http://2013.igem.org/wiki/index.php?title=Special:Contributions/Nidhogg&feed=atom&limit=50&target=Nidhogg&year=&month=2013.igem.org - User contributions [en]2024-03-28T15:09:51ZFrom 2013.igem.orgMediaWiki 1.16.5http://2013.igem.org/Team:Nanjing-China/degTeam:Nanjing-China/deg2013-09-28T03:14:34Z<p>Nidhogg: </p>
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Atrazine is quite hard to degrade naturally in the soil, which makes it persist for a long time in the environment once used. This phenomenon can be considerably severe in that atrazine would cause metabolic disorders both in animals and humankind. For many years, we didn't have an efficient solution to this problem.<br><br><br />
However, the discovery of the super power of Arthrobacter aurescens provides us with a light of hope. This amazing species is capable of utilizing atrazine as a sole source of carbon with the help of a series of degrading enzymes which can metabolize atrazine into a kind of nontoxic substance. In our project, we utilize the most useful degrading enzyme TrzN to degrade atrazine. We even mutated the TrzN to make it degrade faster. At the same time, we found a transmembrane transporter TRM, which involves in transporting atrazine from the outside to the inside of the bacteria so that atrazine can be better degraded.<br><br><br />
<strong>Experiments and Results</strong><br />
<br><br />
We took 12 15mL-tubes, divided them into 4 groups: Group 0; Group Wild Type; Group TrzN- and Group TrzN+, 3 tubes each. With the same protocol used to test the function of TRM, we found that TrzN could distinctively degrade atrazine with the bacteria cultivated 24 hours in 37℃ (Fig. 3-6-1). The difference between wild type and TrzN+ was distinctive, suggesting that TrzN is a degrading enzyme of atrazine.<br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/7/75/HPLC_TrzN_Col.jpg"width="400"></p><br/><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-6-1</strong> TrzN can distinctively degrade atrazine. This figure shows the atrazine concentration in supernatant of bacterial cultures, which were cultured for 24 hours in 37℃. Group 0 means no bacteria in the cultures; cultures in Group Wild Type contained the wild type K12 bacteria; cultures in Group Vector contained the strain K12 with pGFP; and cultures in Group TrzN+ contained the bacteria which are able to express TrzN. "*" means the difference between two groups which are connected by half square brackets is distinctive. From this chart we can conclude that TrzNis an efficient degrading enzyme in solving the problem of TrzN.</div><br><br><br />
We also combined the TRM and TrzN together can make the degradation more efficient (more information about <a href="https://2013.igem.org/Team:Nanjing-China/tran"><strong>TRM</strong></a>). This time, we combined the TRM and TrzN together, under the same method of testing the function of TRM and TrzN, we found that the combination of TRM and TrzN could decrease the atrazine concentration in the bacteria culture to a greater extent (Fig. 3-6-2).<br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/e/e8/HPLC_all_Col.jpg"width="550"></p><br/><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-6-2</strong> Combining TRM and TrzN together can make the degradation more efficient. shows the atrazine concentration in supernatant of bacterial cultures, which were cultured for 24 hours in 37℃. Apart from groups shown above, cultures in Group TRM+TrzN+ contained the bacteria which are able to express TRM as well as TrzN. "*" means the difference between two groups which are connected by half square brackets is distinctive. From this chart we can see that the atrazine concentration in supernatant of Group TRM+TrzN+ was even distinctively less than that in supernatant of TRM+. This means combining TRM and TrzN is indeed a wise choice to polish up our system.</div><br><br><br />
According to these data, we find that TRM can indeed transport atrazine from the outside to the inside of the bacteria, and of course, TrzN can degrade atrazine well and fast. Therefore, we could be confident of the fact that we may one day utilize our system to solve the problem of atrazine in real life. <br/><br/> <br />
<br />
<br />
<strong> Reference</strong><br/><br />
[1] 邱玮. 阿特拉津脱氯水解酶 (TrzN) 的定向进化[D]. 南京农业大学, 2011.<br />
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</html></div>Nidhogghttp://2013.igem.org/Team:Nanjing-China/degTeam:Nanjing-China/deg2013-09-28T03:13:40Z<p>Nidhogg: </p>
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Atrazine is quite hard to degrade naturally in the soil, which makes it persist for a long time in the environment once used. This phenomenon can be considerably severe in that atrazine would cause metabolic disorders both in animals and humankind. For many years, we didn't have an efficient solution to this problem.<br><br><br />
However, the discovery of the super power of Arthrobacter aurescens provides us with a light of hope. This amazing species is capable of utilizing atrazine as a sole source of carbon with the help of a series of degrading enzymes which can metabolize atrazine into a kind of nontoxic substance. In our project, we utilize the most useful degrading enzyme TrzN to degrade atrazine. We even mutated the TrzN to make it degrade faster. At the same time, we found a transmembrane transporter TRM, which involves in transporting atrazine from the outside to the inside of the bacteria so that atrazine can be better degraded.<br><br><br />
<strong>Experiments and Results</strong><br />
<br><br />
We took 12 15mL-tubes, divided them into 4 groups: Group 0; Group Wild Type; Group TrzN- and Group TrzN+, 3 tubes each. With the same protocol used to test the function of TRM, we found that TrzN could distinctively degrade atrazine with the bacteria cultivated 24 hours in 37℃ (Fig. 3-6-1). The difference between wild type and TrzN+ was distinctive, suggesting that TrzN is a degrading enzyme of atrazine.<br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/7/75/HPLC_TrzN_Col.jpg"width="400"></p><br/><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-6-1</strong> TrzN can distinctively degrade atrazine. This figure shows the atrazine concentration in supernatant of bacterial cultures, which were cultured for 24 hours in 37℃. Group 0 means no bacteria in the cultures; cultures in Group Wild Type contained the wild type K12 bacteria; cultures in Group Vector contained the strain K12 with pGFP; and cultures in Group TrzN+ contained the bacteria which are able to express TrzN. "*" means the difference between two groups which are connected by half square brackets is distinctive. From this chart we can conclude that TrzNis an efficient degrading enzyme in solving the problem of TrzN.</div><br><br><br />
