Cartonchou (Talk | contribs) |
|||
(251 intermediate revisions by 4 users not shown) | |||
Line 1: | Line 1: | ||
+ | |||
{{NYMU-Taipei}} | {{NYMU-Taipei}} | ||
{{NYMU-Taipei-css}} | {{NYMU-Taipei-css}} | ||
Line 5: | Line 6: | ||
<head> | <head> | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
<style> | <style> | ||
body{ | body{ | ||
− | background-color:# | + | background-color:#f6ffb2; |
} | } | ||
.Nitrogen_Starvation{ | .Nitrogen_Starvation{ | ||
− | width: | + | width: 81%; |
padding:2%; | padding:2%; | ||
display:block; | display:block; | ||
float:right; | float:right; | ||
− | background-color:#396031; | + | background-color:#396031; |
+ | background-image:url('https://static.igem.org/mediawiki/2017/b/b7/T--NYMU-Taipei--nitrogen_starvation_background.png'); | ||
+ | background-size: cover; | ||
+ | background-attachment:fixed; | ||
font-family:'Fresca',sans-serif; | font-family:'Fresca',sans-serif; | ||
+ | font-size:20px; | ||
padding-bottom:250px; | padding-bottom:250px; | ||
+ | } | ||
+ | |||
+ | .Nitrogen_Starvation_top{ | ||
+ | width: 85%; | ||
+ | display:block; | ||
+ | float:right; | ||
+ | background-color:#000000; | ||
+ | font-family:'Fresca',sans-serif; | ||
+ | font-size:20px; | ||
} | } | ||
Line 38: | Line 43: | ||
.Nitrogen_Starvation h4{ | .Nitrogen_Starvation h4{ | ||
− | padding: | + | padding:10px 0px 11px 20px; |
− | color: # | + | color: #3dba32; |
− | font-size: | + | font-size: 24px; |
− | font-family: ' | + | font-family: 'Acme', sans-serif; |
} | } | ||
.Nitrogen_Starvation p { | .Nitrogen_Starvation p { | ||
− | padding: | + | padding:0px 0px 0px 20px; |
font-size: 20px; | font-size: 20px; | ||
− | color: # | + | color: #205e1a; |
} | } | ||
− | + | .mark_backbone { | |
− | . | + | font-family: 'Acme', sans-serif; |
− | width: | + | font-weight: bold; |
− | + | } | |
+ | |||
+ | .half_column { | ||
+ | width:50%; | ||
+ | float:left; | ||
+ | } | ||
+ | |||
+ | .half_column img { | ||
+ | width:94.5%; | ||
display:block; | display:block; | ||
− | + | margin:auto; | |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
} | } | ||
− | . | + | .full_column { |
− | + | width:96%; | |
− | width: | + | clear: both; |
− | + | } | |
− | + | ||
− | + | .clear { | |
− | + | clear:both; | |
+ | height:1vw; | ||
+ | } | ||
+ | /* Show & hide */ | ||
+ | .expandable { | ||
+ | /* overflow set to hidden to hide the expanded text */ | ||
+ | overflow: hidden; | ||
+ | /* all style changes will ease-in-out for 1s */ | ||
+ | -moz-transition: all 1s ease-in-out; | ||
+ | -ms-transition: all 1s ease-in-out; | ||
+ | -o-transition: all 1s ease-in-out; | ||
+ | -webkit-transition: all 1s ease-in-out; | ||
+ | transition: all 1s; | ||
} | } | ||
+ | |||
+ | .panel { | ||
+ | background-color: rgba(255, 255, 255, 0.9); | ||
+ | border: 1px solid transparent; | ||
+ | border-radius: 4px; | ||
+ | border-color: #ddd; | ||
+ | -webkit-box-shadow: 0 1px 1px rgba(0, 0, 0, .05); | ||
+ | box-shadow: 0 1px 1px rgba(0, 0, 0, .05); | ||
+ | padding:15px; | ||
+ | width:920px; | ||
+ | margin: auto; | ||
+ | margin-top:10px; | ||
+ | } | ||
+ | |||
+ | /* IF THE SCREEN IS LESS THAN 1300PX */ | ||
+ | @media only screen and (max-width: 1300px) { | ||
+ | .panel {width:92%} | ||
+ | } | ||
+ | |||
+ | |||
+ | /* IF THE SCREEN IS LESS THAN 1200PX */ | ||
+ | @media only screen and (max-width: 1200px) { | ||
+ | .panel {width:96%} | ||
+ | } | ||
+ | |||
/* IF THE SCREEN IS LESS THAN 800PX */ | /* IF THE SCREEN IS LESS THAN 800PX */ | ||
@media only screen and (max-width: 800px) { | @media only screen and (max-width: 800px) { | ||
.igem_2017_footer_wrapper {width:100%; margin-left:0px;} | .igem_2017_footer_wrapper {width:100%; margin-left:0px;} | ||
− | + | .Nitrogen_Starvation {width:96%; margin-left:0px;} | |
− | + | .Nitrogen_Starvation_top {width:100%; margin-left:0px;} | |
− | + | ||
} | } | ||
+ | |||
+ | |||
</style> | </style> | ||
− | < | + | <script> |
+ | function toggleHeight1(e, maxHeight) { | ||
+ | e = document.getElementById("s1"); // e = the gray div | ||
+ | |||
+ | if(e.style.height != '30px') { | ||
+ | e.style.height = '30px'; // height of one line: 20px | ||
+ | } else { | ||
+ | e.style.height = maxHeight + 'px'; | ||
