-FRET.png|600px]]</center><br />
<br />
<p><b>Figure 5. Maximum fluorescence intensity of overexpression cell lysates.</b> Intensities of cell lysates after excitation at 430 nm and monitoring at 475 nm (CFP fluorescence, blue bars) and excitation at 510 nm and emission at 527 nm (YFP fluorescence, yellow bars).</p><br><br />
<br />
<p>When the peak intensities from each sample were analyzed individually (Fig. 5), more prominent fluorescence measurements were observed for the PK401 cell lysates than for the control cell lysates. Excitation at 430 nm resulted in a fluorescence emission at 475 nm, which is a characteristic emission maximum of CFP, for the PK401 samples, but not for the control samples. Additionally, direct excitation and emission from YFP was seen for the PK401 cell lysates, but no fluorescence emission was seen for the control samples after excitation at 510 nm. This further indicates that the PK401 pseudoknot induced a -1 frameshift during translation of the PK401 transcript to produce both CFP and YFP.</p><br><br />
<br />
<h2>Generating a Pseudoknot Library</h2><br />
<p><b>Generating a Library of Pseudoknots with Variable Frameshifting Frequencies</b></p><br />
<br />
<p>To make better use of the frameshifting capability of the pseudoknot, we are also generating a randomized library of pseudoknot sequences that can be characterized for frameshifting frequency. It is our goal to create a library of pseudoknots that have a range of frameshifting frequencies to better facilitate broad application of this part. To do this, the PK401 sequence will be mutagenized using error-prone PCR to generate a library of primers that will be used to amplify the plasmid containing the original sequence. The newly generated plasmids contained mutated PK401 sequences will be transformed into E. coli cells, sequenced, and characterized for their specific frameshifting efficiency using a construct such as BBa_K1210000.</p><br><br />
<br />
<p><b>Optimizing Conditions for the Error-Prone PCR</b></p><br />
<br />
<p>To generate a library with high variability of sequence, conditions for the error-prone PCR had to first be optimized. We wanted to disrupt the PCR experiment enough to produce a large variety of primer sequences, but not too much so that the PCR was no longer successful. To do this, we used a temperature gradient to determine the annealing temperature that would best facilitate the amplification of the expected product. Figure 6 shows the resulting PCR products from the temperature gradient reactions, using temperatures from 41.9-62.1°C. The expected size of the PCR product is 93 bp, which was seen in the first three reaction samples. Various unspecific PCR products were seen in the remaining reactions samples. For this reason, the temperature chosen for the subsequent error-prone PCRs was 43.6°C, the temperature used to produce the PCR product in lane 3.</p><br />
<br />
<br><br />
<center>[[Image:ULeth2013iGEM tempgradient.png|400px]]</center><br><br />
<br />
<p><b>Figure 6. Determining the ideal temperature for error-prone PCR reactions.</b> PCR reactions were performed using various annealing temperatures from 41.9-62.1°C (lanes 1-12). The primers used in the reaction flank the PK401 pseudoknot, making the expected product size 93 bp. The temperature from the reaction in lane 3 (43.6°C) was selected for subsequent error-prone PCRs.</p><br><br />
<br />
<p>Next, we modulated a few of the typical PCR conditions to induce errors during the reaction. First, we used Taq polymerase, which is known to have higher error rates than higher fidelity polymerases commonly used in PCRs. Second, we added MnCl2 to the reaction to further hinder the accuracy of the polymerase. Finally, different ratios of deoxynucleotides were used in the reactions, either by increasing the dGTP concentration or by using a higher concentration of both dTTP and dCTP. A number of these conditions worked to produce the expected PCR product at 93 bp (Fig. 7, lanes 1-4, 6). The conditions tested in lanes 1-4 included MnCl2 at concentrations of 0-0.45 mM, and lane 6 tested the effect of increasing dGTP concentrations to 1 mM while the other dNTPs were at a concentration of 0.2 mM.</p><br />
<br />
<br><br />
<center>[[Image:ULeth2013iGEM_errorpronepcr.png|400px]]</center><br><br />
<br />
<p><b>Figure 7. Error-prone PCR of PK401 sequence.</b> PCR conditions were adjusted to induce errors in the PK401 sequence. Reactions were performed with the following modified condtions: lanes 1-6 used MnCl2 concentrations from 0-0.65 mM, lanes 4-6 used dGTP concentrations that were increased to 0.7-1.2 mM, lane 7 used increased concentrations of dTTP and dCTP, lane 8 used increased concentrations of dGTP only, and lane 9 used 0.65 mM MnCl2 only.</p><br><br />
<br />
<p>Now that we have determined the conditions for the error-prone PCR, we will perform a large-scale amplification so that we can purify the resulting PK401 primers by extracting it from an agarose gel. The primers will then be used in a high fidelity PCR reaction to amplify the entire plasmid, which will create the library of PK401 sequences. The final steps will be to transform the final PCR products into E. coli cells, screen them for frameshifting frequency using a similar construct as BBa_K1210000, and sequence the pseudoknot before submitting the parts to the registry to complete the library of frameshifting elements.</p></div>Dustinhttp://2013.igem.org/Team:Lethbridge/resultsTeam:Lethbridge/results2013-09-28T03:00:58Z<p>Dustin: </p>
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</html><br />
<br />
<BLOCKQUOTE><br />
=<font color="black">Results=<br />
<br />
<br />
<br />
<h2>Team Parts Sandbox</h2><br />
<center><br />
<table border="0" style="background-color:#FFF" width="100%" cellpadding="0" cellspacing="5"><br />
<tr><br />
<td><strong>Name</strong></td><br />
<td><strong>Type</strong></td><br />
<td><strong>Description</strong></td><br />
<td><strong>Designer</strong></td><br />
<td><strong>Length</strong></td><br />
<td><strong>Favorite Part</strong></td><br />
</tr><br />
<tr><br />
<td>[http://parts.igem.org/Part:BBa_K1210000 BBa_K1210000]</td><br />
<td>Device</td><br />
<td>LacI-CFP-PK401-YFP</td><br />
<td>Zak Stinson</td><br />
<td>1955</td><br />
<td><center>Yes</center></td><br />
</tr><br />
<tr><br />
<td>[http://parts.igem.org/Part:BBa_K1210001 BBa_K1210001]</td><br />
<td>RNA Part</td><br />
<td>PK401 Pseudoknot</td><br />
<td>Zak Stinson</td><br />
<td>93</td><br />
<td> </td><br />
</tr><br />
<tr><br />
<td>[http://parts.igem.org/Part:BBa_K1210002 BBa_K1210002]</td><br />
<td>Device</td><br />
<td>Enhanced Lumazine Synthase (ELS) Expression Construct</td><br />
<td>Harland Brandon</td><br />
<td>828</td><br />
<td> </td><br />
</tr><br />
</table><br />
<br />
</center><br />
<br />
<br><br />
<center>[[File:ULeth2013_Construct1.jpg|800px]]</center><br />
<br />
<br><br />
<h2>Expression and Characterization of PK401 (BBa_K1210000)</h2><br />
<br />
<p><b>PK401 Overexpression</b></p><br />
<p>The PK401 construct was overexpressed to test and characterize the construct for frameshifting ability. To do this, cultures of E. coli DH5α containing either the PK401 plasmid or an empty control plasmid were grown from glycerol stocks overnight at 37°C in 50 mL LB media containing Kanamycin (100 µg/mL). These cultures were used the next day to inoculate 500 mL of LB media to a starting optical density at 600 nm (OD600) near 0.05. The OD600 was monitored and induced with 1 mM IPTG once reaching an OD600 of 0.6 (Fig. 1). Growth was monitored hourly, and equivalent amounts of cells were taken as samples for SDS-PAGE analysis up to 5 h after induction. The cultures were harvested by centrifuging at 5000 x g and shock frozen to store at -80°C.</p><br />
<br />
<center>[[File:ULeth2013_PK401-overexpression.png|600px]]</center><br />
<br />
<p><b>Figure 1. Growth curve of PK401 construct.</b> E. coli DH5α cells containing the PK401 plasmid or a control plasmid were grown at 37°C in LB media. The OD600 was monitored and the cultures were induced with 1 mM IPTG when the OD600 had reached 0.6. The cultures were grown for an additional 5 h, and a sample of 1 OD600 equivalent of cells were taken at each hour after induction.</p><br><br />
<br />