We also combined the TRM and TrzN together can make the degradation more efficient (more information about <a href="https://2013.igem.org/Team:Nanjing-China/tran"><strong>TRM</strong></a>). This time, we combined the TRM and TrzN together, under the same method of testing the function of TRM and TrzN, we found that the combination of TRM and TrzN could decrease the atrazine concentration in the bacteria culture to a greater extent (Fig. 3-6-2).<br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/e/e8/HPLC_all_Col.jpg"width="550"></p><br/><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-6-2</strong> Combining TRM and TrzN together can make the degradation more efficient. shows the atrazine concentration in supernatant of bacterial cultures, which were cultured for 24 hours in 37℃. Apart from groups shown above, cultures in Group TRM+TrzN+ contained the bacteria which are able to express TRM as well as TrzN. "*" means the difference between two groups which are connected by half square brackets is distinctive. From this chart we can see that the atrazine concentration in supernatant of Group TRM+TrzN+ was even distinctively less than that in supernatant of TRM+. This means combining TRM and TrzN is indeed a wise choice to polish up our system.</div><br><br><br />
According to these data, we find that TRM can indeed transport atrazine from the outside to the inside of the bacteria, and of course, TrzN can degrade atrazine well and fast. Therefore, we could be confident of the fact that we may one day utilize our system to solve the problem of atrazine in real life. <br />
<br />
<br />
<strong> Reference</strong><br/><br />
[1] 邱玮. 阿特拉津脱氯水解酶 (TrzN) 的定向进化[D]. 南京农业大学, 2011.<br />
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</html></div>Nidhogghttp://2013.igem.org/Team:Nanjing-China/achievementTeam:Nanjing-China/achievement2013-09-27T15:25:53Z<p>Nidhogg: </p>
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<dt><a>Achievements</a></dt><br />
<dd class="dd_1"><br />
1. We formed a very tuned team where we all enjoyed working with each other and had fun.<br/><br />
2. For our human practice part, we successfully took a great chance to propagate iGEM, our project as well as synthetic biology to advanced teenagers from all around the nation at China Adolescents Science and Technology Innovation Contest, which is a splendid gala for all brilliant teenagers in China to take part in and show their wisdom.<br/><br />
3. Our bacteria can effectively recognize the presence of atrazine.<br/><br />
4. For the biobrick measurement approach part, we successfully used HPLC to test the function of BBa_K1145001 and BBa_K1145002. The controls were set scientifically and the result was very satisfying.<br/><br />
5. We submitted 7standard biobrick parts with high quality and great performance.<br/><br />
6. We added a LVA-tag at the C-terminal of the primitive CI coding region. This tag will facilitate the turnover of CI in the cells.<br/><br />
7. For our model part, we have introduced the space lattice into our model to better describe the movement of bacteria and the distribution of micromolecule like AHL.<br/><br />
8. We successfully utilized the QS system to recruit more bacteria members to move towards atrazine to degrade it. <br />
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<dt><a>Achievements</a></dt><br />
<dd class="dd_1"><br />
1. We formed a very tuned team where we all enjoyed working with each other and had fun.<br/><br />
2. For our human practice part, we successfully took a great chance to propagate iGEM, our project as well as synthetic biology to advanced teenagers from all around the nation at China Adolescents Science and Technology Innovation Contest, which is a splendid gala for all brilliant teenagers in China to take part in and show their wisdom.<br/><br />
3. Our bacteria can effectively recognize the presence of atrazine.<br/><br />
4. For the biobrick measurement approach part, we successfully used HPLC to test the function of BBa_K1145001 and BBa_K1145002. The controls were set scientifically and the result was very satisfying.<br/><br />
5. We submitted 7standard biobrick parts with high quality and great performance.<br/><br />
6. We added a LVA-tag at the C-terminal of the primitive CI coding region. This tag will facilitate the turnover of CI in the cells.<br />
7. For our model part, we have introduced the space lattice into our model to better describe the movement of bacteria and the distribution of micromolecule like AHL.<br/><br />
8. We successfully utilized the QS system to recruit more bacteria members to move towards atrazine to degrade it. <br />
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<dt><a name="Basic">Extraction of total RNA</a></dt><br />
<dd class="dd_1"><br />
(1) Pick a single colony from a selective plate and inoculate a culture of 5mL LB medium and incubate until OD600 reaches 0.6 at 37℃ with vigorous shaking.<br/><br />
(2) Harvest the bacteria cells by centrifuge at 12000rpm in 1.5mL microcentrifuge and lyse them by repeatedly inhaling and emitting with a pipette in Buffer RL.<br/><br />
(3) Add 0.2mL chloroform every 1mL RL. Shake violently for 15sec and incubate at room temperature for 3min.<br/><br />
(4) Centrifuge at 12000rpm at 4℃ for 10min. The sample will separate into three layers, the bottom layer is the organic layer, the middle and the top layer are colorless water layers, and RNA persists in the water layers. (The volume of the water layers is approximately 60% of the added RL.) Apply the water layers to a new tube.<br/><br />
(5) Add a volume of 70% ethanol (check if anhydrous ethanol is added) to a volume of the solution from last step and mix thoroughly by inverting. There is possibly some precipitation after this step. Apply the solution the possible precipitation to the column RA.<br/><br />
(7) Centrifuge at 10000 rpm for 45s and discard the flow-through.<br/><br />
(8) Add 500μL Buffer RE. Centrifuge at 12000rpm for 60s and discard the flow-through.<br/><br />
(9) Add 700μL Buffer RW (check if anhydrous ethanol is added). Centrifuge at 12000rpm for 60s and discard the flow-through.<br/><br />
(10) Add 500μL Buffer RW. Centrifuge at 12000rpm for 60s and discard the flow-through.<br/><br />
(11) Centrifuge at 12000rpm for 2min and discard the flow-through.<br/><br />
(12) Place the column in a new clean 1.5mL microcentrifuge tube and add 50-80μL ddH2O (better preheated at 65℃) to the center of the column. Stand at room temperature for 2min. Centrifuge at 12000rpm for 1min.<br/><br />
(13) Apply the flow-through from the last step to the center of the column and centrifuge at 12000rpm for 1min. <br />
</dd><br />
</dl><br />
<br />
<dl><br />
<dt><a name="">RT-PCR</a></dt><br />
<dd class="dd_1"><br />
(1) Prepare the following mixture in a microtube:<br><br />
<pre style="width:auto; height:auto; background:none; border:none; color:#676767; font-size:13px; font-family:'Arial';"><br />
Random 6 mers (50μM) 1<br/><br />
dNTP Mixture (10 mM each) 1<br/><br />
Template RNA total RNA less than 5μg<br/><br />
RNase free dH2O up to 10μL<br/><br />
</pre><br />
(2) Keep for 5min at 65℃ and cool immediately on ice.<br><br />
(3) Prepare the reaction mixture by combining the following reagents to a total volume of 20μL:<br><br />
