Team:XMU-China/Project PSystem

From 2014.igem.org

(Difference between revisions)
 
(30 intermediate revisions not shown)
Line 6: Line 6:
Remove this if you use the .htaccess -->
Remove this if you use the .htaccess -->
<meta http-equiv="X-UA-Compatible" content="IE=edge,chrome=1">
<meta http-equiv="X-UA-Compatible" content="IE=edge,chrome=1">
-
 
+
ann
<title>side_bar</title>
<title>side_bar</title>
<meta name="description" content="">
<meta name="description" content="">
Line 465: Line 465:
<img id="Project_Psystem_title" class="Project_title" src="https://static.igem.org/mediawiki/2014/4/48/Xmu_project_P_system_zwei.png"/>
<img id="Project_Psystem_title" class="Project_title" src="https://static.igem.org/mediawiki/2014/4/48/Xmu_project_P_system_zwei.png"/>
<div id="Project_Psystem_wd">
<div id="Project_Psystem_wd">
-
<h2 style="text-align: justify; line-height: 130%; padding-top: 16px; padding-bottom: 4px; font-size: 21px; styleName: Normal; outlineLvl: 1;">
+
 
-
     <span style="font-family: Arial,sans-serif;text-align:center">A reasonable explanation of misfolding GFP under QS oscillation</span>
+
<br />
-
</h2>
+
<p style="text-align: center; line-height: 150%; font-size: 24px;">
 +
     <span style="font-size: 27px; font-weight: 700;">A REASONABLE EXPLANATION OF MISFOLDING GFP UNDER QS OSCILLATION</span>
 +
</p>
 +
<hr width="100%" size="2">
 +
<br/>
<p style="text-align: justify; font-family: Arial,sans-serif; styleName: Normal;">
<p style="text-align: justify; font-family: Arial,sans-serif; styleName: Normal;">
-
     <span style="font-family: Arial,sans-serif;">In</span><span style="font-family: Arial,sans-serif;"> the project </span><span style="font-family: Arial,sans-serif;">of</span><span style="font-family: Arial,sans-serif;"> iGEM</span><span style="font-family: Arial,sans-serif;">13 XMU-China, they</span><span style="font-family: Arial,sans-serif;"> can’t get expected oscillation. H</span><span style="font-family: Arial,sans-serif;">owever</span><span style="font-family: Arial,sans-serif;">,</span><span style="font-family: Arial,sans-serif;"> </span><span style="font-family: Arial,sans-serif;">this year</span><span style="font-family: Arial,sans-serif;"> </span><span style="font-family: Arial,sans-serif;">iGEM14 XMU-China</span><span style="font-family: Arial,sans-serif;"> further </span><span style="font-family: Arial,sans-serif;">investigate the reason of abnormal oscillation. We further review</span><span style="font-family: Arial,sans-serif;"> SDS-PAGE analysis to confirm </span><span style="font-family: Arial,sans-serif;">the circuit </span><span style="font-family: Arial,sans-serif;">at</span><span style="font-family: Arial,sans-serif;"> protein leve</span><span style="font-family: Arial,sans-serif;">l. The SDS-PAGE data is shown in </span><span style="font-family: Arial,sans-serif; font-weight: 700;">Figure 1</span><span style="font-family: Arial,sans-serif;">.</span><span style="font-family: Arial,sans-serif;"> Based on that, we make a reasonable assumption that the unexpected behavior of the LuxR Promoter lead</span><span style="font-family: Arial,sans-serif;">s</span><span style="font-family: Arial,sans-serif;"> to the misfolding proteins </span><span style="font-family: Arial,sans-serif;">hence the</span><span style="font-family: Arial,sans-serif;"> abnormal oscillation.</span>
+
     <span style="font-family: Arial,sans-serif;">In</span><span style="font-family: Arial,sans-serif;"> the project </span><a href="https://2013.igem.org/Team:XMU-China" target="_blank">iGEM13_XMU-China</a>, they<span style="font-family: Arial,sans-serif;"> couldn't get expected oscillation. However</span><span style="font-family: Arial,sans-serif;">,</span><span style="font-family: Arial,sans-serif;"> </span><span style="font-family: Arial,sans-serif;">this year</span> <a href="https://2014.igem.org/Team:XMU-China" target="_blank">iGEM14_XMU-China</a><span style="font-family: Arial,sans-serif;"> further </span><span style="font-family: Arial,sans-serif;">investigated the reason of abnormal oscillation. We further reviewed</span><span style="font-family: Arial,sans-serif;"> SDS-PAGE analysis to confirm </span><span style="font-family: Arial,sans-serif;">the circuit </span><span style="font-family: Arial,sans-serif;">at</span><span style="font-family: Arial,sans-serif;"> protein leve</span><span style="font-family: Arial,sans-serif;">l. The SDS-PAGE data is shown in </span><span style="font-family: Arial,sans-serif; font-weight: 700;">Figure 1</span><span style="font-family: Arial,sans-serif;">.