+ | } | ||
+ | } | ||
+ | |||
+ | function toggleHeight2(e, maxHeight) { | ||
+ | e = document.getElementById("s2"); // e = the gray div | ||
+ | |||
+ | if(e.style.height != '30px') { | ||
+ | e.style.height = '30px'; // height of one line: 20px | ||
+ | } else { | ||
+ | e.style.height = maxHeight + 'px'; | ||
+ | } | ||
+ | } | ||
+ | |||
+ | function toggleHeight3(e, maxHeight) { | ||
+ | e = document.getElementById("s3"); // e = the gray div | ||
+ | |||
+ | if(e.style.height != '30px') { | ||
+ | e.style.height = '30px'; // height of one line: 20px | ||
+ | } else { | ||
+ | e.style.height = maxHeight + 'px'; | ||
+ | } | ||
+ | } | ||
+ | |||
+ | function toggleHeight4(e, maxHeight) { | ||
+ | e = document.getElementById("s4"); // e = the gray div | ||
+ | |||
+ | if(e.style.height != '30px') { | ||
+ | e.style.height = '30px'; // height of one line: 20px | ||
+ | } else { | ||
+ | e.style.height = maxHeight + 'px'; | ||
+ | } | ||
+ | } | ||
+ | |||
+ | function toggleHeight5(e, maxHeight) { | ||
+ | e = document.getElementById("s5"); // e = the gray div | ||
+ | |||
+ | if(e.style.height != '30px') { | ||
+ | e.style.height = '30px'; // height of one line: 20px | ||
+ | } else { | ||
+ | e.style.height = maxHeight + 'px'; | ||
+ | } | ||
+ | } | ||
+ | |||
+ | function toggleHeight6(e, maxHeight) { | ||
+ | e = document.getElementById("s6"); // e = the gray div | ||
+ | |||
+ | if(e.style.height != '30px') { | ||
+ | e.style.height = '30px'; // height of one line: 20px | ||
+ | } else { | ||
+ | e.style.height = maxHeight + 'px'; | ||
+ | } | ||
+ | } | ||
+ | </script> | ||
− | <div class=" | + | </head> |
+ | |||
+ | |||
+ | <body> | ||
+ | <div class="Nitrogen_Starvation_top"> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/2/20/T--NYMU-Taipei--nitrogen_starvation.jpg" width="100%"> | ||
+ | </div> | ||
− | + | <div class="Nitrogen_Starvation"> | |
− | + | ||
− | <div class="panel- | + | <!--background--> |
+ | <div class='panel'> | ||
+ | <div id="s1" class="expandable" style='height: 30px;padding-top:15px;'> | ||
+ | <a href="#!" onclick="toggleHeight1(this, 1100); return false" | ||
+ | style="font-family:'Acme', sans-serif;font-size:30px;color:#205e1a;height: 30px;"> | ||
+ | Background | ||
+ | </a> | ||
+ | <p style="padding-top:10px;"></p> | ||
+ | <p> With the development of global economy in the latest demands of energy in world, an average Taiwanese person produces around 2.58 metric tons of carbon emission a year. | ||
+ | This number far surpasses those of China, Japan and South Korea. | ||
+ | Our project, as a result, works to use biofuel as an alternative to fossil fuel to reduce the current energy crisis. | ||
+ | In our lab, we <font class='mark_backbone'>used microalgae as the source of biofuel</font> since they have the greatest capability of producing large amount of oil. </p> | ||
+ | <p> Our project aims to increase oil accumulation through <font class='mark_backbone'>nitrogen starvation</font>, which was brought up in a research we came upon. | ||
+ | Under nitrogen starvation, de novo synthesis of triacylglycerol from acyl-CoA increases. | ||
+ | Acyl moieties derived from the degradation of membrane lipids then recycle into triacylglycerol, increasing carbon flux towards glycerol-3 -phosphate and acyl-CoA for fatty acid synthesis. | ||
+ | As such, <font class='mark_backbone'>the oil accumulation under nitrogen starvation will increase</font>.</p> | ||
+ | <center><img src="https://static.igem.org/mediawiki/2017/8/85/T--NYMU-Taipei--NS_pathway.jpg" style="width:70%;"></center> | ||
+ | <br> | ||
+ | <p> There are currently two types of culture systems: <font class='mark_backbone'>open-pond</font> and <font class='mark_backbone'>closed bioreactors</font>. | ||
+ | While open-ponds cost way lower than closed bioreactors, open-pond-cultivated microalgae is with significantly lower oil contents. | ||
+ | Many microalgae farms today cultivate microalgae in closed ponds, where regulations are made to keep the nitrogen level low. | ||
+ | This method, though, consumes lots of energy due to the need to maintain proper temperature, nutrition, light, and other growing factors. | ||