<pre style="width:auto; height:auto; background:none; border:none; color:#676767; font-size:13px; font-family:'Arial';"><br />
Template RNA and Primer Mixture 10μL<br/><br />
5× PrimeScript II Buffer 4μL<br/><br />
RNase Inhibitor (40U/μL) 0.5μl<br/><br />
PrimeScript II RTase( 200U/μl) 1μL<br/><br />
RNase free dH2O up to 20μL<br />
</pre><br />
(4) Mix gently.<br><br />
(5) Incubate the reaction mixture immediately under the following conditions<br><br />
42℃ 60min<br />
(6) Inactivate the enzymes by incubation at 95℃ for 5min, followed by cooling on ice. <br />
</dd><br />
</dl><br />
<br />
<dl><br />
<dt><a name="">PCR</a></dt><br />
<dd class="dd_1"><br />
<strong>3-1 Colony PCR</strong><br><br />
<table cellpadding="0" cellspacing="0" border="0" style="color:#676767; font-size:13px; font-family:'Arial';"><br />
<tr><td style="padding-right:10px">(1) System: </td><td style="padding-right:20px">2×PrimeStar</td><td style="padding-right:20px">12.5μL</td></tr><br />
<tr><td></td><td style="padding-right:20px">Forward primer</td><td style="padding-right:20px">1~2.5μL</td></tr><br />
<tr><td></td><td style="padding-right:20px">Reverse primer</td><td style="padding-right:20px">1~2.5μL</td></tr><br />
<tr><td></td><td style="padding-right:20px">Bacteria solution</td><td style="padding-right:20px">1~2μL</td></tr><br />
<tr><td></td><td style="padding-right:20px">dd water</td><td style="padding-right:20px">up to 25μLL</td></tr><br />
</table><br />
<table cellpadding="0" cellspacing="0" border="0" style="color:#676767; font-size:13px; font-family:'Arial';"><br />
<tr><td style="padding-right:10px">(2) Steps:</td><td style="padding-right:20px">Step 1</td><td style="padding-right:20px">94℃</td></tr><br />
<tr><td></td><td style="padding-right:20px">Step 2</td><td style="padding-right:20px">94℃</td></tr><br />
<tr><td></td><td style="padding-right:20px">Step 3</td><td style="padding-right:20px">Annealing temperature</td></tr><br />
<tr><td></td><td style="padding-right:20px">Step 4</td><td style="padding-right:20px">72℃</td></tr><br />
<tr><td></td><td style="padding-right:20px">Step 5</td><td style="padding-right:20px">go to Step 2, 25~35 cycles</td></tr><br />
<tr><td></td><td style="padding-right:20px">Step 6</td><td style="padding-right:20px">72℃</td></tr><br />
<tr><td></td><td style="padding-right:20px">Step 4</td><td style="padding-right:20px">Pause at 4℃</td></tr><br />
</table><br/><br />
<br />
<strong>3-2 PCR with DNA as Template</strong><br> <br />
<table cellpadding="0" cellspacing="0" border="0" style="color:#676767; font-size:13px; font-family:'Arial';"><br />
<tr><td style="padding-right:10px">(1) System:</td><td style="padding-right:20px">2×PrimeStar</td><td style="padding-right:20px">12.5μL</td></tr><br />
<tr><td></td><td style="padding-right:20px">Forward primer</td><td style="padding-right:20px">1μL</td></tr><br />
<tr><td></td><td style="padding-right:20px">Reverse primer</td><td style="padding-right:20px">1μL</td></tr><br />
<tr><td></td><td style="padding-right:20px">Template DNA</td><td style="padding-right:20px">0.5μL</td></tr><br />
<tr><td></td><td style="padding-right:20px">dd water</td><td style="padding-right:20px">up to 25μL</td></tr><br />
</table><br />
<table cellpadding="0" cellspacing="0" border="0" style="color:#676767; font-size:13px; font-family:'Arial';"><br />
<tr><td style="padding-right:10px">(2) Steps: </td><td style="padding-right:20px">Step 1</td><td style="padding-right:20px">94℃</td></tr><br />
<tr><td></td><td style="padding-right:20px">Step 2</td><td style="padding-right:20px">94℃</td></tr><br />
<tr><td></td><td style="padding-right:20px">Step 3</td><td style="padding-right:20px">Annealing temperature</td></tr><br />
<tr><td></td><td style="padding-right:20px">Step 4</td><td style="padding-right:20px">72℃</td></tr><br />
<tr><td></td><td style="padding-right:20px">Step 5</td><td style="padding-right:20px">go to Step 2, 25~35 cycles</td></tr><br />
<tr><td></td><td style="padding-right:20px">Step 6</td><td style="padding-right:20px">72 ℃</td></tr><br />
<tr><td></td><td style="padding-right:20px">Step 7</td><td style="padding-right:20px">Pause at 4℃</td></tr><br />
</table> <br />
</dd><br />
</dl><br />
<dl><br />
<dt><a href="###">Double Digestion (40μL System)</a></dt><br />
<dd class="dd_1"> <br />
(1) Mix the following in a PCR tube:<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Enzyme A&nbsp;&nbsp;&nbsp;&nbsp;2μL<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Enzyme B&nbsp;&nbsp;&nbsp;&nbsp;2μL<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;10Xbuffer&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;2 or 4μL (depend on the buffer used)<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;BSA&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;0 or 4μL (depend on the buffer used)<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;DNA&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;20μL (insert DNA) or 10μl (vector DNA)<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;dd water&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;up to 40μL<br><br />
(2) Incubate at appropriate temperature (30 or 37℃, depend on the enzymes used) for 8~10h (shorter time if the enzymes used have star activity).<br><br />
(3) Pause at 4 ℃.<br><br />
(4) Gel running and purify the digestion product. <br> <br />
</dd><br />
</dl><br />
<br />
<dl><br />
<dt><a href="###">Agarose gel electrophoresis</a></dt><br />
<dd class="dd_1"><br />
(1) Weigh agarose powder according to the volume of the gel wanted (0.1%) and measure the volume of 1×TAE. Add them to a flask.<br><br />
(2) Melt the mixture in a microwave until the solution becomes clear.<br><br />
(3) Let the solution cool to about 50℃. Add corresponding volume of DuRed (1:10000, V/V) and mix thoroughly.<br><br />
(4) Pour the solution into the gel casting tray with appropriate comb.<br><br />
(5) Let the gel cool until it is solid.<br><br />
(6) Pull out the comb and place the gel in the electrophoresis chamber carefully.<br><br />
(7) Add enough 1×TAE to ensure that there is a thin layer of buffer above the surface of the gel.<br><br />
(8) Pipette DNA samples mixed with appropriate amount of loading buffer and DNA marker into wells on the gel.<br><br />
(9) Run the gel at 135V for about 20min.<br />
<br />
</dd><br />
</dl><br />
<br />
<dl><br />
<dt><a href="###">Gel purification with Axygen Gel Extraction Kit</a></dt><br />
<dd class="dd_1"><br />
<br />
(1) Excise the DNA fragment from the agarose gel with a clean shaver blade.<br><br />
(2) Weigh the gel slice in a 1.5mL microcentrifuge tube and add 3 volumes of Buffer DE-A to 1 volume of gel.<br><br />
(3) Incubate at 75°C for 10min (or until the gel slice has completely dissolved). To help dissolve gel, mix by inverting the tube several times every 2~3 min during the incubation.<br><br />
(4) Add a half volume of Buffer DE-B to 1volume of Buffer DE-A.<br><br />
(5) Add a volume of isopropanol to 1 volume of the gel if the DNA fragment is shorter than 400bp.<br><br />