</span><span style="font-family: Arial,sans-serif;"> Based on that, we made a reasonable assumption that the unexpected behavior of the LuxR promoter lead</span><span style="font-family: Arial,sans-serif;">s</span><span style="font-family: Arial,sans-serif;"> to the misfolding proteins Hence result in the abnormal oscillation result in the abnormal oscillation.</span>
</p>
</p>
<p style="text-align: justify; font-family: Arial,sans-serif; styleName: Normal;">
<p style="text-align: justify; font-family: Arial,sans-serif; styleName: Normal;">
Line 486: Line 490:
             <td style="border-width: medium 1px 1px; border-color: currentColor rgb(0, 0, 0) rgb(0, 0, 0); padding: 0px 7px; width: 588px; vertical-align: top;">
             <td style="border-width: medium 1px 1px; border-color: currentColor rgb(0, 0, 0) rgb(0, 0, 0); padding: 0px 7px; width: 588px; vertical-align: top;">
                 <p style="font-family:Times New Roman;line-height:1.2;">
                 <p style="font-family:Times New Roman;line-height:1.2;">
-
                     <span style="font-family: Times New Roman; font-weight: 700;">Figure 1. </span>SDS-PAGE analysis of E.coli K strain (<span style="font-family: Times New Roman; font-style: italic;">DH5α</span>). </span><span style="font-family: Times New Roman; font-weight: 700;">(a)</span> Lane 1-2: supernatant and pellet of original </span><span style="font-family: Times New Roman; font-style: italic;">DH5α</span>; Lane 3-4: supernatant and pellet of strain with single plasmid A1 (BBa_K1036003); Lane 5-6: supernatant and pellet of strain with both plasmids A1</span> </span>(BBa_K1036003) and B</span> (BBa_K1036000)</span>. The red arrows indicate the </span><span style="font-family: Times New Roman; font-weight: 700;">misfolding</span> GFP-LVA protein (27.6 kDa) in the precipitation. </span><span style="font-family: Times New Roman; font-weight: 700;">(b)</span> Lane 1-2: supernatant and pellet of original </span><span style="font-family: Times New Roman; font-style: italic;">BL21</span>; Lane 3-4: supernatant and pellet of strain with single plasmid A1</span> (BBa_K1036003)</span>; Lane 5-6: supernatant and pellet of strain with both plasmids A1</span> (BBa_K1036003) </span>and B</span> (BBa_K1036000)</span>. The blue arrows indicate LuxR (27.5 kDa), GFP-LVA (27.6 kDa) and AiiA-LVA (28.7 kDa) in the supernatant. The orange arrows indicate LuxI-LVA (22.4 kDa) in the supernatant. (The marker of b was not in right position, however, the proteins were confirmed by MALDI-TOF-TOF .)</span>
+
                     <span style="font-family: Times New Roman; font-weight: 700;">Figure 1. </span>SDS-PAGE analysis of <em>E. coli</em> K strain (<span style="font-family: Times New Roman;">DH5α</span>). </span><span style="font-family: Times New Roman; font-weight: 700;">(a)</span> Lane 1-2: supernatant and pellet of original </span><span style="font-family: Times New Roman; font-style: ">DH5α</span>; Lane 3-4: supernatant and pellet of strain with single plasmid A1 (<span>BBa_K1036003</span>); Lane 5-6: supernatant and pellet of strain with both plasmids A1</span> </span>(<span>BBa_K1036003</span>) and B</span> (<span>BBa_K1036000</span>)</span>. The red arrows indicate the </span><span style="font-family: Times New Roman; font-weight: 700;">misfolding</span> GFP-LVA protein (27.6 kDa) in the precipitation. </span><span style="font-family: Times New Roman; font-weight: 700;">(b)</span> Lane 1-2: supernatant and pellet of original </span><span style="font-family: Times New Roman; font-style: italic;">BL21</span>; Lane 3-4: supernatant and pellet of strain with single plasmid A1</span> (<span>BBa_K1036003</span>)</span>; Lane 5-6: supernatant and pellet of strain with both plasmids A1</span> (<span>BBa_K1036003</span>) </span>and B</span> (<span>BBa_K1036000</span>)</span>. The blue arrows indicate LuxR (27.5 kDa), GFP-LVA (27.6 kDa) and AiiA-LVA (28.7 kDa) in the supernatant. The orange arrows indicate LuxI-LVA (22.4 kDa) in the supernatant. (The marker of b was not in right position, however, the proteins were confirmed by MALDI-TOF-TOF .)</span>