<p>The samples taken for SDS-PAGE analysis were pelleted and resuspended in 80 µL 0.1 M Tris-HCl pH 8.5 containing 5 M urea and 20 µL SDS-PAGE gel-loading buffer. The samples were then analyzed on a 12% SDS-PAGE and stained with Coomassie blue to confirm overexpression of the non-frameshifted and -1 frameshifted protein products from the PK401 construct (Fig. 2). The non-frameshifted product (CFP) has an expected size of 29 kD, and the -1 frameshifted product (a fusion protein of CFP-PK401-YFP) has an expected size of 60 kD. Bands of increasing intensity after induction with IPTG were seen at approximately 30 kD and 60 kD, corresponding to both the non-frameshifted and -1 frameshifted product. These same bands were seen in the uninduced samples, however this could be due to the expression being controlled by the pLacI promoter, which is known to give leaky expression.</p><br />
<br />
<p>To estimate the frameshifting frequency of PK401, the relative band intensities of the 60 kD protein were compared to that of the 29 kD protein. This resulted in a calculated frameshift efficiency of 42 ± 5%, indicating that 42% of the translated product was frameshifted into the -1 frame to produce the CFP-PK401-YFP fusion protein.</p><br />
<br />
<br><br />
<center>[[Image:ULeth2013_PK401-overexpressiongel.png|400px]]</center><br />
<br><br />
<br />
<p><b>Figure 2. Over-expression of non-frameshifted and -1 frameshifted protein products from the PK401 construct.</b> Equivalent amounts of cells at 0, 1, 3, and 5 h after induction with IPTG (PK401 +IPTG) were analyzed by 12% SDS-PAGE. The same time samples from the uninduced culture were also analyzed (PK401 –IPTG). Black boxes indicate bands of increasing intensity that migrated with an approximate molecular weight of 60 kD and 30 kD, corresponding to the -1 frameshifted CFP-PK401-YFP fusion product and the non-frameshifted CFP product, respectively.</p><br><br />
<br />
<p><b>CFP and YFP Fluorescence from PK401 Overexpression</b></p><br />
<p>To confirm that the protein products seen in the overexpression of the PK401 construct corresponded to the expected fluorescent proteins, fluorescence spectra of the cell lysates from overexpression were measured. The cells from the overexpression were lysed using 1 mg/mL lysozyme and 12.5 mg/g sodium deoxycholate. DNase was added to degrade the DNA followed by centrifugation at 3000 x g for 30 min. The supernatant was collected and centrifuged further at 30 000 x g for 45 min. This supernatant was then used for fluorescence measurements.</p><br />
<br />
<p>The supernatants were diluted 100-fold in the buffer used for cell opening. Excitation and emission scans were measured for all samples in the CFP and YFP range in order to confirm the presence of both fluorescent proteins. CFP was excited at 430 nm, and emission was monitored from 445-650 nm (Fig. 3). YFP was excited at 510 nm, and emission was monitored from 525-650 nm (Fig. 4). These spectra were measured for the cell opening buffer and for the induced and uninduced samples of the cells that contained the PK401 construct, as well as the cells containing the control plasmid.</p><br />
<br />
<br><br />
<center>[[Image:ULeth2013_PK401-CFPexcitation.png|600px]]</center><br />
<br />
<p><b>Figure 3. Emission spectra after excitation at 430 nm.</b> Cell lysates from the cultures used for overexpression were excited near the excitation maximum of CFP (at 430 nm) and the emission was monitored from 445-650 nm. The spectra shown include the uninduced PK401 construct (blue), induced PK401 construct (yellow), uninduced control (dark green), induced control (lightgreen), and cell opening buffer (grey).</p><br><br />
<br />
<p>The cell lysates from the PK401 overexpression show the characteristic emission spectrum of CFP upon excitation at 430 nm. There is an additional shoulder in the spectrum near 527 nm, which is the emission maximum of YFP. When YFP was excited directly in the PK401 overexpression samples, the characteristic emission spectrum of YFP was seen, indicating that both CFP and YFP were expressed in these samples. The fluorescence from YFP could only result from a frameshift during expression of the PK401 transcript, indicating that the pseudoknot worked as expected to induce a -1 frameshift during translation. </p><br />
<br />
<br><br />
<center>[[Image:ULeth2013_PK401-YFPexcitation.png|600px]]</center><br />
<br />
<p><b>Figure 4. Emission spectra after excitation at 510 nm.</b> Cell lysates from the cultures used for overexpression were excited near the excitation maximum of YFP (at 510 nm) and the emission was monitored from 525-650 nm. The spectra shown include the uninduced PK401 construct (blue), induced PK401 construct (yellow), uninduced control (dark green), induced control (lightgreen), and cell opening buffer (grey). The characteristic emission maximum of YFP is at 527 nm, which was observed in the spectra from the PK401 samples.</p><br><br />
<br />
<p>Upon excitation of the cell lysates from the control plasmid cultures at 430 nm, a fluorescence signal of half the intensity of the PK401 cell lysates was observed with a plateau from approximately 500-520 nm. This did not correspond to the emission maximum of CFP or YFP, so the signal was likely due to other cellular components. Additionally, when the sample was excited at 510 nm, there was no detectable emission signal, indicating that YFP was not present in these samples. </p><br />
<br />
<br><br />
<center>[[Image:ULeth2013_PK401-FRET.png|600px]]</center><br />
<br />
<p><b>Figure 5. Maximum fluorescence intensity of overexpression cell lysates.</b> Intensities of cell lysates after excitation at 430 nm and monitoring at 475 nm (CFP fluorescence, blue bars) and excitation at 510 nm and emission at 527 nm (YFP fluorescence, yellow bars).</p><br><br />
<br />
<p>When the peak intensities from each sample were analyzed individually (Fig. 5), more prominent fluorescence measurements were observed for the PK401 cell lysates than for the control cell lysates. Excitation at 430 nm resulted in a fluorescence emission at 475 nm, which is a characteristic emission maximum of CFP, for the PK401 samples, but not for the control samples. Additionally, direct excitation and emission from YFP was seen for the PK401 cell lysates, but no fluorescence emission was seen for the control samples after excitation at 510 nm. This further indicates that the PK401 pseudoknot induced a -1 frameshift during translation of the PK401 transcript to produce both CFP and YFP.</p><br />
<br />
<h2>Generating a Pseudoknot Library</h2><br />
<p><b>Generating a Library of Pseudoknots with Variable Frameshifting Frequencies</b></p><br />
<br />
<p>To make better use of the frameshifting capability of the pseudoknot, we are also generating a randomized library of pseudoknot sequences that can be characterized for frameshifting frequency. It is our goal to create a library of pseudoknots that have a range of frameshifting frequencies to better facilitate broad application of this part. To do this, the PK401 sequence will be mutagenized using error-prone PCR to generate a library of primers that will be used to amplify the plasmid containing the original sequence. The newly generated plasmids contained mutated PK401 sequences will be transformed into E. coli cells, sequenced, and characterized for their specific frameshifting efficiency using a construct such as BBa_K1210000.</p><br><br />
<br />
<p><b>Optimizing Conditions for the Error-Prone PCR</b></p><br />
<br />
<p>To generate a library with high variability of sequence, conditions for the error-prone PCR had to first be optimized. We wanted to disrupt the PCR experiment enough to produce a large variety of primer sequences, but not too much so that the PCR was no longer successful. To do this, we used a temperature gradient to determine the annealing temperature that would best facilitate the amplification of the expected product. Figure 6 shows the resulting PCR products from the temperature gradient reactions, using temperatures from 41.9-62.1°C. The expected size of the PCR product is 93 bp, which was seen in the first three reaction samples. Various unspecific PCR products were seen in the remaining reactions samples. For this reason, the temperature chosen for the subsequent error-prone PCRs was 43.6°C, the temperature used to produce the PCR product in lane 3.</p><br />
<br />
<br><br />
<center>[[Image:ULeth2013iGEM tempgradient.png|400px]]</center><br><br />
<br />
<p><b>Figure 6. Determining the ideal temperature for error-prone PCR reactions.</b> PCR reactions were performed using various annealing temperatures from 41.9-62.1°C (lanes 1-12). The primers used in the reaction flank the PK401 pseudoknot, making the expected product size 93 bp. The temperature from the reaction in lane 3 (43.6°C) was selected for subsequent error-prone PCRs.</p><br><br />