(6) Apply the solution from the last step to the column.<br><br />
(7) Centrifuge at 12000rpm for 1min and discard the flow-through.<br><br />
(8) Wash the column by adding 500μL Buffer W1 and centrifuge at 12000rpm for 30sec. Discard the flow-through.<br><br />
(9) Wash the column by adding 700μL Buffer W2 and centrifuge at 12000 rpm for 30sec. Discard the flow-through.<br><br />
(10) Wash the column by adding 700μL Buffer W2 and centrifuge at 12000 rpm for 1min. Discard the flow-through.<br><br />
(11) Centrifuge at 12000rpm for 1min to remove the residual wash buffer.<br><br />
(12) Place the column in a new clean 1.5mL microcentrifuge tube and add 30μL ddH2O to the center of the column. Stand for 1min (or longer for better elution).<br><br />
(13) Centrifuge at 12000rpm for 1min (or longer for better elution).<br><br />
(14) Apply the flow-through from the last step to the center of the column. Stand for 1min (or longer for better elution).<br><br />
(15) Centrifuge at 12000rpm for 1min (or longer for better elution) and discard the column.<br><br />
<br />
</dd><br />
</dl><br />
<br />
<dl><br />
<dt><a href="###">Ligation</a></dt><br />
<dd class="dd_1"><br />
(1) Test the concentration of the DNA samples.<br><br />
(2) Pipet the following into a PCR tube:<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Linearized vector DNA <br><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Insert DNA<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Solution I<br><br />
(3) Mix thoroughly and incubate the mixture at 16℃ for 2~3h.<br><br />
(4) Pause at 4℃. <br> <br />
</dd><br />
</dl> <br />
<br />
<dl><br />
<dt><a href="###">Tranformatiom</a></dt><br />
<dd class="dd_1"><br />
(1) Get the competent cell from -80℃ and wait for its fusion on ice.<br><br />
(2) Add the entire ligation product or 1μL plasmid into the tube.<br><br />
(3) Mix and incubate on ice for 30min.<br><br />
(4) Heat pulse at 42℃ for 90sec.<br><br />
(5) Put back the tube on ice and incubate for 5min.<br><br />
(6) Add 1mL LB and incubate at 37℃ with vigorous shaking for 1h.<br><br />
(7) Centrifuge at 4000rpm for 5min and remove most of the supernatant with about 100μL left.<br><br />
(8) Resuspend the compact cells and plate the culture on LB plate containing corresponding antibiotics. <br><br />
</dd><br />
</dl> <br />
<br />
<dl><br />
<dt><a href="###">Mini-prep with Axygen Mini-prep Kit</a></dt><br />
<dd class="dd_1"><br />
(1) Pick a single colony from a selective plate and inoculate a culture of 5mL LB medium containing the appropriate antibiotic. Incubate for proper time (depending on strains and antibiotic) at 37℃ with vigorous shaking.<br><br />
(2) Harvest the bacteria cells by centrifuge at 12000 rpm in 1.5mL microcentrifuge for 1min at room temperature and remove all traces of supernatant.<br><br />
(3) Repeat the last step.<br><br />
(4) Resuspend compact cells in 250μL buffer S1.<br><br />
(5) Add 250μL buffer S2 and mix thoroughly by inverting the tubes 4~6 times.<br><br />
(6) Add 350μL buffer S3 and mix thoroughly by inverting the tubes 6~8 times.<br><br />
(7) Centrifuge at 12000rpm for 10min.<br><br />
(8) Apply the supernatant from the last step to the column.<br><br />
(9) Centrifuge at 12000rpm for 1min and discard the flow-through.<br><br />
(10) Wash the column by adding 500μL buffer W1 and centrifuge at 12000rpm for 1min. Discard the flow-through.<br><br />
(11) Wash the column by adding 700μL buffer W2 and centrifuge at 12000rpm for 1min. Discard the flow-through.<br><br />
(12) Repeat the last step.<br><br />
(13) Centrifuge at 12000rpm for 1min to remove the residual wash buffer.<br><br />
(14) Place the column in a new clean 1.5mL microcentrifuge tube and add 30μL ddH2O to the center of the column. Stand for 1min (or longer for better elution).<br><br />
(15) Centrifuge at 12000rpm for 1min (or longer for better elution).<br><br />
(16) Apply the flow-through from the last step to the center of the column. Stand for 1min (or longer for better elution).<br><br />
(17) Centrifuge at 12000rpm for 1min (or longer for better elution) and discard the column.<br> <br />
</dd><br />
</dl><br />
<br />
<dl><br />
<dt><a href="###">Preparation of competent cells</a></dt><br />
<dd class="dd_1"><br />
(1) Pick a single colony from a selective plate and inoculate a culture of 20mL LB medium. Incubate overnight at 37℃ with vigorous shaking.<br><br />
(2) Add 2mL of the incubated bacteria solution into 50mL LB medium. Incubate for about 1.5h by shaking at 220rpm until OD600 grows to between 0.5 and 0.6.<br><br />
(3) Incubate on ice for 15min. Harvest the bacteria cells by centrifuge at 6000rpm for 5min at 4℃.<br><br />
(4) Remove all traces of supernatant and add 10 ml precooled buffer I on ice. Shake slightly to resuspend the cells.<br><br />
(5) Put the tube on ice for 2h. Harvest the bacteria cells by centrifuge at 6000rpm for 5min at 4℃.<br><br />
(6) Remove all traces of supernatant and resuspend the cells with 3.5mL precooled buffer II.<br><br />
(7) For every 100μL, separate the solution into precooled sterile Eppendorf tube.<br><br />
(8) Store at -80℃ after liquid nitrogen freezing.<br><br />
<table cellpadding="0" cellspacing="0" border="0" style="color:#676767; font-size:13px; font-family:'Arial';"><br />
<tr><td style="padding-right:10px">Buffer I:</td><td style="padding-right:20px">100mM RbCl </td><td style="padding-right:20px">1.2g/100mL</td></tr><br />
<tr><td></td><td style="padding-right:20px">45mM MnCl2·4H2O</td><td style="padding-right:20px">0.891g/100mL</td></tr><br />
<tr><td></td><td style="padding-right:20px">30mM KAc(pH 7.5) </td><td style="padding-right:20px">0.294g/100mL</td></tr><br />
<tr><td></td><td style="padding-right:20px">10mM CaCl2·2H2O</td><td style="padding-right:20px">0.147g/100mL</td></tr><br />
<tr><td></td><td style="padding-right:20px">glycerol (pH 6.8)</td><td style="padding-right:20px">15% (w/v)</td></tr><br />
</table><br />
<table cellpadding="0" cellspacing="0" border="0" style="color:#676767; font-size:13px; font-family:'Arial';"><br />
<tr><td style="padding-right:10px">Buffer II: </td><td style="padding-right:20px">10mM MOPS </td><td style="padding-right:20px">0.209g/100mL</td></tr><br />
<tr><td></td><td style="padding-right:20px">10mM RbCl </td><td style="padding-right:20px">0.12g/100mL</td></tr><br />
<tr><td></td><td style="padding-right:20px">75mM CaCl2·2H2O</td><td style="padding-right:20px">1.1025g/100mL</td></tr><br />
<tr><td></td><td style="padding-right:20px">glycerol (pH 6.8)</td><td style="padding-right:20px">15% (w/v)</td></tr><br />
</table><br />
</pre> <br />
</dd><br />
</dl><br />
<br />
<dl><br />
<dt><a name="Observation">Measuring GFP expression with ELISA</a></dt><br />
<dd class="dd_1"><br />
(1) Pick a single colony with required plasmids from a selective plate and inoculate a culture of 5mL LB medium containing the appropriate antibiotic.<br><br />
(2) Incubate at 37℃ for 20h with vigorous shaking.<br><br />