+ | We want to develop a new method that allows microalgae to reach nitrogen starvation in open-pond, and thus reaching <font class='mark_backbone'>the same effectiveness of closed bioreactors with the affordable price</font>. </p> | ||
+ | <!--close--> | ||
+ | <center> | ||
+ | <a href="#!" onclick="toggleHeight1(this, 1100);" style='color:#56914a'> | ||
+ | click to close | ||
+ | </a> | ||
+ | </center> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | <!--design--> | ||
+ | <div class='panel'> | ||
+ | <div id="s2" class="expandable" style='height: 30px;padding-top:15px;'> | ||
+ | <a href="#!" onclick="toggleHeight2(this, 920); return false" | ||
+ | style="font-family:'Acme', sans-serif;font-size:30px;color:#205e1a;height: 30px;"> | ||
+ | Design | ||
+ | </a> | ||
+ | <p style="padding-top:10px;"></p> | ||
+ | <p> NrtA protein sticks to the periplasmic membrane through a flexible linker to capture nitrite or nitrate in the periplasm. | ||
+ | Then delivery to the transmembrane complex that made by NrtB. | ||
+ | In our project, we try to <font class='mark_backbone'>transform NrtA gene from cyanobacteria <i>Synechocystis</i> sp. PCC 6803 to<i> E.coli</i>, and then co-culture the engineered <i>E.coli</i> with microalgae</font>. | ||
+ | Engineered <i>E.coli</i> will be capable of clutching nitrite or nitrate present in the environment. | ||
+ | They will not intake nitrate or nitrite since the accumulation may be lethal to <i>E. coli</i> | ||
+ | But the amount of microalgae that contains nitrate and/or nitrite will decrease. | ||
+ | Therefore, the microalgae will undergo <font class='mark_backbone'>nitrogen starvation</font> and produce oil more efficiently.</p> | ||
+ | <p> After building up the nitrogen starvation and extracting oil from microalgae, we need to kill the <i>E.coli</i> to prevent contamination. | ||
+ | So we planned to <font class='mark_backbone'>use endolysin and holin for cell lysis</font>, which is similar to the mechanism used by team PeKing (2014 iGEM Beijing). | ||
+ | Holin can trigger the formation of holes on cell membrane. | ||
+ | When holin successfully forms holes on cell membrane, endolysin can pass through the membrane to decompose peptidoglycan. | ||
+ | Then, <i>E.coli</i> is lysed after the cell membrane and cell wall are destroyed. | ||
+ | To control the suicide timing, we designed an inducible promoter for holin, so that we can induce the suicide of the <i>E.coli</i> at the exact time we want.</p> | ||
+ | <center><img src='https://static.igem.org/mediawiki/2017/7/75/T--NYMU-Taipei--nitrogen_starvation_animation.gif' style='width:70%'></center> | ||
+ | <br> | ||
+ | <br> | ||
+ | <!--close--> | ||
+ | <center> | ||
+ | <a href="#!" onclick="toggleHeight2(this, 920);" style='color:#56914a'> | ||
+ | click to close | ||
+ | </a> | ||
+ | </center> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | <!--experiments--> | ||
+ | <div class='panel'> | ||
+ | <div id="s3" class="expandable" style='height: 30px;padding-top:15px;'> | ||
+ | <a href="#!" onclick="toggleHeight3(this, 7300); return false" | ||
+ | style="font-family:'Acme', sans-serif;font-size:30px;color:#205e1a;height: 30px;"> | ||
+ | Experiments | ||
+ | </a> | ||
+ | <p style="padding-top:10px;"></p> | ||
+ | <h4>NrtA Expression</h4> | ||
+ | <div class="full_column"> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/1/15/T--NYMU-Taipei--NS_NrtA_snapgene.png" style="width:60%;float:left;margin:0 3vw 0 3vw;"> | ||
+ | <p> We successfully transformed NrtA gene from cyanobacteria <i>Synechocystis</i> sp. PCC 6803 to <i>E.coli</i>.</p> | ||
+ | </div> | ||
+ | <div class="clear"></div> | ||
+ | <div class="full_column"> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/d/da/T--NYMU-Taipei--NS_NrtA_RE.png" style="width:60%;float:left;margin:0 3vw 0 3vw;"> | ||
+ | <p> This figure shows restriction enzyme check electrophoresis result of NrtA construct. | ||
+ | We use XhoI and HindIII to digest the plasmid, and the expected lengths are 892 bp, 1169 bp, 1540 bp. | ||
+ | M represents 1 Kb marker, and the result shows that numbers 2, 4, 6, 8, 10, 12 are correct. | ||
+ | </p> | ||
+ | </div> | ||
− | <div class=" | + | <div class="clear"></div> |