                 </p>
                 </p>
             </td>
             </td>
Line 499: Line 503:
</p>
</p>
<p>
<p>
-
     As the SDS-PAGE shows, a large amount of GFP-LVA and LuxI-LVA appear in pellet where misfolding proteins often exist. Both proteins directly affect the oscillation result. And it is critical to find out the reason for misfolding proteins. iGEM14 XMU-China make the following assumption:
+
     As the SDS-PAGE shows, a large amount of GFP-LVA and LuxI-LVA appear in pellet where misfolding proteins often exist. Both proteins directly affect the oscillation result. And it is critical to find out the reason for misfolding proteins. <a href="https://2014.igem.org/Team:XMU-China" target="_blank">iGEM14_XMU-China</a> made the following assumption:
</p>
</p>
<p style="font-family: Arial,sans-serif;">
<p style="font-family: Arial,sans-serif;">
Line 505: Line 509:
</p>
</p>
<p>
<p>
-
     The research reveals an unexpected behavior<sup>[1]</sup> of Lux pR (BBa_R0062). In the absence of autoinducer 3OC6 (AHL), LuxR binds to Plux (Lux pR) and activates backwards transcription (<strong>Figure 2</strong>).
+
     The research<sup>[1]</sup> reveals an unexpected behavior of Lux pR (<span>BBa_R0062</span>). In the absence of autoinducer 3OC6 (AHL), LuxR binds to plux (Lux pR) and activates backwards transcription (<strong>Figure 2</strong>).
</p>
</p>
<p style="font-family: Arial,sans-serif;">
<p style="font-family: Arial,sans-serif;">
Line 522: Line 526:
             <td style="border-width: medium 1px 1px; border-color: currentColor rgb(0, 0, 0) rgb(0, 0, 0); padding: 0px 7px; width: 567px; vertical-align: top;">
             <td style="border-width: medium 1px 1px; border-color: currentColor rgb(0, 0, 0) rgb(0, 0, 0); padding: 0px 7px; width: 567px; vertical-align: top;">
                 <p style="font-family: Times New Roman;line-height:1.2;">
                 <p style="font-family: Times New Roman;line-height:1.2;">
-
                     <span style="font-weight: 700;">Figure 2. Relative RFP fluorescence for a control construct designed to measure backwards transcription from Lux pR. Addition of LuxR and 3OC6 90 (AHL) as indicated. Error bars in all panels are one standard deviation.</span>
+
                     <span style="font-weight: 700;"><b>Figure 2.</b> Relative RFP fluorescence for a control construct designed to measure backwards transcription from Lux pR. Addition of LuxR and 3OC6 90 (AHL) as indicated. Error bars in all panels are one standard deviation.</span>
                 </p>
                 </p>
             </td>
             </td>
Line 538: Line 542:
</p>
</p>
<p>
<p>
-
     From the original design by Jeff Hasty, Lux pR and Lux pL are set in opposite directions (<strong>Figure 3A</strong>). In the absence of AHL, LuxR could activate backward transcription of Lux pR <strong>leading to more expression of LuxR</strong> which is critical to meet the oscillation conditions. However, present literature don’t consider the backwards transcription which have effect on quorum sensing oscillation.
+
     From the original design by Jeff Hasty, Lux pR and Lux pL were set in opposite directions (<strong>Figure 3A</strong>). In the absence of AHL, LuxR could activate backwards transcription of Lux pR leading to more expression of LuxR which was critical to meet the oscillation conditions. However, present literature did’t consider the backwards transcription which had effect on quorum sensing oscillation.
</p>
</p>
<p style="font-family: Arial,sans-serif; font-weight: 700;">
<p style="font-family: Arial,sans-serif; font-weight: 700;">
Line 558: Line 562:
             <td style="border-width: medium 1px 1px; border-color: currentColor rgb(0, 0, 0) rgb(0, 0, 0); padding: 0px 7px; width: 553px; vertical-align: top;">
             <td style="border-width: medium 1px 1px; border-color: currentColor rgb(0, 0, 0) rgb(0, 0, 0); padding: 0px 7px; width: 553px; vertical-align: top;">