(3) Sample 1mL of the bacteria solution every 2h until 10h and sample 1mL of the bacteria solution after 20h since the start of the incubation.<br><br />
(4) Measure the fluorescence (excitation: 485nm, detection: 535nm) <br> <br />
</dd><br />
</dl><br />
<br />
<dl><br />
<dt><a href="###">Observation GFP expression with Confocal Microscope</a></dt><br />
<dd class="dd_1"><br />
(1) Pick a single colony with required plasmids from a selective plate and inoculate a culture of 5mL LB medium containing the appropriate antibiotic.<br><br />
(2) Incubate at 37℃ for 20h with vigorous shaking.<br><br />
(3) Sample 1mL of the bacteria solution every 2h until 10h and sample 1mL of the bacteria solution after 20h since the start of the incubation.<br><br />
(4) Observation GFP expression with Confocal Microscope. <br> <br />
</dd><br />
</dl><br />
<br />
<dl><br />
<dt><a name="Drawing">Drawing growth curve</a></dt><br />
<dd class="dd_1"><br />
<br />
(1) Prepare the mother solution of atrazine (200mmol/L) with ethanol.<br><br />
(2) Dilute the mother solution with LB into four different concentrations of atrazine: 200μmol/L; 500μmol/L; 1000μmol/L.<br><br />
(3) Take 52 15mL tubes, divide them into four groups: Group 0, Group 200, Group 500 and Group 1000, 14 tubes each. <br><br />
(4) Label number 0-13 on each tube in each group. (Each group has tubes labeled 1-13.)<br><br />
(5) Add 10mL diluted atrazine solutions into Group 0, 200, 500 and 1000. 0μmol/L atrazine solution for Group 0, 200μmol/L atrazine solution for Group 200, 500μmol/L atrazine solution for Group 500 and 1000μmol/L atrazine solution for Group 1000.<br><br />
(6) Add 1μL Bacteria culture (K12) into each tube, and put them all together into the shaker.<br><br />
(7) Set the speed of the shaker to 200rpm.<br><br />
(8) Take corresponding tubes out of the shaker every 1h. For example, tubes which are labeled 4 should be taken out after four hours, tubes which are labeled 13 should be taken out after 13 hours.<br><br />
(9) Test the OD of the solution in the tubes immediately. Each tube has to be tested for three times. (Use the bacteria-free atrazine solution with corresponding concentration as control.)<br><br />
(10) Record the data and analyze them. <br> <br />
</dd><br />
</dl> <br />
<br />
<dl><br />
<dt><a name="HPLC">HPLC</a></dt><br />
<dd class="dd_1"><br />
(1) Prepare the mother solution of atrazine (100mmol/L) with ethanol.<br><br />
(2) Dilute the mother solution with LB into 500μmol/L.<br><br />
(3) Take 24 15mL tubes, divide them into 8 groups: 0, K12, GFP, TRM, TrzN-, TrzN+, TRM+TrzN-, TRM+TrzN+, 3 tubes each.<br><br />
(4) Add 10mL 500μmol/L atrazine solution into each tube.<br><br />
(5) Do nothing to Group 0; add 1μL bacteria culture with corresponding features into each group of tubes.<br> <br />
(6) Put all the tubes into the shaker, set the speed to 200rpm.<br><br />
(7) Add 12μL solution IPTG into Group TrzN+ and Group TRM+TrzN+ to induce the expression of TrzN after 5 hours of culture.<br><br />
(8) After 24h of culture, take all the tubes out and put them on the ice.<br><br />
(9) Treat all the tubes with centrifuge for 10000rpm, 2min.<br><br />
(10) Filtrate the 1mL supernate in each tube with 0.22μm-filter membrane into EP tubes.<br><br />
(11) Analyze the samples with HPLC. <br> <br />
</dd><br />
</dl> <br />
<br />
<dl><br />
<dt><a name="Verification">Verification of the motility from phenotype</a></dt><br />
<dd class="dd_1"><br />
(1) Pick a single colony with required plasmids from a selective plate and inoculate a culture of 5mL LB medium containing the appropriate antibiotic.<br><br />
(2) Incubate at 37℃ for appropriate time with vigorous shaking until the bacteria reach their plateau phase.<br><br />
(3) Add 5μL bacteria solution from the last step into 5mL LB medium and incubate at 37℃ for 3h with vigorous shaking.<br><br />
(4) Inoculate 2μL suspension from the last step on semisolid (the concentration of agar or agarose depends) LB plate.<br><br />
(5) Incubate at 37℃ for 20h.<br><br />
(6) Take photos with Gel Analysis System.<br><br />
</pre> <br />
</dd><br />
</dl> <br />
<br />
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<dl><br />
<dt><a name="Basic">Extraction of total RNA</a></dt><br />
<dd class="dd_1"><br />
(1) Pick a single colony from a selective plate and inoculate a culture of 5mL LB medium and incubate until OD600 reaches 0.6 at 37℃ with vigorous shaking.<br/><br />
(2) Harvest the bacteria cells by centrifuge at 12000rpm in 1.5mL microcentrifuge and lyse them by repeatedly inhaling and emitting with a pipette in Buffer RL.<br/><br />
(3) Add 0.2mL chloroform every 1mL RL. Shake violently for 15sec and incubate at room temperature for 3min.<br/><br />
(4) Centrifuge at 12000rpm at 4℃ for 10min. The sample will separate into three layers, the bottom layer is the organic layer, the middle and the top layer are colorless water layers, and RNA persists in the water layers. (The volume of the water layers is approximately 60% of the added RL.) Apply the water layers to a new tube.<br/><br />
(5) Add a volume of 70% ethanol (check if anhydrous ethanol is added) to a volume of the solution from last step and mix thoroughly by inverting. There is possibly some precipitation after this step. Apply the solution the possible precipitation to the column RA.<br/><br />
(7) Centrifuge at 10000 rpm for 45s and discard the flow-through.<br/><br />
(8) Add 500μL Buffer RE. Centrifuge at 12000rpm for 60s and discard the flow-through.<br/><br />
(9) Add 700μL Buffer RW (check if anhydrous ethanol is added). Centrifuge at 12000rpm for 60s and discard the flow-through.<br/><br />
(10) Add 500μL Buffer RW. Centrifuge at 12000rpm for 60s and discard the flow-through.<br/><br />
(11) Centrifuge at 12000rpm for 2min and discard the flow-through.<br/><br />
(12) Place the column in a new clean 1.5mL microcentrifuge tube and add 50-80μL ddH2O (better preheated at 65℃) to the center of the column. Stand at room temperature for 2min. Centrifuge at 12000rpm for 1min.<br/><br />
(13) Apply the flow-through from the last step to the center of the column and centrifuge at 12000rpm for 1min. <br />
</dd><br />
</dl><br />
<br />
<dl><br />
<dt><a name="">RT-PCR</a></dt><br />
<dd class="dd_1"><br />
(1) Prepare the following mixture in a microtube:<br><br />
<pre style="width:auto; height:auto; background:none; border:none; color:#676767; font-size:13px; font-family:'Arial';"><br />
Random 6 mers (50μM) 1<br/><br />
dNTP Mixture (10 mM each) 1<br/><br />
Template RNA total RNA less than 5μg<br/><br />
RNase free dH2O up to 10μL<br/><br />
</pre><br />
(2) Keep for 5min at 65℃ and cool immediately on ice.<br><br />
(3) Prepare the reaction mixture by combining the following reagents to a total volume of 20μL:<br><br />
<pre style="width:auto; height:auto; background:none; border:none; color:#676767; font-size:13px; font-family:'Arial';"><br />
Template RNA and Primer Mixture 10μL<br/><br />
5× PrimeScript II Buffer 4μL<br/><br />
RNase Inhibitor (40U/μL) 0.5μl<br/><br />
PrimeScript II RTase( 200U/μl) 1μL<br/><br />