− | + | <h4></h4> | |
− | + | <h4></h4> | |
− | + | <h4>Endolysin Construct</h4> | |
− | + | <div class="full_column"> | |
− | + | <img src="https://static.igem.org/mediawiki/2017/c/c7/T--NYMU-Taipei--NS_endolysin_snapgene.png" style="width:40%;float:left;margin:0 3vw 0 4vw;"> | |
− | + | <p> We successfully constructed <font class='mark_backbone'>J23106-B0034-Endolysin-B0010-B0012</font>. The endolysin in this part is from iGEM released part, BBa_K112806, which is the endolysin from the enterobacteria phage T4. Besides the endolysin, in this composite part, we choose BBa_J23106 as the constitutive promoter, BBa_B0034 as the ribosome binding site, BBa_B0010 and BBa_B0012 as the double terminator, all of which are widely used parts in iGEM.</p> | |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
</div> | </div> | ||
− | + | <div class="clear"> | |
− | <div class=" | + | |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
</div> | </div> | ||
− | + | <div class="half_column"> | |
− | <div class=" | + | |
− | < | + | <img src="https://static.igem.org/mediawiki/2017/c/c4/T--NYMU-Taipei--NS_endolysin_PCR.png" style="width:76%"> |
− | + | <p>This figure is the gel electrophoresis result of endolysin PCR product. | |
− | + | The marker is 100 bp. The expected length is 514 bp. | |
− | + | </p> | |
− | + | </div > | |
− | + | <div class="half_column"> | |
− | + | <img src="https://static.igem.org/mediawiki/2017/2/22/T--NYMU-Taipei--NS_endolysin_BB.png" > | |
− | + | <p>This figure is the gel electrophoresis result of endolysin backbone PCR product. | |
− | + | The marker is 100 bp. The expected length is 2268 bp. | |
− | + | </p> | |
− | + | ||
− | </ | + | |
</div> | </div> | ||
− | + | <div class="clear"></div> | |
− | <div class=" | + | |
− | + | <h4></h4> | |
− | + | <h4>Holin Construct</h4> | |
− | + | <img src="https://static.igem.org/mediawiki/2017/6/62/T--NYMU-Taipei--NS_holin_snapgene.png" style="width:35%;float:left;margin:0 3vw 0 1vw;"> | |
− | + | <p> We successfully constructed <font class='mark_backbone'>R0010-B0034-Holin-B0010-B0012</font>. To control the precise suicide timing, we choose a lactose-inducible promoter, BBa_R0010, to regulate this suicide mechanism. Besides holin coding sequence and the inducible promoter, in this composite part, we also choose BBa_B0034 as ribosome binding site, and BBa_B0010 and BBa_B0012 as double terminator.</p> | |
− | + | ||
− | + | <div class="clear"></div> | |
− | + | ||
− | + | <div class="half_column"> | |
− | + | <img src="https://static.igem.org/mediawiki/2017/9/9a/T--NYMU-Taipei--NS_holin_PCR.png" > | |
− | + | <p>This figure is the gel electrophoresis result of holin PCR product. | |
− | + | The marker is 100 bp. The expected length is 657 bp. | |
− | </ | + | </p> |
</div> | </div> | ||
− | + | ||
− | <div class=" | + | <div class="half_column"> |
− | < | + | <img src="https://static.igem.org/mediawiki/2017/3/34/T--NYMU-Taipei--NS_ex_holin_bb_new.png" style="width:80%;"> |
− | + | <p>This figure is the gel electrophoresis result of holin backbone (from BBa_J04450) PCR product. The marker is 1 Kb. The expected length is 2458 bp. | |
− | + | </p> | |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | </ | + | |
</div> | </div> | ||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | + | <div class="clear"></div> | |
+ | |||
+ | <h4></h4> | ||
+ | <h4>Endolysin-Holin Construct</h4> | ||
+ | <div class="half_column"> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/e/e9/T--NYMU-Taipei--NS_endolysin_holin_snapgene.png" > | ||
+ | <p> We successfully constructed <font class='mark_backbone'>R0010-B0034-Holin-B0010-B0012-J23106-B0034-Endolysin-B0010-B0012</font>. We combined holin and endolysin for suicide mechanism. In this composite part, holin functions as an important regulator.</p> | ||
</div> | </div> | ||
+ | |||
+ | <div class="half_column"> | ||
+ | <img src="https://static.igem.org/mediawiki/parts/e/e2/T--NYMU-Taipei%E2%80%94Holin-Endolysin-REcheck.png" style="width:50%;"> | ||
+ | <p>This is the restriction enzyme check gel electrophoresis result of holin-endolysin construct. | ||
+ | The marker is 1 Kb. We used XmaI to digest the sample. The expected total length is 3843 bp. | ||