                 <p style="text-align: justify; text-indent: -24px; font-family: Arial,sans-serif; font-weight: 700; margin-left: 24px; styleName: Normal;">
                 <p style="text-align: justify; text-indent: -24px; font-family: Arial,sans-serif; font-weight: 700; margin-left: 24px; styleName: Normal;">
-
                     <span style="font-weight: 700;">B.</span><span style="font-weight: 700;"> </span><span style="font-family: Arial,sans-serif; font-weight: 400;">iGEM13 XMU-China Design</span>
+
                     <span style="font-weight: 700;">B.</span><span style="font-weight: 700;"> </span><span style="font-family: Arial,sans-serif; font-weight: 400;"><a href="https://2013.igem.org/Team:XMU-China" target="_blank">iGEM13_XMU-China</a> Design</span>
                 </p>
                 </p>
                 <p style="font-family: Arial,sans-serif; font-weight: 700;">
                 <p style="font-family: Arial,sans-serif; font-weight: 700;">
Line 568: Line 572:
             <td style="border-width: medium 1px 1px; border-color: currentColor rgb(0, 0, 0) rgb(0, 0, 0); padding: 0px 7px; width: 553px; vertical-align: top;">
             <td style="border-width: medium 1px 1px; border-color: currentColor rgb(0, 0, 0) rgb(0, 0, 0); padding: 0px 7px; width: 553px; vertical-align: top;">
                 <p style="font-family: Times New Roman; font-weight: 400;line-height:1.2;">
                 <p style="font-family: Times New Roman; font-weight: 400;line-height:1.2;">
-
                     <span style="font-weight: 700;">Figure 3A.</span>Top row is the original design by Jeff Hasty.<span style="font-weight: 700;">B.</span> Bottom row is the simplified design which sets lux pL and lux pR in the same direction.
+
                     <span style="font-weight: 700;">Figure 3A.</span>Top row is the original design by Jeff Hasty.<span style="font-weight: 700;">B.</span> Bottom row is the simplified design which sets Lux pL and Lux pR in the same direction.
                 </p>
                 </p>
             </td>
             </td>
Line 581: Line 585:
</p>
</p>
<p style="font-family: Arial,sans-serif; font-weight: 700;">
<p style="font-family: Arial,sans-serif; font-weight: 700;">
-
     <span style="font-family: Arial,sans-serif; font-weight: 400;">In the simplif</span><span style="font-family: Arial,sans-serif; font-weight: 400;">ied</span><span style="font-family: Arial,sans-serif; font-weight: 400;"> design (</span><span style="font-family: Arial,sans-serif; font-weight: 700;">Figure 3B</span><span style="font-family: Arial,sans-serif; font-weight: 400;">), when LuxR activates the backward transcription, RNA polymerase will be blocked by the terminators B0015. So that this simplification doesn’t perform as same as original design. Actually, the reverse terminated efficiency of B0015 is 0.295(CC)[2] which may lead to leakage transcription. However, the correct sequence of GFP-LAA can’t be transcribed during the backwards transcription, even if the minus-strand of GFP-LAA could be transcribed, the sequence of the RNA is not in the right direction of GFP-LAA, hence incorrect amino acid sequences may be translated, resulting in misfolding GFP just as the SDS-PAGE shows (</span><span style="font-family: Arial,sans-serif; font-weight: 700;">Figure 1</span><span style="font-family: Arial,sans-serif; font-weight: 400;">). </span>
+
     <span style="font-family: Arial,sans-serif; font-weight: 400;">In the simplif</span><span style="font-family: Arial,sans-serif; font-weight: 400;">ied</span><span style="font-family: Arial,sans-serif; font-weight: 400;"> design (</span><span style="font-family: Arial,sans-serif; font-weight: 700;">Figure 3B</span><span style="font-family: Arial,sans-serif; font-weight: 400;">), when LuxR activates the backwards transcription, RNA polymerase will be blocked by the terminators B0015. So this simplification will not  perform as same as original design. Actually, the reverse terminated efficiency of B0015 is 0.295(CC)<sup>[2]</sup> which may lead to leakage transcription. However, the correct sequence of GFP-LAA couldn’t be transcribed during the backwards transcription, even if the minus-strand of GFP-LAA could be transcribed, the sequence of the RNA is not in the right direction of GFP-LAA, hence incorrect amino acid sequences might be translated, resulting in misfolding GFP just as what the SDS-PAGE shows (</span><span style="font-family: Arial,sans-serif; font-weight: 700;">Figure 1</span><span style="font-family: Arial,sans-serif; font-weight: 400;">). </span>