RNase free dH2O up to 20μL<br />
</pre><br />
(4) Mix gently.<br><br />
(5) Incubate the reaction mixture immediately under the following conditions<br><br />
42℃ 60min<br />
(6) Inactivate the enzymes by incubation at 95℃ for 5min, followed by cooling on ice. <br />
</dd><br />
</dl><br />
<br />
<dl><br />
<dt><a name="">PCR</a></dt><br />
<dd class="dd_1"><br />
<strong>3-1 Colony PCR</strong><br><br />
<table cellpadding="0" cellspacing="0" border="0" style="color:#676767; font-size:13px; font-family:'Arial';"><br />
<tr><td style="padding-right:10px">(1) System: </td><td style="padding-right:20px">2×PrimeStar</td><td style="padding-right:20px">12.5μL</td></tr><br />
<tr><td></td><td style="padding-right:20px">Forward primer</td><td style="padding-right:20px">1~2.5μL</td></tr><br />
<tr><td></td><td style="padding-right:20px">Reverse primer</td><td style="padding-right:20px">1~2.5μL</td></tr><br />
<tr><td></td><td style="padding-right:20px">Bacteria solution</td><td style="padding-right:20px">1~2μL</td></tr><br />
<tr><td></td><td style="padding-right:20px">dd water</td><td style="padding-right:20px">up to 25μLL</td></tr><br />
</table><br />
<table cellpadding="0" cellspacing="0" border="0" style="color:#676767; font-size:13px; font-family:'Arial';"><br />
<tr><td style="padding-right:10px">(2) Steps:</td><td style="padding-right:20px">Step 1</td><td style="padding-right:20px">94℃</td></tr><br />
<tr><td></td><td style="padding-right:20px">Step 2</td><td style="padding-right:20px">94℃</td></tr><br />
<tr><td></td><td style="padding-right:20px">Step 3</td><td style="padding-right:20px">Annealing temperature</td></tr><br />
<tr><td></td><td style="padding-right:20px">Step 4</td><td style="padding-right:20px">72℃</td></tr><br />
<tr><td></td><td style="padding-right:20px">Step 5</td><td style="padding-right:20px">go to Step 2, 25~35 cycles</td></tr><br />
<tr><td></td><td style="padding-right:20px">Step 6</td><td style="padding-right:20px">72℃</td></tr><br />
<tr><td></td><td style="padding-right:20px">Step 4</td><td style="padding-right:20px">Pause at 4℃</td></tr><br />
</table><br/><br />
<br />
<strong>3-2 PCR with DNA as Template</strong><br> <br />
<table cellpadding="0" cellspacing="0" border="0" style="color:#676767; font-size:13px; font-family:'Arial';"><br />
<tr><td style="padding-right:10px">(1) System:</td><td style="padding-right:20px">2×PrimeStar</td><td style="padding-right:20px">12.5μL</td></tr><br />
<tr><td></td><td style="padding-right:20px">Forward primer</td><td style="padding-right:20px">1μL</td></tr><br />
<tr><td></td><td style="padding-right:20px">Reverse primer</td><td style="padding-right:20px">1μL</td></tr><br />
<tr><td></td><td style="padding-right:20px">Template DNA</td><td style="padding-right:20px">0.5μL</td></tr><br />
<tr><td></td><td style="padding-right:20px">dd water</td><td style="padding-right:20px">up to 25μL</td></tr><br />
</table><br />
<table cellpadding="0" cellspacing="0" border="0" style="color:#676767; font-size:13px; font-family:'Arial';"><br />
<tr><td style="padding-right:10px">(2) Steps: </td><td style="padding-right:20px">Step 1</td><td style="padding-right:20px">94℃</td></tr><br />
<tr><td></td><td style="padding-right:20px">Step 2</td><td style="padding-right:20px">94℃</td></tr><br />
<tr><td></td><td style="padding-right:20px">Step 3</td><td style="padding-right:20px">Annealing temperature</td></tr><br />
<tr><td></td><td style="padding-right:20px">Step 4</td><td style="padding-right:20px">72℃</td></tr><br />
<tr><td></td><td style="padding-right:20px">Step 5</td><td style="padding-right:20px">go to Step 2, 25~35 cycles</td></tr><br />
<tr><td></td><td style="padding-right:20px">Step 6</td><td style="padding-right:20px">72 ℃</td></tr><br />
<tr><td></td><td style="padding-right:20px">Step 7</td><td style="padding-right:20px">Pause at 4℃</td></tr><br />
</table> <br />
</dd><br />
</dl><br />
<dl><br />
<dt><a href="###">Double Digestion (40μL System)</a></dt><br />
<dd class="dd_1"> <br />
(1) Mix the following in a PCR tube:<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Enzyme A&nbsp;&nbsp;&nbsp;&nbsp;2μL<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Enzyme B&nbsp;&nbsp;&nbsp;&nbsp;2μL<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;10Xbuffer&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;2 or 4μL (depend on the buffer used)<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;BSA&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;0 or 4μL (depend on the buffer used)<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;DNA&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;20μL (insert DNA) or 10μl (vector DNA)<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;dd water&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;up to 40μL<br><br />
(2) Incubate at appropriate temperature (30 or 37℃, depend on the enzymes used) for 8~10h (shorter time if the enzymes used have star activity).<br><br />
(3) Pause at 4 ℃.<br><br />
(4) Gel running and purify the digestion product. <br> <br />
</dd><br />
</dl><br />
<br />
<dl><br />
<dt><a href="###">Agarose gel electrophoresis</a></dt><br />
<dd class="dd_1"><br />
(1) Weigh agarose powder according to the volume of the gel wanted (0.1%) and measure the volume of 1×TAE. Add them to a flask.<br><br />
(2) Melt the mixture in a microwave until the solution becomes clear.<br><br />
(3) Let the solution cool to about 50℃. Add corresponding volume of DuRed (1:10000, V/V) and mix thoroughly.<br><br />
(4) Pour the solution into the gel casting tray with appropriate comb.<br><br />
(5) Let the gel cool until it is solid.<br><br />
(6) Pull out the comb and place the gel in the electrophoresis chamber carefully.<br><br />
(7) Add enough 1×TAE to ensure that there is a thin layer of buffer above the surface of the gel.<br><br />
(8) Pipette DNA samples mixed with appropriate amount of loading buffer and DNA marker into wells on the gel.<br><br />
(9) Run the gel at 135V for about 20min.<br />
<br />
</dd><br />
</dl><br />
<br />
<dl><br />
<dt><a href="###">Gel purification with Axygen Gel Extraction Kit</a></dt><br />
<dd class="dd_1"><br />
<br />
(1) Excise the DNA fragment from the agarose gel with a clean shaver blade.<br><br />
(2) Weigh the gel slice in a 1.5mL microcentrifuge tube and add 3 volumes of Buffer DE-A to 1 volume of gel.<br><br />
(3) Incubate at 75°C for 10min (or until the gel slice has completely dissolved). To help dissolve gel, mix by inverting the tube several times every 2~3 min during the incubation.<br><br />
(4) Add a half volume of Buffer DE-B to 1volume of Buffer DE-A.<br><br />
(5) Add a volume of isopropanol to 1 volume of the gel if the DNA fragment is shorter than 400bp.<br><br />
(6) Apply the solution from the last step to the column.<br><br />
(7) Centrifuge at 12000rpm for 1min and discard the flow-through.<br><br />
(8) Wash the column by adding 500μL Buffer W1 and centrifuge at 12000rpm for 30sec. Discard the flow-through.<br><br />