+ | </p> | ||
+ | </div> | ||
+ | <div class="clear"></div> | ||
+ | |||
+ | <h4></h4> | ||
+ | <h4>Endolysin-Holin-NrtA Construct</h4> | ||
+ | <div class="half_column"> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/c/ca/T--NYMU-Taipei--endolysin_holin_NrtA_snapgene.png"> | ||
+ | <p> We successfully constructed <font class='mark_backbone'>R0010-B0034-Holin-B0010-B0012-J23106-B0034-Endolysin-B0010-B0012-J23118-B0034-NrtA-B0015</font>. This part contains holin, endolysin, and NrtA and has <font class='mark_backbone'>nitrate-capturing</font> function and <font class='mark_backbone'>suicide</font> function.</p> | ||
+ | </div> | ||
+ | |||
+ | <div class="half_column"> | ||
+ | <img src="https://static.igem.org/mediawiki/parts/b/b0/Holin-Endolysin-NrtA_REcheck.png" style="width:70%;"> | ||
+ | <p>This figure is the restriction enzyme check gel electrophoresis result of endolysin-holin-NrtA construct. | ||
+ | We used XmaI to digest sample 1~3. The result showed that 2 and 3 are correct. M represents 1 Kb marker. | ||
+ | </p> | ||
+ | </div> | ||
+ | |||
+ | <div class="clear"></div> | ||
+ | <h4></h4> | ||
+ | <h4 style="color:#000000;">*See our parts: <a href="https://2017.igem.org/Team:NYMU-Taipei/Parts" target="_blank">click</a></h4> | ||
+ | <h4 style="color:#000000;">*See our experiments protocols: <a href="https://2017.igem.org/Team:NYMU-Taipei/Notebook" target="_blank">click</a></h4> | ||
+ | |||
+ | <h4></h4> | ||
+ | <h4></h4> | ||
+ | <h4>Growth Curve Measurement</h4> | ||
+ | <p> In order to determine <font class='mark_backbone'>the correct timing to co-culture our modified <i>E.coli</i> with <i>Chlorella vulgaris</i></font>, we evaluated growth curve of <i>Chlorella vulgaris</i>. We measured the OD value at 680nm by using the spectrophotometer and plotted the growth curve with R software. | ||
+ | <br> However, we encountered some difficulties such as irregular measurement time and personal errors. Therefore, we decided to search for a better measurement method. Later, we borrowed a photo-bioreactor from Professor Ya Tang Yang, Department of Electrical Engineering, National Tsing Hua University, and used it to measure OD values from microalgae cultures. | ||
+ | </p> | ||
+ | <center><img src='https://static.igem.org/mediawiki/2017/4/48/T--NYMU-Taipei--NS_growth_curve_2.png' style='width:60%'></center> | ||
+ | <ol style="color: #205e1a;"> | ||
+ | <li>The above line chart presents growth curve of <i>Chlorella vulgaris</i>. This measurement lasted more than 216 hours and the picture roughly meets our expectation. | ||
+ | <li>Although the later part of growth curve shows violent fluctuating data, it may be due to effects from environmental nitrogen metabolites of <i>Chlorella vulgaris</i>. | ||
+ | <li>The measurement result helps us determine the lipid production duration of <i>Chlorella vulgaris</i>, and the timing to add NrtA-transformed <i>E. coli</i> into the medium. | ||
+ | </ol> | ||
+ | |||
+ | |||
+ | <h4></h4> | ||
+ | <h4>Automatic Measurement</h4> | ||
+ | <p> In the <font class='mark_backbone'>photo-bioreactor</font>, there are four light-emitting diode sources and two photodetectors. Once calibrated, the device can <font class='mark_backbone'>cultivate microbial cells and record their growth expression without human intervention</font>. We measured two different kinds of microalgae during cell growth in the same culture medium, BG-11, respectively. | ||
+ | </p> | ||
+ | <center><img src='https://static.igem.org/mediawiki/2017/7/76/T--NYMU-Taipei--NS_growth_curve_1.jpg' style='width:60%'></center> | ||
+ | <p> The photo-bioreactor was designed by Professor Yang. It was made with Arduino and some circuit components. Yang’s students also helped us assemble the device and taught us how to use this device. The photo-bioreactor itself can detect multiple units of organisms at the same time, it has pumps, fans, stir bars and some light bars. In Yang’s laboratory, we created a calibration curve for correcting and we confirmed that there was a very high degree of correlation between voltage and OD value. This can be observed in the charts below. | ||
+ | </p> | ||