</p>
</p>
<p style="font-family: Arial,sans-serif; font-weight: 700;">
<p style="font-family: Arial,sans-serif; font-weight: 700;">
Line 587: Line 591:
</p>
</p>
<p style="font-family: Arial,sans-serif; font-weight: 700;">
<p style="font-family: Arial,sans-serif; font-weight: 700;">
-
     <span style="font-family: Arial,sans-serif; font-weight: 400;">Because of the </span><span style="font-family: Arial,sans-serif; font-weight: 400;">imperfect simplified</span><span style="font-family: Arial,sans-serif; font-weight: 400;"> design doesn’t follow the original function completely, the abnormal oscillation is justifiable. </span><span style="font-family: Arial,sans-serif; font-weight: 400;">M</span><span style="font-family: Arial,sans-serif; font-weight: 400;">isfolding protein is a</span><span style="font-family: Arial,sans-serif; font-weight: 400;">n</span><span style="font-family: Arial,sans-serif; font-weight: 400;"> evidence to support our assumption.</span>
+
     <span style="font-family: Arial,sans-serif; font-weight: 400;">Because of the </span><span style="font-family: Arial,sans-serif; font-weight: 400;">imperfect simplified</span><span style="font-family: Arial,sans-serif; font-weight: 400;"> design didn’t follow the original function completely, the abnormal oscillation was justifiable. </span><span style="font-family: Arial,sans-serif; font-weight: 400;">M</span><span style="font-family: Arial,sans-serif; font-weight: 400;">isfolding protein were</span><span style="font-family: Arial,sans-serif; font-weight: 400;">n</span><span style="font-family: Arial,sans-serif; font-weight: 400;"> evidence to support our assumption.</span>
</p>
</p>
<p style="font-family: Arial,sans-serif; font-weight: 700;">
<p style="font-family: Arial,sans-serif; font-weight: 700;">
Line 593: Line 597:
</p>
</p>
<p style="font-family: Arial,sans-serif; font-weight: 700;">
<p style="font-family: Arial,sans-serif; font-weight: 700;">
-
     <span style="font-family: Arial,sans-serif; font-weight: 400;">iGEM14 XMU-China involved sequence comparison to investigate the difference between the original and </span><span style="font-family: Arial,sans-serif; font-weight: 400;">the registry parts.</span><span style="font-family: Arial,sans-serif; font-weight: 400;"> We find that</span><span style="font-family: Arial,sans-serif; font-weight: 400;"> the original Lux pR has 20bp overlapping sequence with origin</span><span style="font-family: Arial,sans-serif; font-weight: 400;">al Lux p</span><span style="font-family: Arial,sans-serif; font-weight: 400;">R</span><span style="font-family: Arial,sans-serif; font-weight: 400;">. </span><span style="font-family: Arial,sans-serif; font-weight: 400;">There is </span><span style="font-family: Arial,sans-serif; font-weight: 400;">a r</span><span style="font-family: Arial,sans-serif; font-weight: 400;">estriction e</span><span style="font-family: Arial,sans-serif; font-weight: 400;">nzyme cutting site (EcoR I)</span><span style="font-family: Arial,sans-serif; font-weight: 400;"> at the 56bp of original Lux pR</span><span style="font-family: Arial,sans-serif; font-weight: 400;"> (</span><span style="font-family: Arial,sans-serif; font-weight: 700;">Figure 4</span><span style="font-family: Arial,sans-serif; font-weight: 400;">)</span><span style="font-family: Arial,sans-serif; font-weight: 400;">.</span>
+