(9) Wash the column by adding 700μL Buffer W2 and centrifuge at 12000 rpm for 30sec. Discard the flow-through.<br><br />
(10) Wash the column by adding 700μL Buffer W2 and centrifuge at 12000 rpm for 1min. Discard the flow-through.<br><br />
(11) Centrifuge at 12000rpm for 1min to remove the residual wash buffer.<br><br />
(12) Place the column in a new clean 1.5mL microcentrifuge tube and add 30μL ddH2O to the center of the column. Stand for 1min (or longer for better elution).<br><br />
(13) Centrifuge at 12000rpm for 1min (or longer for better elution).<br><br />
(14) Apply the flow-through from the last step to the center of the column. Stand for 1min (or longer for better elution).<br><br />
(15) Centrifuge at 12000rpm for 1min (or longer for better elution) and discard the column.<br><br />
<br />
</dd><br />
</dl><br />
<br />
<dl><br />
<dt><a href="###">Ligation</a></dt><br />
<dd class="dd_1"><br />
(1) Test the concentration of the DNA samples.<br><br />
(2) Pipet the following into a PCR tube:<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Linearized vector DNA <br><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Insert DNA<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Solution I<br><br />
(3) Mix thoroughly and incubate the mixture at 16℃ for 2~3h.<br><br />
(4) Pause at 4℃. <br> <br />
</dd><br />
</dl> <br />
<br />
<dl><br />
<dt><a href="###">Tranformatiom</a></dt><br />
<dd class="dd_1"><br />
(1) Get the competent cell from -80℃ and wait for its fusion on ice.<br><br />
(2) Add the entire ligation product or 1μL plasmid into the tube.<br><br />
(3) Mix and incubate on ice for 30min.<br><br />
(4) Heat pulse at 42℃ for 90sec.<br><br />
(5) Put back the tube on ice and incubate for 5min.<br><br />
(6) Add 1mL LB and incubate at 37℃ with vigorous shaking for 1h.<br><br />
(7) Centrifuge at 4000rpm for 5min and remove most of the supernatant with about 100μL left.<br><br />
(8) Resuspend the compact cells and plate the culture on LB plate containing corresponding antibiotics. <br><br />
</dd><br />
</dl> <br />
<br />
<dl><br />
<dt><a href="###">Mini-prep with Axygen Mini-prep Kit</a></dt><br />
<dd class="dd_1"><br />
(1) Pick a single colony from a selective plate and inoculate a culture of 5mL LB medium containing the appropriate antibiotic. Incubate for proper time (depending on strains and antibiotic) at 37℃ with vigorous shaking.<br><br />
(2) Harvest the bacteria cells by centrifuge at 12000 rpm in 1.5mL microcentrifuge for 1min at room temperature and remove all traces of supernatant.<br><br />
(3) Repeat the last step.<br><br />
(4) Resuspend compact cells in 250μL buffer S1.<br><br />
(5) Add 250μL buffer S2 and mix thoroughly by inverting the tubes 4~6 times.<br><br />
(6) Add 350μL buffer S3 and mix thoroughly by inverting the tubes 6~8 times.<br><br />
(7) Centrifuge at 12000rpm for 10min.<br><br />
(8) Apply the supernatant from the last step to the column.<br><br />
(9) Centrifuge at 12000rpm for 1min and discard the flow-through.<br><br />
(10) Wash the column by adding 500μL buffer W1 and centrifuge at 12000rpm for 1min. Discard the flow-through.<br><br />
(11) Wash the column by adding 700μL buffer W2 and centrifuge at 12000rpm for 1min. Discard the flow-through.<br><br />
(12) Repeat the last step.<br><br />
(13) Centrifuge at 12000rpm for 1min to remove the residual wash buffer.<br><br />
(14) Place the column in a new clean 1.5mL microcentrifuge tube and add 30μL ddH2O to the center of the column. Stand for 1min (or longer for better elution).<br><br />
(15) Centrifuge at 12000rpm for 1min (or longer for better elution).<br><br />
(16) Apply the flow-through from the last step to the center of the column. Stand for 1min (or longer for better elution).<br><br />
(17) Centrifuge at 12000rpm for 1min (or longer for better elution) and discard the column.<br> <br />
</dd><br />
</dl><br />
<br />
<dl><br />
<dt><a href="###">Preparation of competent cells</a></dt><br />
<dd class="dd_1"><br />
(1) Pick a single colony from a selective plate and inoculate a culture of 20mL LB medium. Incubate overnight at 37℃ with vigorous shaking.<br><br />
(2) Add 2mL of the incubated bacteria solution into 50mL LB medium. Incubate for about 1.5h by shaking at 220rpm until OD600 grows to between 0.5 and 0.6.<br><br />
(3) Incubate on ice for 15min. Harvest the bacteria cells by centrifuge at 6000rpm for 5min at 4℃.<br><br />
(4) Remove all traces of supernatant and add 10 ml precooled buffer I on ice. Shake slightly to resuspend the cells.<br><br />
(5) Put the tube on ice for 2h. Harvest the bacteria cells by centrifuge at 6000rpm for 5min at 4℃.<br><br />
(6) Remove all traces of supernatant and resuspend the cells with 3.5mL precooled buffer II.<br><br />
(7) For every 100μL, separate the solution into precooled sterile Eppendorf tube.<br><br />
(8) Store at -80℃ after liquid nitrogen freezing.<br><br />
<table cellpadding="0" cellspacing="0" border="0" style="color:#676767; font-size:13px; font-family:'Arial';"><br />
<tr><td style="padding-right:10px">Buffer I:</td><td style="padding-right:20px">100mM RbCl </td><td style="padding-right:20px">1.2g/100mL</td></tr><br />
<tr><td></td><td style="padding-right:20px">45mM MnCl2·4H2O</td><td style="padding-right:20px">0.891g/100mL</td></tr><br />
<tr><td></td><td style="padding-right:20px">30mM KAc(pH 7.5) </td><td style="padding-right:20px">0.294g/100mL</td></tr><br />
<tr><td></td><td style="padding-right:20px">10mM CaCl2·2H2O</td><td style="padding-right:20px">0.147g/100mL</td></tr><br />
<tr><td></td><td style="padding-right:20px">glycerol (pH 6.8)</td><td style="padding-right:20px">15% (w/v)</td></tr><br />
</table><br />
<table cellpadding="0" cellspacing="0" border="0" style="color:#676767; font-size:13px; font-family:'Arial';"><br />
<tr><td style="padding-right:10px">Buffer II: </td><td style="padding-right:20px">10mM MOPS </td><td style="padding-right:20px">0.209g/100mL</td></tr><br />
<tr><td></td><td style="padding-right:20px">10mM RbCl </td><td style="padding-right:20px">0.12g/100mL</td></tr><br />
<tr><td></td><td style="padding-right:20px">75mM CaCl2·2H2O</td><td style="padding-right:20px">1.1025g/100mL</td></tr><br />
<tr><td></td><td style="padding-right:20px">glycerol (pH 6.8)</td><td style="padding-right:20px">15% (w/v)</td></tr><br />
</table><br />
</pre> <br />
</dd><br />
</dl><br />
<br />
<dl><br />
<dt><a name="Observation">Measuring GFP expression with ELISA</a></dt><br />
<dd class="dd_1"><br />
(1) Pick a single colony with required plasmids from a selective plate and inoculate a culture of 5mL LB medium containing the appropriate antibiotic.<br><br />
(2) Incubate at 37℃ for 20h with vigorous shaking.<br><br />
(3) Sample 1mL of the bacteria solution every 2h until 10h and sample 1mL of the bacteria solution after 20h since the start of the incubation.<br><br />
(4) Measure the fluorescence (excitation: 485nm, detection: 535nm) <br> <br />
</dd><br />
</dl><br />
<br />
<dl><br />
<dt><a href="###">Observation GFP expression with Confocal Microscope</a></dt><br />