<br />
<p>Next, we modulated a few of the typical PCR conditions to induce errors during the reaction. First, we used Taq polymerase, which is known to have higher error rates than higher fidelity polymerases commonly used in PCRs. Second, we added MnCl2 to the reaction to further hinder the accuracy of the polymerase. Finally, different ratios of deoxynucleotides were used in the reactions, either by increasing the dGTP concentration or by using a higher concentration of both dTTP and dCTP. A number of these conditions worked to produce the expected PCR product at 93 bp (Fig. 7, lanes 1-4, 6). The conditions tested in lanes 1-4 included MnCl2 at concentrations of 0-0.45 mM, and lane 6 tested the effect of increasing dGTP concentrations to 1 mM while the other dNTPs were at a concentration of 0.2 mM.</p><br />
<br />
<br><br />
<center>[[Image:ULeth2013iGEM_errorpronepcr.png|400px]]</center><br><br />
<br />
<p><b>Figure 7. Error-prone PCR of PK401 sequence.</b> PCR conditions were adjusted to induce errors in the PK401 sequence. Reactions were performed with the following modified condtions: lanes 1-6 used MnCl2 concentrations from 0-0.65 mM, lanes 4-6 used dGTP concentrations that were increased to 0.7-1.2 mM, lane 7 used increased concentrations of dTTP and dCTP, lane 8 used increased concentrations of dGTP only, and lane 9 used 0.65 mM MnCl2 only.</p><br><br />
<br />
<p>Now that we have determined the conditions for the error-prone PCR, we will perform a large-scale amplification so that we can purify the resulting PK401 primers by extracting it from an agarose gel. The primers will then be used in a high fidelity PCR reaction to amplify the entire plasmid, which will create the library of PK401 sequences. The final steps will be to transform the final PCR products into E. coli cells, screen them for frameshifting frequency using a similar construct as BBa_K1210000, and sequence the pseudoknot before submitting the parts to the registry to complete the library of frameshifting elements.</p></div>Dustinhttp://2013.igem.org/Team:Lethbridge/attributionsTeam:Lethbridge/attributions2013-09-28T02:56:00Z<p>Dustin: </p>
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<BLOCKQUOTE><br />
<br />
=<font color="black">Attributions=<br />
All planning and wet lab work was performed by the undergraduate members of our iGEM team.<br />
<br />
<h2>Contributions</h2><br />
<p><b>Dallas Thomas</b> (University of Lethbridge, Lethbridge Agriculture and Agri-Food Canada): For developing the software code to overlap protein coding sequences</p><br />
<p><b>Dipankar Goyal</b> (University of Lethbridge): For assistance with our Wiki design</p><br />
<p><b>Kelsey Kristensen</b> (University of Lethbridge): For taking our team photos, please visit her [http://seventhousandplus.weebly.com/index.html website]</p><br />
<p>The Department of Chemistry and Biochemistry, the Alberta RNA Research and Training Institute, the Wieden Lab, the Kothe Lab, and the Selinger Lab at the University of Lethbridge for providing us with equipment and laboratory space.</p><br />
<br />
<h2>Sponsors</h2><br />
<p>We would also like to acknowledge our sponsors for the following support of the 2013 Lethbridge iGEM team.</p><br><br />
<br />
<html><br />
<p><center><image src="https://static.igem.org/mediawiki/2013/0/00/ULeth2013_Sponsors_-_Platinum.png"; width="400px"; height="200px" /></center></p><br><br />
<br />
<p><center><image src="https://static.igem.org/mediawiki/2013/1/1e/ULeth2013_Sponsors_-_Silver.png"; width="400px"; height="200px" /></center></p><br><br />
<br />
<p><center><image src="https://static.igem.org/mediawiki/2013/4/4c/ULeth2013_Sponsors_-_Bronze.png"; width="600px"; height="200px" /></center></p><br />
</html><br />
<br />
<br />
<p><b>1. Platinum - $5000+ or gift in kind</b></p><br />
<p>Logo on front of team shirts, large logo on scientific poster, large logo on team wiki and verbal recognition during team project presentations/media interviews.</p><br />
<p><b>2. Gold - $2000-$4999 or gift in kind</b></p><br />
<p>Logo on team shirts, medium logo on scientific poster, medium logo on team wiki and written recognition at end of team project presentations.</p><br />
<p><b>3. Silver - $1000-$1999 or gift in kind</b></p><br />
<p>Medium logo on scientific poster, medium logo on team wiki and written recognition at end of team project presentations.</p><br />
<p><b>4. Bronze - <$999 or gift in kind</b></p><br />
<p>Small logo on scientific poster, small logo on team wiki and written recognition at end of team project presentations.</p></div>Dustinhttp://2013.igem.org/Team:Lethbridge/attributionsTeam:Lethbridge/attributions2013-09-28T02:55:45Z<p>Dustin: </p>
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<div>{{TeamLethbridgeHead}}<br />
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<br />
</html><br />
<BLOCKQUOTE><br />
<br />
=<font color="black">Attributions=<br />
All planning and wet lab work was performed by the undergraduate members of our iGEM team.<br />
<br />
<h2>Contributions</h2><br />
<p><b>Dallas Thomas</b> (University of Lethbridge, Lethbridge Agriculture and Agri-Food Canada): For developing the software code to overlap protein coding sequences</p><br />
<p><b>Dipankar Goyal</b> (University of Lethbridge): For assistance with our Wiki design</p><br />
<p><b>Kelsey Kristensen</b> (University of Lethbridge): For taking our team photos, please visit her [http://seventhousandplus.weebly.com/index.html website]</p><br />
<p>The Department of Chemistry and Biochemistry, the Alberta RNA Research and Training Institute, the Wieden Lab, the Kothe Lab, and the Selinger Lab at the University of Lethbridge for providing us with equipment and laboratory space.</p><br />
<br />
<h2>Sponsors</h2><br />
<p>We would also like to acknowledge our sponsors for the following support of the 2013 Lethbridge iGEM team.</p><br><br />
<br />
<html><br />
<p><center><image src="https://static.igem.org/mediawiki/2013/0/00/ULeth2013_Sponsors_-_Platinum.png"; width="400px"; height="200px" /></center></p><br><br />
<br />
<p><center><image src="https://static.igem.org/mediawiki/2013/1/1e/ULeth2013_Sponsors_-_Silver.png"; width="400px"; height="200px" /></center></p><br><br />
<br />
<p><center><image src="https://static.igem.org/mediawiki/2013/4/4c/ULeth2013_Sponsors_-_Bronze.png"; width="600px"; height="200px" /></center></p><br />
</html><br />
<br />
<br />
<p><b>1. Platinum - $5000+ or gift in kind</b></p><br />
<p>Logo on front of team shirts, large logo on scientific poster, large logo on team wiki and verbal recognition during team project presentations/media interviews.</p><br />
<p><b>2. Gold - $2000-$4999 or gift in kind</b></p><br />
<p>Logo on team shirts, medium logo on scientific poster, medium logo on team wiki and written recognition at end of team project presentations.</p><br />
<p><b>3. Silver - $1000-$1999 or gift in kind</b></p><br />
<p>Medium logo on scientific poster, medium logo on team wiki and written recognition at end of team project presentations.</p><br />
<p><b>4. Bronze - <$999 or gift in kind</b></p><br />
<p>Small logo on scientific poster, small logo on team wiki and written recognition at end of team project presentations.</p></div>Dustinhttp://2013.igem.org/Team:Lethbridge/attributionsTeam:Lethbridge/attributions2013-09-28T02:54:21Z<p>Dustin: </p>
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<br />
</html><br />
<BLOCKQUOTE><br />
<br />
=<font color="black">Attributions=<br />
All planning and wet lab work was performed by the undergraduate members of our iGEM team.<br />
<br />
<h2>Contributions</h2><br />
<p><b>Dallas Thomas</b> (University of Lethbridge, Lethbridge Agriculture and Agri-Food Canada): For developing the software code to overlap protein coding sequences</p><br />
<p><b>Dipankar Goyal</b> (University of Lethbridge): For assistance with our Wiki design</p><br />
<p><b>Kelsey Kristensen</b> (University of Lethbridge): For taking our team photos, please visit her [http://seventhousandplus.weebly.com/index.html website]</p><br />
<p>The Department of Chemistry and Biochemistry, the Alberta RNA Research and Training Institute, the Wieden Lab, the Kothe Lab, and the Selinger Lab at the University of Lethbridge for providing us with equipment and laboratory space.</p><br />
<br />
<h2>Sponsors</h2><br />