     <span style="font-family: Arial,sans-serif; font-weight: 400;"><a href="https://2014.igem.org/Team:XMU-China" target="_blank">iGEM14_XMU-China</a> involved sequence comparison to investigate the difference between the original and </span><span style="font-family: Arial,sans-serif; font-weight: 400;">the registry parts.</span><span style="font-family: Arial,sans-serif; font-weight: 400;"> We found that</span><span style="font-family: Arial,sans-serif; font-weight: 400;"> the original Lux pR had 20bp overlapping sequence with original</span><span style="font-family: Arial,sans-serif; font-weight: 400;">al Lux p</span><span style="font-family: Arial,sans-serif; font-weight: 400;">R</span><span style="font-family: Arial,sans-serif; font-weight: 400;">. </span><span style="font-family: Arial,sans-serif; font-weight: 400;">There was </span><span style="font-family: Arial,sans-serif; font-weight: 400;">a r</span><span style="font-family: Arial,sans-serif; font-weight: 400;">estriction e</span><span style="font-family: Arial,sans-serif; font-weight: 400;">nzyme cutting site (EcoR I)</span><span style="font-family: Arial,sans-serif; font-weight: 400;"> at the 56bp of original Lux pR</span><span style="font-family: Arial,sans-serif; font-weight: 400;"> (</span><span style="font-family: Arial,sans-serif; font-weight: 700;">Figure 4</span><span style="font-family: Arial,sans-serif; font-weight: 400;">)</span><span style="font-family: Arial,sans-serif; font-weight: 400;">.</span>
</p>
</p>
<p style="font-family: Arial,sans-serif; font-weight: 700;">
<p style="font-family: Arial,sans-serif; font-weight: 700;">
     &nbsp;
     &nbsp;
</p>
</p>
-
<table style="border: currentColor; border-image: none; margin-left: 0px; border-collapse: collapse; styleName: Normal Table; border-insideH: 1px none #000000; border-insideV: 1px none #000000; cellpadding: 0px 7px 0px 7px;">
+
<table style="border: currentColor; border-image: none; margin-right: auto; margin-left: auto; border-collapse: collapse; styleName: Normal Table; border-insideH: 1px none #000000; border-insideV: 1px none #000000; cellpadding: 0px 7px 0px 7px;">
     <tbody>
     <tbody>
         <tr class="firstRow">
         <tr class="firstRow">
Line 623: Line 627:
</p>
</p>
<p style="font-family: Arial,sans-serif;">
<p style="font-family: Arial,sans-serif;">
-
         <span style="font-family: Arial,sans-serif; font-weight: 400;">Parts registry truncate the original Lux pR at 56bp to get the 55bp Lux pR (BBa_R0062). On the contrary, Lux pL (BBa_R0063) is longer the original Lux pL, and at the end of BBa_R0063 is initial part of 41bp LuxR (BBa_C0062). Thus new problems arise—is the modification of original QS promoter reasonable? Does the modification result in the unexpected backward transcription?</span>
+
         <span style="font-family: Arial,sans-serif; font-weight: 400;">Parts registry truncated the original Lux pR at 56bp to get the 55bp Lux pR (BBa_R0062). On the contrary, Lux pL (BBa_R0063) is longer than the original Lux pL, and at the end of BBa_R0063 is initial part of 41bp LuxR (BBa_C0062). Thus new problems arised—was the modification of original QS promoter reasonable? Did the modification result in the unexpected backwards transcription?</span>
</p>
</p>
<p style="font-family: Arial,sans-serif;">
<p style="font-family: Arial,sans-serif;">
Line 629: Line 633:
</p>
</p>
<p style="font-family: Arial,sans-serif; font-weight: 700;">
<p style="font-family: Arial,sans-serif; font-weight: 700;">
-
     <span style="font-family: Arial,sans-serif; font-weight: 400;">Quorum sensing system is so widely used in the synthetic biology, we think it’s remarkable to make it clear. We highlight the abnormal phenomenon of QS oscillation which may be caused by imperfect simplification for the very first time. We hope that more efforts could be made to figure out the interaction between QS oscillation parts.[3]</span>
+
     <span style="font-family: Arial,sans-serif; font-weight: 400;">Quorum sensing system is so widely used in the synthetic biology, we thought it’s remarkable to make it clear. We highlighted the abnormal phenomenon of QS oscillation which might be caused by imperfect simplification for the very first time. We hope that more efforts could be made to figure out the interaction between QS oscillation parts.<sup>[3]</sup></span>
</p>
</p>
<p style="font-family: Arial,sans-serif; font-weight: 700;">
<p style="font-family: Arial,sans-serif; font-weight: 700;">
Line 641: Line 645:
</p>
</p>
<p>
<p>
-
     <span style="font-size:14px"> <a href="http://parts.igem.org/Part:BBa_B0015" target="_self">2.http://parts.igem.org/Part:BBa_B0015</a></span>
+
     <span style="font-size:14px"> <a href="http://parts.igem.org/Part:BBa_B0015" target="_self">2. http://parts.igem.org/Part:BBa_B0015</a></span>
</p>
</p>
<p>
<p>