+ | <center><img src='https://static.igem.org/mediawiki/2017/1/13/T--NYMU-Taipei--NS_growth_curve_3.png' style='width:60%'></center> | ||
+ | <br> | ||
+ | <center><img src='https://static.igem.org/mediawiki/2017/2/2f/T--NYMU-Taipei--NS_growth_curve_4.png' style='width:60%'></center> | ||
+ | <br> | ||
+ | <p>And here are our results.</p> | ||
+ | <center><img src='https://static.igem.org/mediawiki/2017/6/6d/T--NYMU-Taipei--NS_growth_curve_5.png' style='width:60%'></center> | ||
+ | <ol style="color: #205e1a;"> | ||
+ | <li>The above line chart presents growth curve of <i>Chlorella vulgaris</i>, under measurement near 10000 minutes made by the photo-bioreactor. | ||
+ | <li>In comparison to the stimulation <font class='mark_backbone'>model</font> we made, the result meets our expectation. | ||
+ | </ol> | ||
+ | <center><img src='https://static.igem.org/mediawiki/2017/6/67/T--NYMU-Taipei--NS_growth_curve_6.png' style='width:60%'></center> | ||
+ | |||
+ | |||
+ | |||
+ | <!--close--> | ||
+ | <br> | ||
+ | <br> | ||
+ | <center> | ||
+ | <a href="#!" onclick="toggleHeight3(this, 7300);" style='color:#56914a'> | ||
+ | click to close | ||
+ | </a> | ||
+ | </center> | ||
+ | </div> | ||
+ | </div> | ||
+ | <!--functional test NrtA--> | ||
+ | <div class='panel'> | ||
+ | <div id="s4" class="expandable" style='height: 30px;padding-top:15px;'> | ||
+ | <a href="#!" onclick="toggleHeight4(this, 2900); return false" | ||
+ | style="font-family:'Acme', sans-serif;font-size:30px;color:#205e1a;height: 30px;"> | ||
+ | NrtA Functional Test | ||
+ | </a> | ||
+ | <p style="padding-top:10px;"></p> | ||
+ | <p> We expect the engineered <i>E.coli</i> with NrtA gene could express NrtA protein. When we co-cultured it with microalgae, the NrtA protein will capture nitrate and nitrite, and the microalgae will undergo nitrogen starvation and produce more oil. To test our hypothesis, first, we should verify whether NrtA protein could capture nitrate and nitrite or not, and then, second, we need to test whether the engineered <i>E.coli</i> could decrease nitrate and nitrite in the medium. </p> | ||
+ | <h4></h4> | ||
+ | <h4>NrtA Protein</h4> | ||
+ | <p> To test whether NrtA protein could capture nitrate and nitrite, we used <font class='mark_backbone'>French Pressure Press</font> to lyse the control group (Negative control: CC) and the NrtA-expressing group, after dialysis in BG-11 medium. We used <font class='mark_backbone'>Cayman Nitrate/Nitrite Colorimetric Assay Kit</font> to measure the nitrate and nitrite concentration in the medium with/without NrtA protein. (You can see the detail in our Notebook page: <a href="https://2017.igem.org/Team:NYMU-Taipei/Notebook">click</a>.)</p> | ||
+ | <p> Here are our results.</p> | ||
+ | <div style="width:60%;margin:auto"> | ||
+ | <img src="https://static.igem.org/mediawiki/parts/8/8c/T--NYMU-Taipei--NS_NrtA_func_1.png" style="width:95%"> | ||
+ | <p style="font-size:16px;">Figure1. Nitrate concentration of cell lysate. | ||
+ | <br>Blank: nitrate concentration assay kit assay buffer. | ||
+ | <br>BG11: microalgae culture medium buffer. | ||
+ | <br>CC: competent cell. | ||
+ | <br>NrtA: NrtA-expressing cell. | ||
+ | </p> | ||
+ | <br> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/e/ec/NYMU_2017_awards_basicpart_3.jpg" style="width:95%"> | ||
+ | <p style="font-size:16px;">Table1. Dunnett’s T3 test of nitrate concentration of cell lysate. | ||
+ | <br>Blank: nitrate concentration assay kit assay buffer. | ||
+ | <br>BG11: microalgae culture medium buffer. | ||
+ | <br>CC: competent cell. | ||
+ | <br>NrtA: NrtA-expressing cell. | ||
+ | </p> | ||
+ | </div> | ||
+ | <br> | ||
+ | <p> The nitrate and nitrite concentration of competent cell and NrtA is significant different. The results indicate that <font class='mark_backbone'>NrtA protein can capture nitrite and nitrate</font>! This is a milestone of our project!</p> | ||
+ | |||
+ | |||
+ | <h4></h4> | ||
+ | <h4>The Engineered <i>E.coli</i> with NrtA Gene</h4> | ||
+ | <p> Then we wanted to know whether the engineered <i>E.coli</i> could decrease nitrate and nitrite in the environment. We used Cayman Nitrate/Nitrite Colorimetric Assay Kit to measure the nitrate and nitrite concentration of the BG-11 medium submerged several hours with/without the NrtA-engineered <i>E.coli</i>.</p> | ||