<p>We would also like to acknowledge our sponsors for the following support of the 2013 Lethbridge iGEM team.</p><br><br />
<br />
<html><br />
<p><center><image src="https://static.igem.org/mediawiki/2013/0/00/ULeth2013_Sponsors_-_Platinum.png"; width="400px"; height="200px" /></center></p><br><br />
<br />
<p><center><image src="https://static.igem.org/mediawiki/2013/1/1e/ULeth2013_Sponsors_-_Silver.png"; width="400px"; height="200px" /></center></p><br><br />
<br />
<p><center><image src="https://static.igem.org/mediawiki/2013/4/4c/ULeth2013_Sponsors_-_Bronze.png"; width="600px"; height="200px" /></center></p><br />
</html><br />
<br />
<br />
<p><b>1. Platinum - $5000+ or gift in kind</b></p><br />
<p>Logo on front of team shirts, large logo on scientific poster, large logo on team wiki and verbal recognition during team project presentations/media interviews.</p><br />
<p><b>2. Gold - $2000-$4999 or gift in kind</b></p><br />
<p>Logo on team shirts, medium logo on scientific poster, medium logo on team wiki and written recognition at end of team project presentations.</p><br />
<p><b>3. Silver - $1000-$1999 or gift in kind</b></p><br />
<p>Medium logo on scientific poster, medium logo on team wiki and written recognition at end of team project presentations.</p><br />
<p><b>4. Bronze - <$999 or gift in kind</b></p><br />
<p>Small logo on scientific poster, small logo on team wiki and written recognition at end of team project presentations.</p></div>Dustinhttp://2013.igem.org/Team:Lethbridge/attributionsTeam:Lethbridge/attributions2013-09-28T02:53:42Z<p>Dustin: </p>
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<div>{{TeamLethbridgeHead}}<br />
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<br />
</html><br />
<BLOCKQUOTE><br />
<br />
=<font color="black">Attributions=<br />
All planning and wet lab work was performed by the undergraduate members of our iGEM team.<br />
<br />
<h2>Contributions</h2><br />
<p><b>Dallas Thomas</b> (University of Lethbridge, Lethbridge Agriculture and Agri-Food Canada): For developing the software code to overlap protein coding sequences</p><br />
<p><b>Dipankar Goyal</b> (University of Lethbridge): For assistance with our Wiki design</p><br />
<p><b>Kelsey Kristensen</b> (University of Lethbridge): For taking our team photos, please visit her [http://seventhousandplus.weebly.com/index.html website]</p><br />
<p>The Department of Chemistry and Biochemistry, the Alberta RNA Research and Training Institute, the Wieden Lab, the Kothe Lab, and the Selinger Lab at the University of Lethbridge for providing us with equipment and laboratory space.</p><br />
<br />
<h2>Sponsors</h2><br />
<p>We would also like to acknowledge our sponsors for the following support of the 2013 Lethbridge iGEM team.</p><br><br />
<br />
<html><br />
<p><center><image src="https://static.igem.org/mediawiki/2013/0/00/ULeth2013_Sponsors_-_Platinum.png"; width="400px"; height="200px" /></center></p><br><br />
<br />
<p><center><image src="https://static.igem.org/mediawiki/2013/1/1e/ULeth2013_Sponsors_-_Silver.png"; width="400px"; height="200px" /></center></p><br><br />
<br />
<p><center><image src="https://static.igem.org/mediawiki/2013/4/4c/ULeth2013_Sponsors_-_Bronze.png"; width="600px"; height="200px" /></center></p><br />
</html><br />
<br />
<br />
<p><b>1. Platinum - $5000+ or gift in kind</b></p><br />
<p>Logo on front of team shirts, large logo on scientific poster, large logo on team wiki and verbal recognition during team project presentations/media interviews.</p><br />
<p><b>2. Gold - $2000-$4999 or gift in kind</b></p><br />
<p>Logo on team shirts, medium logo on scientific poster, medium logo on team wiki and written recognition at end of team project presentations.</p><br />
<p><b>3. Silver - $1000-$1999 or gift in kind</b></p><br />
<p>Medium logo on scientific poster, medium logo on team wiki and written recognition at end of team project presentations.</p><br />
<p><b>4. Bronze - <$999 or gift in kind</b></p><br />
<p>Small logo on scientific poster, small logo on team wiki and written recognition at end of team project presentations.</p></div>Dustinhttp://2013.igem.org/File:Attributions_uofl_2013.pngFile:Attributions uofl 2013.png2013-09-28T02:50:28Z<p>Dustin: </p>
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<div></div>Dustinhttp://2013.igem.org/Team:LethbridgeTeam:Lethbridge2013-09-28T02:49:26Z<p>Dustin: </p>
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<center><a href="https://2013.igem.org/Team:Lethbridge/project"><image src="https://static.igem.org/mediawiki/2013/0/05/ULeth2013iGEM_Mainpage_PKFig.png" width="600px" height="400px"/></a></center><br />
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<br />
<h2>Project Overview</h2><br />
<p style="color:black">The current growth in synthetic biology research promises more complex and useful engineered systems. However, increased complexity often requires more genetic material that can be difficult to introduce into organisms. We propose the development of a new library of regulatory gene expression elements that allow for compression of multiple coding sequences into a smaller amount of genetic space. Using a pseudoknot RNA structural motif, commonly used by viruses to minimize their genome size, we will show the utility of dual-coding gene sequences to give useful protein products whose expression can be regulated by the pseudoknot’s ability to induce ribosomal frameshifting. A software tool will also be used to zip multiple coding sequences into different reading frames. Ultimately, this library of standardized parts will be available for use in a variety of engineered systems requiring minimal coding space and multiple protein expression.</p><br><br />
<br />
<b style="color:black"><u>WHAT?</u></b><br />
<ul style="color:black"><li>Our project is directed towards standardizing pseudoknots to make a new class of parts available to the synthetic biology community</li></ul> <br />
<br />
<br><br />
<b style="color:black"><u>WHY?</u></b><br />
<ul style="color:black"><li>As the field of synthetic biology grows, so should its toolset. By introducing a standardized method of implementing programmed ribosomal frameshifts in synthetic gene networks, we could not only enable others to reduce plasmid size and regulate operon expression, but also enable them to come up with new, exciting applications</li></ul><br />
<br />
<br><br />
<b style="color:black"><u>HOW?</b></u><br />
<ul style="color:black"><li>We have brought pseudoknots to the iGEM community by: <br />
<ul><li>Characterizing their function in a biobrick system</li><br />
<li>Designing software that enables others to dual code proteins</li><br />
<li>Ensuring that the release of these tools to the wider public does not pose a significant risk to the rest of the world</li></li></ul></ul><br><br />
<br />
<br />
<h2>Sponsors</h2><br />
<br><br />
<p><center><image src="https://static.igem.org/mediawiki/2013/0/00/ULeth2013_Sponsors_-_Platinum.png"; width="200px"; height="100px" />&nbsp;&nbsp;&nbsp;&nbsp;<br />
<br />
<image src="https://static.igem.org/mediawiki/2013/1/1e/ULeth2013_Sponsors_-_Silver.png"; width="200px"; height="100px" />&nbsp;&nbsp;&nbsp;&nbsp;<br />
<br />
<image src="https://static.igem.org/mediawiki/2013/4/4c/ULeth2013_Sponsors_-_Bronze.png"; width="300px"; height="100px" /></center></p>&nbsp;&nbsp;&nbsp;&nbsp;<br />
</html></div>Dustinhttp://2013.igem.org/Team:Lethbridge/safetyTeam:Lethbridge/safety2013-09-28T02:42:08Z<p>Dustin: </p>
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<h3><b>Basic Safety Questions for Lethbridge iGEM 2013</b></h3><br />
<ol><li>a. Please describe the chassis organism(s) you will be using for this project. If you will be using more than one chassis organism, provide information on each of them: <br />
<ul><li>Species: Escherichia coli </li><br />
<li>Strain no/name: DHα and BL21 DE3 </li><br />
<li>Risk Group: 1</li><br />
<li>Risk group source link: www.cmu.edu/osp/regulatory-compliance/rDNA_Documents/</li><br />
<li>Disease risk to humans? If so, which disease? Very low risk, not associated with causing disease in humans</li></ul><br />
<li> Highest Risk Group listed: 1</li><br />
<br />
<li> List and describe all new or modified coding regions you will be using in your project. (If you use parts from the 2013 iGEM Distribution without modifying them, you do not need to list those parts.)<br />
<ol><li> Part number: BBa_K1210000<br />
<ul><li>Where did you get the physical DNA for this part (which lab, synthesis company, etc) : Synthesized, GENEWIZ</li><br />