Latest revision as of 03:26, 18 October 2014

ann side_bar


A REASONABLE EXPLANATION OF MISFOLDING GFP UNDER QS OSCILLATION



In the project iGEM13_XMU-China, they couldn't get expected oscillation. However, this year iGEM14_XMU-China further investigated the reason of abnormal oscillation. We further reviewed SDS-PAGE analysis to confirm the circuit at protein level. The SDS-PAGE data is shown in Figure 1. Based on that, we made a reasonable assumption that the unexpected behavior of the LuxR promoter leads to the misfolding proteins Hence result in the abnormal oscillation result in the abnormal oscillation.

 

Figure 1. SDS-PAGE analysis of E. coli K strain (DH5α). (a) Lane 1-2: supernatant and pellet of original DH5α; Lane 3-4: supernatant and pellet of strain with single plasmid A1 (BBa_K1036003); Lane 5-6: supernatant and pellet of strain with both plasmids A1 (BBa_K1036003) and B (BBa_K1036000). The red arrows indicate the misfolding GFP-LVA protein (27.6 kDa) in the precipitation. (b) Lane 1-2: supernatant and pellet of original BL21; Lane 3-4: supernatant and pellet of strain with single plasmid A1 (BBa_K1036003); Lane 5-6: supernatant and pellet of strain with both plasmids A1 (BBa_K1036003) and B (BBa_K1036000). The blue arrows indicate LuxR (27.5 kDa), GFP-LVA (27.6 kDa) and AiiA-LVA (28.7 kDa) in the supernatant. The orange arrows indicate LuxI-LVA (22.4 kDa) in the supernatant. (The marker of b was not in right position, however, the proteins were confirmed by MALDI-TOF-TOF .)