+ | <p>Here are our results.</p> | ||
+ | <div style="width:60%;margin:auto"> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/0/0d/NYMU_2017_awards_basicpart_2.jpg" style="width:95%"> | ||
+ | <p style="font-size:16px;">Figure 2. Nitrate concentration of cell. | ||
+ | <br>Blank: nitrate concentration assay kit assay buffer. | ||
+ | <br>BG11: microalgae culture medium buffer. | ||
+ | <br>CC: Supernatant of BG-11 submerged competent cell. | ||
+ | <br>NrtA: Supernatant of BG-11 submerged NrtA-engineered <i>E.coli</i>. | ||
+ | </p> | ||
+ | <br> | ||
+ | <img src="https://static.igem.org/mediawiki/parts/0/0d/S_NRTABBB.jpg" style="width:95%"> | ||
+ | <p style="font-size:16px;">Table 2. Dunnett’s T3 test of nitrate concentration of cell. | ||
+ | <br>Blank: nitrate concentration assay kit assay buffer. | ||
+ | <br>BG11: microalgae culture medium buffer. | ||
+ | <br>CC: Supernatant of BG-11 submerged competent cell. | ||
+ | <br>NrtA: Supernatant of BG-11 submerged NrtA-engineered <i>E.coli</i> liquid culture. | ||
+ | </p> | ||
+ | </div> | ||
+ | <br> | ||
+ | <p> The nitrate and nitrite concentration of NrtA and competent cell has slightly but not significant difference. The results imply that NrtA protein could capture nitrate and nitrite while the engineered NrtA-expressing <i>E.coli</i> cannot. The engineered <i>E.coli</i> with NrtA gene can express NrtA protein, while NrtA protein might be in the cytosol, so nitrate and nitrite concentration in the medium cannot be decreased. To make NrtA protein become a secreted protein, we are trying to construct <font class='mark_backbone'>signal peptide sequence</font> into it, so that NrtA protein can be released to the medium.</p> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <!--close--> | ||
+ | <center> | ||
+ | <a href="#!" onclick="toggleHeight4(this, 2900);" style='color:#56914a'> | ||
+ | click to close | ||
+ | </a> | ||
+ | </center> | ||
+ | </div> | ||
+ | </div> | ||
+ | <!--functional test suicide--> | ||
+ | <div class='panel'> | ||
+ | <div id="s6" class="expandable" style='height: 30px;padding-top:15px;'> | ||
+ | <a href="#!" onclick="toggleHeight6(this, 1950); return false" | ||
+ | style="font-family:'Acme', sans-serif;font-size:30px;color:#205e1a;height: 30px;"> | ||
+ | Suicide Mechanism Functional Test | ||
+ | </a> | ||
+ | <p style="padding-top:10px;"></p> | ||
+ | |||
+ | <p> Before we construct our suicide mechanism with NrtA, we want to <font class='mark_backbone'>make sure that our suicide mechanism, endolysin and holin, can be induced by lactose efficiently</font>. Therefore, we tested our suicide mechanism by adding different concentrations of lactose in order to find out the minimum effective concentration. Furthermore, to induce <i>E.coli</i> suicide at the time we want, we also want to know the changing degree of the OD value of bacteria at different time points after the lactose is added.</p> | ||
+ | <p> We measured the OD<sub>600</sub> value of the sample, and then aliquot liquid culture into several cuvettes. Next, we added a different amount of 0.5 mM lactose into cuvettes to form different concentrations of lactose, and measured the OD<sub>600</sub> value per hour.</p> | ||
+ | <center><img src="https://static.igem.org/mediawiki/2017/4/4d/T--NYMU-Taipei--NS_suicide_func.png" style="width:85%"></center> | ||
+ | <p> As the figure shows, <font class='mark_backbone'>the suicide mechanism is induced immediately when lactose is added into the samples</font>. Besides, we can see that the lactose concentration and the degree of decline of OD value are positively correlated.</p> | ||
+ | <p> We also collaborate with team <font class='mark_backbone'>TAS Taipei</font>. They helped us testing our holin-endolysin-NrtA construct.</p> | ||
+ | <center><img src="https://static.igem.org/mediawiki/2017/8/8b/NYMU_2017_collaboration_TFG_.jpg" style="width:70%"></center> | ||
+ | <p> As the figure shows, the trend of the relative absorbance is downward as the lactose is added to induce the suicide mechanism. The concentration of lactose is also positively correlated with the declining degree of the relative absorbance.</p> | ||