<li>What species does this part originally come from?: Virus</li><br />
<li>What is the Risk Group of the species?: 2</li><br />
<li>What is the function of this part, in its parent species?: Programmed -1 ribosomal frameshifting</li></li></ul><br />
<br />
<li>Part number: BBa_K1210001<br />
<ul><li>Where did you get the physical DNA for this part (which lab, synthesis company, etc) : Synthesized, GENEWIZ</li><br />
<li>What species does this part originally come from?: IBV- Infectious bronchitis virus</li><br />
<li>What is the Risk Group of the species?: 2</li><br />
<li>What is the function of this part, in its parent species?: Programmed -1 ribosomal frameshifting</li></li></ul></ol><br />
<br />
<li> Do the biological materials used in your lab work pose any of the following risks? Please describe.<br />
<ul><li>a. Risks to the safety and health of team members or others working in the lab?<br />
<p>There is very minimal risk to safety as we only work with laboratory strains of Escherichia coli, and all team members have previously undergone laboratory safety training. </p></li><br />
<li>b. Risks to the safety and health of the general public, if released by design or by accident?<br />
<p>None of the parts that we have made raise any safety issues.</p></li><br />
<li>c. Risks to the environment, if released by design or by accident?<br />
<p>There are no direct risks to the environment if the parts are released by design or by accident. The organisms containing these parts are not currently designed for environmental applications. Therefore, risk of environmental contamination is very low. </p></li><br />
<li>d. Risks to security through malicious misuse by individuals, groups, or countries?<br />
<p>Our project only poses safety issues if it was to be used to aid in the expression of dangerous sequences making harmful proteins. However, we intend to create software to help genes synthesis companies detect harmful sequences hidden in another frame, preventing the synthesis of dangerous sequences. </p></li></ul></li><br />
<br />
<li> If your project moved from a small-scale lab study to become widely used as a commercial/industrial product, what new risks might arise? (Consider the different categories of risks that are listed in parts a-d of the previous question.) Also, what risks might arise if the knowledge you generate or the methods you develop became widely available? (Note: This is meant to be a somewhat open-ended discussion question.)<br />
<p>The parts do not cause any harm for the lab worker, public, or the environment. If individuals make use of the frameshifting parts, with the intent of hiding dangerous sequences, synthesis companies may have to modify or develop new screening methods for detecting these dangerous sequences.</p></li><br />
<li> Does your project include any design features to address safety risks? (For example: kill switches, auxotrophic chassis, etc.) Note that including such features is not mandatory to participate in iGEM, but many groups choose to include them.<br />
<p>Our parts do not include any design features to address safety risks. However, we are creating software which will help gene synthesis companies prevent the misuse of our parts for malicious intent. </p></li><br />
<li> What safety training have you received (or plan to receive in the future)? Provide a brief description, and a link to your institution’s safety training requirements, if available.<br />
<p>All iGEM team members have been fully trained in WHMIS as well as an introduction to biosafety basics. http://www.uleth.ca/hum/riskandsafetyservices/pages/Laboratory%20Forms<p></li><br />
<li> Under what biosafety provisions will / do you work?<br />
<ul><li>a. Please provide a link to your institution biosafety guidelines<br />
<p>http://www.uleth.ca/hum/riskandsafetyservices/</p></li><br />
<li>b. Does your institution have an Institutional Biosafety Committee, or an equivalent group? If yes, have you discussed your project with them? Describe any concerns they raised with your project, and any changes you made to your project plan based on their review.<br />
<p>The University of Lethbridge Risk and Safety Services department has appointed a committee for biosafety. This committee ensures that biological materials are used safely on campus. We have discussed our project with them and they forsee no problems with the Lethbridge 2013 iGEM project. </p></li><br />
<li>c. Does your country have national biosafety regulations or guidelines? If so, please provide a link to these regulations or guidelines if possible.<br />
<p>Yes, http://canadianbiosafetystandards.collaboration.gc.ca/</p></li><br />
<li>d. According to the WHO Biosafety Manual, what is the BioSafety Level rating of your lab? (Check the summary table on page 3, and the fuller description that starts on page 9.) If your lab does not fit neatly into category 1, 2, 3, or 4, please describe its safety features [see 2013.igem.org/Safety for help].<br />
<p>BSL1, we are using a RNA sequence that is from a risk group 2 organism, however, we do not require the use of the actual organism to utilize this element. </p></li><br />
<li>e. What is the Risk Group of your chassis organism(s), as you stated in question 1? If it does not match the BSL rating of your laboratory, please explain what additional safety measures you are taking.<br />
<p>Risk Group 1</p></li></ol><br />
<br />
<h3><b>Safety Form 2 Questions for Lethbridge iGEM 2013</b></h3><br />
<ol><li>Organism name and strain name or number: Infectious bronchitis virus</li><br />
<li>Organism Risk Group: Group 2</li><br />
<li>If you are using this organism as a chassis, write "chassis". If you are using a genetic part from <br />
the organism, give the name of the part and a brief description of what it does and why you are <br />
using it.<br />
<p>BBa_K120001: includes the sequence for a pseudoknot secondary structural RNA element that can cause programmed -1 frameshifting during translation. We plan to characterize the frameshifting frequency of this pseudoknot and create a library of frameshifting elements.</p><br />
<br />
<p>BBa_K120000: includes reporter genes upstream and downstream of the pseudoknot, as a construct for testing the frameshift frequency.</p><br />
</li><br />
<li>How did you physically acquire the organism or part? <br />
<p>The part was synthesized by GENEWIZ.</p></li><br />
<br />
<li>What potential safety/health risks to team members, other people at your institution, or the <br />
general public could arise from your use of this organism/part?<br />
<p>The part itself is innocuous and not infectious. Since it is just a short sequence of DNA that, when transcribed into RNA, folds into a specific secondary structure, it does not pose a safety threat to our team members, the university, or the general public.</p></li><br />
<br />
<li>What measures do you intend to take to ensure that your project is safe for team members, <br />
other people at your institution, and the general public?<br />
<p>Since the part itself does not pose a safety threat, standard laboratory safety procedures will be followed, including ensuring that the part or the chassis containing the part does not leave the laboratory space. Proper personal protective equipment will be worn at all times when working with the part and all team members are fully trained in WHMIS and biosafety before being allowed to work in the laboratory.</p></li><br />
<br />
<li> If you are using only a part from the organism, and you believe the part by itself is not <br />
dangerous, explain why you believe it is not dangerous.<br />
<p>Use of the part does not require handling of the virus from which it comes. Therefore, the part itself is not dangerous. Pseudoknots are common RNA elements found in organisms besides viruses, and are not dangerous outside of the context of their host.</p></li><br />
<br />
<li>Why do you need to use this organism/part? Is there an organism/part from a less dangerous <br />
Risk Group that would accomplish the same purpose?<br />
<p>We use standard E. coli strains as our chassis for the expression of our constructs. The pseudoknot from infectious bronchitis virus (IBV) has been shown to cause frameshifting in E. coli cells. Therefore, the sequence of the IBV pseudoknot was used. Most pseudoknot sequences that have been characterized for frameshifting activity originate from viruses, so the pseudoknot sequence could not be retrieved from a Risk Level 1 organism.</p></li><br />