 

As the SDS-PAGE shows, a large amount of GFP-LVA and LuxI-LVA appear in pellet where misfolding proteins often exist. Both proteins directly affect the oscillation result. And it is critical to find out the reason for misfolding proteins. iGEM14_XMU-China made the following assumption:

   

The research[1] reveals an unexpected behavior of Lux pR (BBa_R0062). In the absence of autoinducer 3OC6 (AHL), LuxR binds to plux (Lux pR) and activates backwards transcription (Figure 2).

 

Figure 2. Relative RFP fluorescence for a control construct designed to measure backwards transcription from Lux pR. Addition of LuxR and 3OC6 90 (AHL) as indicated. Error bars in all panels are one standard deviation.

 

The imperfect simplification of setting lux pL and Lux pR in the same direction: 

From the original design by Jeff Hasty, Lux pR and Lux pL were set in opposite directions (Figure 3A). In the absence of AHL, LuxR could activate backwards transcription of Lux pR leading to more expression of LuxR which was critical to meet the oscillation conditions. However, present literature did’t consider the backwards transcription which had effect on quorum sensing oscillation.

 

A. Original Design

B. iGEM13_XMU-China Design

Figure 3A.Top row is the original design by Jeff Hasty.B. Bottom row is the simplified design which sets Lux pL and Lux pR in the same direction.

 

In the simplified design (Figure 3B), when LuxR activates the backwards transcription, RNA polymerase will be blocked by the terminators B0015. So this simplification will not perform as same as original design. Actually, the reverse terminated efficiency of B0015 is 0.295(CC)[2] which may lead to leakage transcription. However, the correct sequence of GFP-LAA couldn’t be transcribed during the backwards transcription, even if the minus-strand of GFP-LAA could be transcribed, the sequence of the RNA is not in the right direction of GFP-LAA, hence incorrect amino acid sequences might be translated, resulting in misfolding GFP just as what the SDS-PAGE shows (Figure 1).

 

Because of the imperfect simplified design didn’t follow the original function completely, the abnormal oscillation was justifiable. Misfolding protein weren evidence to support our assumption.

 

iGEM14_XMU-China involved sequence comparison to investigate the difference between the original and the registry parts. We found that the original Lux pR had 20bp overlapping sequence with originalal Lux pR. There was a restriction enzyme cutting site (EcoR I) at the 56bp of original Lux pR (Figure 4).

 

Figure 4. Schematic of original QS promoter.

 

Parts registry truncated the original Lux pR at 56bp to get the 55bp Lux pR (BBa_R0062). On the contrary, Lux pL (BBa_R0063) is longer than the original Lux pL, and at the end of BBa_R0063 is initial part of 41bp LuxR (BBa_C0062). Thus new problems arised—was the modification of original QS promoter reasonable? Did the modification result in the unexpected backwards transcription?

 

Quorum sensing system is so widely used in the synthetic biology, we thought it’s remarkable to make it clear. We highlighted the abnormal phenomenon of QS oscillation which might be caused by imperfect simplification for the very first time. We hope that more efforts could be made to figure out the interaction between QS oscillation parts.[3]

 

References

1. Eckdahl T, Sawyer E M, Barta C, et al. Bacterial logic devices reveal unexpected behavior of frameshift suppressor tRNAs[J]. Interdisciplinary Bio Central, 2012, 4(1): 10.

2. http://parts.igem.org/Part:BBa_B0015

3. Danino T, Mondragón-Palomino O, Tsimring L, et al.A synchronized quorum of genetic clocks[J]. Nature, 2010, 463(7279): 326-330.