+ | <p> We need to mention that there are some differences in the process of team TAS Taipei and our functional tests. We used different medium to cultivate the bacteria. For the bacteria with holin-endolysin construct, we used Luria-Bertani (LB) broth with 0.9% glucose to cultivate; team TAS Taipei cultivates the bacteria with holin-endolysin-NrtA construct with only Luria-Bertani (LB) broth. According to our previous experimental results, the bacterium doesn't seem to grow well in only LB broth medium. Therefore, to make the figure look similar, team TAS Taipei <font class='mark_backbone'>normalized their data</font> (absorbance at 0 hour is the background). <font class='mark_backbone'>Despite the culture medium difference, the results both show that our suicide mechanism can work</font>. | ||
+ | <a href="https://2017.igem.org/Team:NYMU-Taipei/Collaborations">(see more detail about collaboration)</a></p> | ||
+ | |||
+ | <!--close--> | ||
+ | <center> | ||
+ | <a href="#!" onclick="toggleHeight6(this, 1950);" style='color:#56914a'> | ||
+ | click to close | ||
+ | </a> | ||
+ | </center> | ||
+ | </div> | ||
+ | </div> | ||
− | + | <!--reference--> | |
− | + | <div class='panel'> | |
+ | <div id="s5" class="expandable" style='height: 30px;padding-top:15px;'> | ||
+ | <a href="#!" onclick="toggleHeight5(this, 660); return false" | ||
+ | style="font-family:'Acme', sans-serif;font-size:30px;color:#205e1a;height: 30px;"> | ||
+ | Reference | ||
+ | </a> | ||
+ | <p></p> | ||
+ | |||
+ | <div style="font-size:16px;"> | ||
+ | <p style="padding-top:5px;"></p> | ||
+ | <ol> | ||
+ | <p> | ||
+ | <li>J.W. Allen et al. (2015). Triacylglycerol synthesis during nitrogen stress involves the prokaryotic lipid synthesis pathway and acyl chain remodeling in the microalgae <i>Coccomyxa subellipsoidea</i>. <i>Algal Research, 10,</i> 110–120. | ||
+ | </p> | ||
+ | <p> | ||
+ | <li>G. Breuer et al. (2012). The impact of nitrogen starvation on the dynamics of triacylglycerol accumulation in nine microalgae strains. <i>Bioresource Technology, 124,</i> 217–226. | ||
+ | </p> | ||
+ | <p> | ||
+ | <li>Valledor et al. (2014). System-level network analysis of nitrogen starvation and recovery in <i>Chlamydomonas reinhardtii</i> reveals potential new targets for increased lipid accumulation. <i>Biotechnology for Biofuels, 7:171.</i> | ||
+ | </p> | ||
+ | <p> | ||
+ | <li>S. Zhu et al. (2014). Metabolic changes of starch and lipid triggered by nitrogen starvation in the microalga <i>Chlorella zofingiensis</i>. <i>Bioresource Technology, 152,</i> 292–298. | ||
+ | </p> | ||
+ | <p> | ||
+ | <li>J. Jia et al. (2015). Molecular mechanisms for photosynthetic carbon partitioning into storage neutral lipids in <i>Nannochloropsis oceanica</i> under nitrogen-depletion conditions. <i>Algal Research, 7, </i> 66–77. | ||
+ | </p> | ||
+ | <p> | ||
+ | <li>S.K. Lenka et al. (2016). Current advances in molecular, biochemical, and computational modeling analysis of microalgal triacylglycerol biosynthesis. <i>Biotechnology Advances, 34,</i> 1046–1063 | ||
+ | </p> | ||
+ | <p> | ||
+ | <li>A.J. Klok et al. (2013). A model for customising biomass composition in continuous microalgae production. <i>Bioresource Technology, 146,</i> 89–100. | ||
+ | </p> | ||
+ | <p> | ||
+ | <li>H. Abedini Najafabadi et al. (2015). Effect of various carbon sources on biomass and lipid production of <i>Chlorella vulgaris</i> during nutrient sufficient and nitrogen starvation conditions. <i>Bioresource Technology, 180,</i> 311–317. | ||
+ | </p> | ||
+ | <p> | ||
+ | <li>Yu et al. (2011). Modifications of the metabolic pathways of lipid and triacylglycerol production in microalgae. <i>Microbial Cell Factories, 10:91.</i> | ||
+ | </p> | ||
+ | </ol> | ||
+ | <!--close--> | ||
+ | <center> | ||
+ | <a href="#!" onclick="toggleHeight5(this, 660);" style='color:#56914a'> | ||
+ | click to close | ||
+ | </a> | ||
+ | </center> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
</div> | </div> | ||
− | |||
</body> | </body> | ||
</html> | </html> |
Latest revision as of 19:07, 1 November 2017