<li>Is the organism/part listed under the Australia Group guidelines, or otherwise restricted for <br />
transport? If so, how will your team ship this part to iGEM and the Jamborees?<br />
<p>The organism that the part originates from is not listed under the Australia Group guidelines, and therefore no additional measures are needed for transport of the BioBrick part.</p></li><br />
<br />
<li>Please describe the BioSafety Level of the lab in which the team works, or description of <br />
safety features of lab (Refer to Basic Safety form, question 8. d.). If you are using organisms with <br />
a BSL level greater than you lab, please explain any additional safety precautions you are taking. <br />
<p>The lab in which the team works is rated as a BioSafety Level 1 laboratory. Prior to starting lab work, all members must complete WHMIS and Biosafety Training. Since use of the part does not require handling of the Risk Level 2 organism that it comes from, no additional safety precautions are necessary.</p></li></ul><br />
<br />
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</html></div>Dustinhttp://2013.igem.org/Team:Lethbridge/safetyTeam:Lethbridge/safety2013-09-28T02:41:36Z<p>Dustin: </p>
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<h3><b>Basic Safety Questions for Lethbridge iGEM 2013</b></h3><br />
<ol><li>a. Please describe the chassis organism(s) you will be using for this project. If you will be using more than one chassis organism, provide information on each of them: <br />
<ul><li>Species: Escherichia coli </li><br />
<li>Strain no/name: DHα and BL21 DE3 </li><br />
<li>Risk Group: 1</li><br />
<li>Risk group source link: www.cmu.edu/osp/regulatory-compliance/rDNA_Documents/</li><br />
<li>Disease risk to humans? If so, which disease? Very low risk, not associated with causing disease in humans</li></ul><br />
<li> Highest Risk Group listed: 1</li><br />
<br />
<li> List and describe all new or modified coding regions you will be using in your project. (If you use parts from the 2013 iGEM Distribution without modifying them, you do not need to list those parts.)<br />
<ol><li> Part number: BBa_K1210000<br />
<ul><li>Where did you get the physical DNA for this part (which lab, synthesis company, etc) : Synthesized, GENEWIZ</li><br />
<li>What species does this part originally come from?: Virus</li><br />
<li>What is the Risk Group of the species?: 2</li><br />
<li>What is the function of this part, in its parent species?: Programmed -1 ribosomal frameshifting</li></li></ul><br />
<br />
<li>Part number: BBa_K1210001<br />
<ul><li>Where did you get the physical DNA for this part (which lab, synthesis company, etc) : Synthesized, GENEWIZ</li><br />
<li>What species does this part originally come from?: IBV- Infectious bronchitis virus</li><br />
<li>What is the Risk Group of the species?: 2</li><br />
<li>What is the function of this part, in its parent species?: Programmed -1 ribosomal frameshifting</li></li></ul></ol><br />
<br />
<li> Do the biological materials used in your lab work pose any of the following risks? Please describe.<br />
<ul><li>a. Risks to the safety and health of team members or others working in the lab?<br />
<p>There is very minimal risk to safety as we only work with laboratory strains of Escherichia coli, and all team members have previously undergone laboratory safety training. </p></li><br />
<li>b. Risks to the safety and health of the general public, if released by design or by accident?<br />
<p>None of the parts that we have made raise any safety issues.</p></li><br />
<li>c. Risks to the environment, if released by design or by accident?<br />
<p>There are no direct risks to the environment if the parts are released by design or by accident. The organisms containing these parts are not currently designed for environmental applications. Therefore, risk of environmental contamination is very low. </p></li><br />
<li>d. Risks to security through malicious misuse by individuals, groups, or countries?<br />
<p>Our project only poses safety issues if it was to be used to aid in the expression of dangerous sequences making harmful proteins. However, we intend to create software to help genes synthesis companies detect harmful sequences hidden in another frame, preventing the synthesis of dangerous sequences. </p></li></ul></li><br />
<br />
<li> If your project moved from a small-scale lab study to become widely used as a commercial/industrial product, what new risks might arise? (Consider the different categories of risks that are listed in parts a-d of the previous question.) Also, what risks might arise if the knowledge you generate or the methods you develop became widely available? (Note: This is meant to be a somewhat open-ended discussion question.)<br />
<p>The parts do not cause any harm for the lab worker, public, or the environment. If individuals make use of the frameshifting parts, with the intent of hiding dangerous sequences, synthesis companies may have to modify or develop new screening methods for detecting these dangerous sequences.</p></li><br />
<li> Does your project include any design features to address safety risks? (For example: kill switches, auxotrophic chassis, etc.) Note that including such features is not mandatory to participate in iGEM, but many groups choose to include them.<br />
<p>Our parts do not include any design features to address safety risks. However, we are creating software which will help gene synthesis companies prevent the misuse of our parts for malicious intent. </p></li><br />
<li> What safety training have you received (or plan to receive in the future)? Provide a brief description, and a link to your institution’s safety training requirements, if available.<br />
<p>All iGEM team members have been fully trained in WHMIS as well as an introduction to biosafety basics. http://www.uleth.ca/hum/riskandsafetyservices/pages/Laboratory%20Forms<p></li><br />
<li> Under what biosafety provisions will / do you work?<br />
<ul><li>a. Please provide a link to your institution biosafety guidelines<br />
<p>http://www.uleth.ca/hum/riskandsafetyservices/</p></li><br />
<li>b. Does your institution have an Institutional Biosafety Committee, or an equivalent group? If yes, have you discussed your project with them? Describe any concerns they raised with your project, and any changes you made to your project plan based on their review.<br />
<p>The University of Lethbridge Risk and Safety Services department has appointed a committee for biosafety. This committee ensures that biological materials are used safely on campus. We have discussed our project with them and they forsee no problems with the Lethbridge 2013 iGEM project. </p></li><br />
<li>c. Does your country have national biosafety regulations or guidelines? If so, please provide a link to these regulations or guidelines if possible.<br />
<p>Yes, http://canadianbiosafetystandards.collaboration.gc.ca/</p></li><br />
<li>d. According to the WHO Biosafety Manual, what is the BioSafety Level rating of your lab? (Check the summary table on page 3, and the fuller description that starts on page 9.) If your lab does not fit neatly into category 1, 2, 3, or 4, please describe its safety features [see 2013.igem.org/Safety for help].<br />
<p>BSL1, we are using a RNA sequence that is from a risk group 2 organism, however, we do not require the use of the actual organism to utilize this element. </p></li><br />
<li>e. What is the Risk Group of your chassis organism(s), as you stated in question 1? If it does not match the BSL rating of your laboratory, please explain what additional safety measures you are taking.<br />
<p>Risk Group 1</p></li></ol><br />
<br />
<h3><b>Safety Form 2 Questions for Lethbridge iGEM 2013</b></h3><br />
<ol><li>Organism name and strain name or number: Infectious bronchitis virus</li><br />
<li>Organism Risk Group: Group 2</li><br />
<li>If you are using this organism as a chassis, write "chassis". If you are using a genetic part from <br />
the organism, give the name of the part and a brief description of what it does and why you are <br />
using it.<br />
<p>BBa_K120001: includes the sequence for a pseudoknot secondary structural RNA element that can cause programmed -1 frameshifting during translation. We plan to characterize the frameshifting frequency of this pseudoknot and create a library of frameshifting elements.</p><br />
<br />
<p>BBa_K120000: includes reporter genes upstream and downstream of the pseudoknot, as a construct for testing the frameshift frequency.</p><br />
</li><br />
<li>How did you physically acquire the organism or part? <br />
<p>The part was synthesized by GENEWIZ.</p></li><br />
<br />
<li>What potential safety/health risks to team members, other people at your institution, or the <br />
general public could arise from your use of this organism/part?<br />
<p>The part itself is innocuous and not infectious. Since it is just a short sequence of DNA that, when transcribed into RNA, folds into a specific secondary structure, it does not pose a safety threat to our team members, the university, or the general public.</p></li><br />
<br />
<li>What measures do you intend to take to ensure that your project is safe for team members, <br />
other people at your institution, and the general public?<br />
<p>Since the part itself does not pose a safety threat, standard laboratory safety procedures will be followed, including ensuring that the part or the chassis containing the part does not leave the laboratory space. Proper personal protective equipment will be worn at all times when working with the part and all team members are fully trained in WHMIS and biosafety before being allowed to work in the laboratory.</p></li><br />
<br />
<li> If you are using only a part from the organism, and you believe the part by itself is not <br />
dangerous, explain why you believe it is not dangerous.<br />
<p>Use of the part does not require handling of the virus from which it comes. Therefore, the part itself is not dangerous. Pseudoknots are common RNA elements found in organisms besides viruses, and are not dangerous outside of the context of their host.</p></li><br />
<br />
<li>Why do you need to use this organism/part? Is there an organism/part from a less dangerous <br />
Risk Group that would accomplish the same purpose?<br />
<p>We use standard E. coli strains as our chassis for the expression of our constructs. The pseudoknot from infectious bronchitis virus (IBV) has been shown to cause frameshifting in E. coli cells. Therefore, the sequence of the IBV pseudoknot was used. Most pseudoknot sequences that have been characterized for frameshifting activity originate from viruses, so the pseudoknot sequence could not be retrieved from a Risk Level 1 organism.</p></li><br />
<li>Is the organism/part listed under the Australia Group guidelines, or otherwise restricted for <br />
transport? If so, how will your team ship this part to iGEM and the Jamborees?<br />
<p>The organism that the part originates from is not listed under the Australia Group guidelines, and therefore no additional measures are needed for transport of the BioBrick part.</p></li><br />
<br />
<li>Please describe the BioSafety Level of the lab in which the team works, or description of <br />
safety features of lab (Refer to Basic Safety form, question 8. d.). If you are using organisms with <br />
a BSL level greater than you lab, please explain any additional safety precautions you are taking. <br />
<p>The lab in which the team works is rated as a BioSafety Level 1 laboratory. Prior to starting lab work, all members must complete WHMIS and Biosafety Training. Since use of the part does not require handling of the Risk Level 2 organism that it comes from, no additional safety precautions are necessary.</p></li></ul><br />
<br />
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</html></div>Dustinhttp://2013.igem.org/File:Safety_uofl_2013.pngFile:Safety uofl 2013.png2013-09-28T02:39:54Z<p>Dustin: </p>
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<div></div>Dustinhttp://2013.igem.org/Team:Lethbridge/safetyTeam:Lethbridge/safety2013-09-28T02:36:26Z<p>Dustin: </p>
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<h3><b>Basic Safety Questions for Lethbridge iGEM 2013</b></h3><br />
<ol><li>a. Please describe the chassis organism(s) you will be using for this project. If you will be using more than one chassis organism, provide information on each of them: <br />
<ul><li>Species: Escherichia coli </li><br />
<li>Strain no/name: DHα and BL21 DE3 </li><br />
<li>Risk Group: 1</li><br />
<li>Risk group source link: www.cmu.edu/osp/regulatory-compliance/rDNA_Documents/</li><br />
<li>Disease risk to humans? If so, which disease? Very low risk, not associated with causing disease in humans</li></ul><br />
<li> Highest Risk Group listed: 1</li><br />
<br />
<li> List and describe all new or modified coding regions you will be using in your project. (If you use parts from the 2013 iGEM Distribution without modifying them, you do not need to list those parts.)<br />
<ol><li> Part number: BBa_K1210000<br />
<ul><li>Where did you get the physical DNA for this part (which lab, synthesis company, etc) : Synthesized, GENEWIZ</li><br />
<li>What species does this part originally come from?: Virus</li><br />
<li>What is the Risk Group of the species?: 2</li><br />
<li>What is the function of this part, in its parent species?: Programmed -1 ribosomal frameshifting</li></li></ul><br />
<br />
<li>Part number: BBa_K1210001<br />
<ul><li>Where did you get the physical DNA for this part (which lab, synthesis company, etc) : Synthesized, GENEWIZ</li><br />
<li>What species does this part originally come from?: IBV- Infectious bronchitis virus</li><br />
<li>What is the Risk Group of the species?: 2</li><br />
<li>What is the function of this part, in its parent species?: Programmed -1 ribosomal frameshifting</li></li></ul></ol><br />
<br />
<li> Do the biological materials used in your lab work pose any of the following risks? Please describe.<br />
<ul><li>a. Risks to the safety and health of team members or others working in the lab?<br />
<p>There is very minimal risk to safety as we only work with laboratory strains of Escherichia coli, and all team members have previously undergone laboratory safety training. </p></li><br />
<li>b. Risks to the safety and health of the general public, if released by design or by accident?<br />
<p>None of the parts that we have made raise any safety issues.</p></li><br />
<li>c. Risks to the environment, if released by design or by accident?<br />
<p>There are no direct risks to the environment if the parts are released by design or by accident. The organisms containing these parts are not currently designed for environmental applications. Therefore, risk of environmental contamination is very low. </p></li><br />
<li>d. Risks to security through malicious misuse by individuals, groups, or countries?<br />
<p>Our project only poses safety issues if it was to be used to aid in the expression of dangerous sequences making harmful proteins. However, we intend to create software to help genes synthesis companies detect harmful sequences hidden in another frame, preventing the synthesis of dangerous sequences. </p></li></ul></li><br />
<br />
<li> If your project moved from a small-scale lab study to become widely used as a commercial/industrial product, what new risks might arise? (Consider the different categories of risks that are listed in parts a-d of the previous question.) Also, what risks might arise if the knowledge you generate or the methods you develop became widely available? (Note: This is meant to be a somewhat open-ended discussion question.)<br />
<p>The parts do not cause any harm for the lab worker, public, or the environment. If individuals make use of the frameshifting parts, with the intent of hiding dangerous sequences, synthesis companies may have to modify or develop new screening methods for detecting these dangerous sequences.</p></li><br />
<li> Does your project include any design features to address safety risks? (For example: kill switches, auxotrophic chassis, etc.) Note that including such features is not mandatory to participate in iGEM, but many groups choose to include them.<br />
<p>Our parts do not include any design features to address safety risks. However, we are creating software which will help gene synthesis companies prevent the misuse of our parts for malicious intent. </p></li><br />
<li> What safety training have you received (or plan to receive in the future)? Provide a brief description, and a link to your institution’s safety training requirements, if available.<br />
<p>All iGEM team members have been fully trained in WHMIS as well as an introduction to biosafety basics. http://www.uleth.ca/hum/riskandsafetyservices/pages/Laboratory%20Forms<p></li><br />
<li> Under what biosafety provisions will / do you work?<br />
<ul><li>a. Please provide a link to your institution biosafety guidelines<br />
<p>http://www.uleth.ca/hum/riskandsafetyservices/</p></li><br />
<li>b. Does your institution have an Institutional Biosafety Committee, or an equivalent group? If yes, have you discussed your project with them? Describe any concerns they raised with your project, and any changes you made to your project plan based on their review.<br />
<p>The University of Lethbridge Risk and Safety Services department has appointed a committee for biosafety. This committee ensures that biological materials are used safely on campus. We have discussed our project with them and they forsee no problems with the Lethbridge 2013 iGEM project. </p></li><br />
<li>c. Does your country have national biosafety regulations or guidelines? If so, please provide a link to these regulations or guidelines if possible.<br />
<p>Yes, http://canadianbiosafetystandards.collaboration.gc.ca/</p></li><br />
<li>d. According to the WHO Biosafety Manual, what is the BioSafety Level rating of your lab? (Check the summary table on page 3, and the fuller description that starts on page 9.) If your lab does not fit neatly into category 1, 2, 3, or 4, please describe its safety features [see 2013.igem.org/Safety for help].<br />
<p>BSL1, we are using a RNA sequence that is from a risk group 2 organism, however, we do not require the use of the actual organism to utilize this element. </p></li><br />
<li>e. What is the Risk Group of your chassis organism(s), as you stated in question 1? If it does not match the BSL rating of your laboratory, please explain what additional safety measures you are taking.<br />
<p>Risk Group 1</p></li></ol><br />
<br />
<h3><b>Safety Form 2 Questions for Lethbridge iGEM 2013</b></h3><br />
<ol><li>Organism name and strain name or number: Infectious bronchitis virus</li><br />
<li>Organism Risk Group: Group 2</li><br />
<li>If you are using this organism as a chassis, write "chassis". If you are using a genetic part from <br />
the organism, give the name of the part and a brief description of what it does and why you are <br />
using it.<br />
<p>BBa_K120001: includes the sequence for a pseudoknot secondary structural RNA element that can cause programmed -1 frameshifting during translation. We plan to characterize the frameshifting frequency of this pseudoknot and create a library of frameshifting elements.</p><br />
<br />
<p>BBa_K120000: includes reporter genes upstream and downstream of the pseudoknot, as a construct for testing the frameshift frequency.</p><br />
</li><br />
<li>How did you physically acquire the organism or part? <br />
<p>The part was synthesized by GENEWIZ.</p></li><br />
<br />
<li>What potential safety/health risks to team members, other people at your institution, or the <br />
general public could arise from your use of this organism/part?<br />
<p>The part itself is innocuous and not infectious. Since it is just a short sequence of DNA that, when transcribed into RNA, folds into a specific secondary structure, it does not pose a safety threat to our team members, the university, or the general public.</p></li><br />
<br />
<li>What measures do you intend to take to ensure that your project is safe for team members, <br />
other people at your institution, and the general public?<br />
<p>Since the part itself does not pose a safety threat, standard laboratory safety procedures will be followed, including ensuring that the part or the chassis containing the part does not leave the laboratory space. Proper personal protective equipment will be worn at all times when working with the part and all team members are fully trained in WHMIS and biosafety before being allowed to work in the laboratory.</p></li><br />
<br />
<li> If you are using only a part from the organism, and you believe the part by itself is not <br />
dangerous, explain why you believe it is not dangerous.<br />
<p>Use of the part does not require handling of the virus from which it comes. Therefore, the part itself is not dangerous. Pseudoknots are common RNA elements found in organisms besides viruses, and are not dangerous outside of the context of their host.</p></li><br />
<br />
<li>Why do you need to use this organism/part? Is there an organism/part from a less dangerous <br />
Risk Group that would accomplish the same purpose?<br />
<p>We use standard E. coli strains as our chassis for the expression of our constructs. The pseudoknot from infectious bronchitis virus (IBV) has been shown to cause frameshifting in E. coli cells. Therefore, the sequence of the IBV pseudoknot was used. Most pseudoknot sequences that have been characterized for frameshifting activity originate from viruses, so the pseudoknot sequence could not be retrieved from a Risk Level 1 organism.</p></li><br />
<li>Is the organism/part listed under the Australia Group guidelines, or otherwise restricted for <br />
transport? If so, how will your team ship this part to iGEM and the Jamborees?<br />
<p>The organism that the part originates from is not listed under the Australia Group guidelines, and therefore no additional measures are needed for transport of the BioBrick part.</p></li><br />
<br />
<li>Please describe the BioSafety Level of the lab in which the team works, or description of <br />
safety features of lab (Refer to Basic Safety form, question 8. d.). If you are using organisms with <br />
a BSL level greater than you lab, please explain any additional safety precautions you are taking. <br />
<p>The lab in which the team works is rated as a BioSafety Level 1 laboratory. Prior to starting lab work, all members must complete WHMIS and Biosafety Training. Since use of the part does not require handling of the Risk Level 2 organism that it comes from, no additional safety precautions are necessary.</p></li></ul><br />
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</html></div>Dustinhttp://2013.igem.org/Team:LethbridgeTeam:Lethbridge2013-09-28T02:04:52Z<p>Dustin: </p>
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<h2>Project Overview</h2><br />
<p style="color:black">The current growth in synthetic biology research promises more complex and useful engineered systems. However, increased complexity often requires more genetic material that can be difficult to introduce into organisms. We propose the development of a new library of regulatory gene expression elements that allow for compression of multiple coding sequences into a smaller amount of genetic space. Using a pseudoknot RNA structural motif, commonly used by viruses to minimize their genome size, we will show the utility of dual-coding gene sequences to give useful protein products whose expression can be regulated by the pseudoknot’s ability to induce ribosomal frameshifting. A software tool will also be used to zip multiple coding sequences into different reading frames. Ultimately, this library of standardized parts will be available for use in a variety of engineered systems requiring minimal coding space and multiple protein expression.</p><br><br />
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<b style="color:black"><u>WHAT?</u></b><br />
<ul style="color:black"><li>Our project is directed towards standardizing pseudoknots to make a new class of parts available to the synthetic biology community</li></ul> <br />
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<b style="color:black"><u>WHY?</u></b><br />
<ul style="color:black"><li>As the field of synthetic biology grows, so should its toolset. By introducing a standardized method of implementing programmed ribosomal frameshifts in synthetic gene networks, we could not only enable others to reduce plasmid size and regulate operon expression, but also enable them to come up with new, exciting applications</li></ul><br />
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<b style="color:black"><u>HOW?</b></u><br />
<ul style="color:black"><li>We have brought pseudoknots to the iGEM community by: <br />
<ul><li>Characterizing their function in a biobrick system</li><br />
<li>Designing software that enables others to dual code proteins</li><br />
<li>Ensuring that the release of these tools to the wider public does not pose a significant risk to the rest of the world</li></li></ul></ul><br><br />
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<h2>Sponsors</h2><br />
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<image src="https://static.igem.org/mediawiki/2013/4/4c/ULeth2013_Sponsors_-_Bronze.png"; width="300px"; height="100px" /></center></p>&nbsp;&nbsp;&nbsp;&nbsp;<br />
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