<dd class="dd_1"><br />
(1) Pick a single colony with required plasmids from a selective plate and inoculate a culture of 5mL LB medium containing the appropriate antibiotic.<br><br />
(2) Incubate at 37℃ for 20h with vigorous shaking.<br><br />
(3) Sample 1mL of the bacteria solution every 2h until 10h and sample 1mL of the bacteria solution after 20h since the start of the incubation.<br><br />
(4) Observation GFP expression with Confocal Microscope. <br> <br />
</dd><br />
</dl><br />
<br />
<dl><br />
<dt><a name="Drawing">Drawing growth curve</a></dt><br />
<dd class="dd_1"><br />
<br />
(1) Prepare the mother solution of atrazine (200mmol/L) with ethanol.<br><br />
(2) Dilute the mother solution with LB into four different concentrations of atrazine: 200μmol/L; 500μmol/L; 1000μmol/L.<br><br />
(3) Take 52 15mL tubes, divide them into four groups: Group 0, Group 200, Group 500 and Group 1000, 14 tubes each. <br><br />
(4) Label number 0-13 on each tube in each group. (Each group has tubes labeled 1-13.)<br><br />
(5) Add 10mL diluted atrazine solutions into Group 0, 200, 500 and 1000. 0μmol/L atrazine solution for Group 0, 200μmol/L atrazine solution for Group 200, 500μmol/L atrazine solution for Group 500 and 1000μmol/L atrazine solution for Group 1000.<br><br />
(6) Add 1μL Bacteria culture (K12) into each tube, and put them all together into the shaker.<br><br />
(7) Set the speed of the shaker to 200rpm.<br><br />
(8) Take corresponding tubes out of the shaker every 1h. For example, tubes which are labeled 4 should be taken out after four hours, tubes which are labeled 13 should be taken out after 13 hours.<br><br />
(9) Test the OD of the solution in the tubes immediately. Each tube has to be tested for three times. (Use the bacteria-free atrazine solution with corresponding concentration as control.)<br><br />
(10) Record the data and analyze them. <br> <br />
</dd><br />
</dl> <br />
<br />
<dl><br />
<dt><a name="HPLC">HPLC</a></dt><br />
<dd class="dd_1"><br />
(1) Prepare the mother solution of atrazine (100mmol/L) with ethanol.<br><br />
(2) Dilute the mother solution with LB into 500μmol/L.<br><br />
(3) Take 24 15mL tubes, divide them into 8 groups: 0, K12, GFP, TRM, TrzN-, TrzN+, TRM+TrzN-, TRM+TrzN+, 3 tubes each.<br><br />
(4) Add 10mL 500μmol/L atrazine solution into each tube.<br><br />
(5) Do nothing to Group 0; add 1μL bacteria culture with corresponding features into each group of tubes.<br> <br />
(6) Put all the tubes into the shaker, set the speed to 200rpm.<br><br />
(7) Add 12μL solution IPTG into Group TrzN+ and Group TRM+TrzN+ to induce the expression of TrzN after 5 hours of culture.<br><br />
(8) After 24h of culture, take all the tubes out and put them on the ice.<br><br />
(9) Treat all the tubes with centrifuge for 10000rpm, 2min.<br><br />
(10) Filtrate the 1mL supernate in each tube with 0.22μm-filter membrane into EP tubes.<br><br />
(11) Analyze the samples with HPLC. <br> <br />
</dd><br />
</dl> <br />
<br />
<dl><br />
<dt><a name="Verification">Verification of the motility from phenotype</a></dt><br />
<dd class="dd_1"><br />
(1) Pick a single colony with required plasmids from a selective plate and inoculate a culture of 5mL LB medium containing the appropriate antibiotic.<br><br />
(2) Incubate at 37℃ for appropriate time with vigorous shaking until the bacteria reach their plateau phase.<br><br />
(3) Add 5μL bacteria solution from the last step into 5mL LB medium and incubate at 37℃ for 3h with vigorous shaking.<br><br />
(4) Inoculate 2μL suspension from the last step on semisolid (the concentration of agar or agarose depends) LB plate.<br><br />
(5) Incubate at 37℃ for 20h.<br><br />
(6) Take photos with Gel Analysis System.<br><br />
</pre> <br />
</dd><br />
</dl> <br />
<br />
</div><br />
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</html></div>Nidhogghttp://2013.igem.org/Team:Nanjing-China/protocolTeam:Nanjing-China/protocol2013-09-27T15:17:47Z<p>Nidhogg: </p>
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<dl><br />
<dt><a name="Basic">Extraction of mRNA</a></dt><br />
<dd class="dd_1"><br />
(1) Pick a single colony from a selective plate and inoculate a culture of 5mL LB medium and incubate until OD600 reaches 0.6 at 37℃ with vigorous shaking.<br/><br />
(2) Harvest the bacteria cells by centrifuge at 12000rpm in 1.5mL microcentrifuge and lyse them by repeatedly inhaling and emitting with a pipette in Buffer RL.<br/><br />
(3) Add 0.2mL chloroform every 1mL RL. Shake violently for 15sec and incubate at room temperature for 3min.<br/><br />
(4) Centrifuge at 12000rpm at 4℃ for 10min. The sample will separate into three layers, the bottom layer is the organic layer, the middle and the top layer are colorless water layers, and RNA persists in the water layers. (The volume of the water layers is approximately 60% of the added RL.) Apply the water layers to a new tube.<br/><br />
(5) Add a volume of 70% ethanol (check if anhydrous ethanol is added) to a volume of the solution from last step and mix thoroughly by inverting. There is possibly some precipitation after this step. Apply the solution the possible precipitation to the column RA.<br/><br />
(7) Centrifuge at 10000 rpm for 45s and discard the flow-through.<br/><br />
(8) Add 500μL Buffer RE. Centrifuge at 12000rpm for 60s and discard the flow-through.<br/><br />
(9) Add 700μL Buffer RW (check if anhydrous ethanol is added). Centrifuge at 12000rpm for 60s and discard the flow-through.<br/><br />
(10) Add 500μL Buffer RW. Centrifuge at 12000rpm for 60s and discard the flow-through.<br/><br />
(11) Centrifuge at 12000rpm for 2min and discard the flow-through.<br/><br />
(12) Place the column in a new clean 1.5mL microcentrifuge tube and add 50-80μL ddH2O (better preheated at 65℃) to the center of the column. Stand at room temperature for 2min. Centrifuge at 12000rpm for 1min.<br/><br />
(13) Apply the flow-through from the last step to the center of the column and centrifuge at 12000rpm for 1min. <br />
</dd><br />
</dl><br />
<br />
<dl><br />
<dt><a name="">RT-PCR</a></dt><br />
<dd class="dd_1"><br />
(1) Prepare the following mixture in a microtube:<br><br />
<pre style="width:auto; height:auto; background:none; border:none; color:#676767; font-size:13px; font-family:'Arial';"><br />
Random 6 mers (50μM) 1<br/><br />
dNTP Mixture (10 mM each) 1<br/><br />
Template RNA total RNA less than 5μg<br/><br />
RNase free dH2O up to 10μL<br/><br />
</pre><br />
(2) Keep for 5min at 65℃ and cool immediately on ice.<br><br />
(3) Prepare the reaction mixture by combining the following reagents to a total volume of 20μL:<br><br />
<pre style="width:auto; height:auto; background:none; border:none; color:#676767; font-size:13px; font-family:'Arial';"><br />
Template RNA and Primer Mixture 10μL<br/><br />
5× PrimeScript II Buffer 4μL<br/><br />
RNase Inhibitor (40U/μL) 0.5μl<br/><br />
PrimeScript II RTase( 200U/μl) 1μL<br/><br />
RNase free dH2O up to 20μL<br />
</pre><br />
(4) Mix gently.<br><br />
(5) Incubate the reaction mixture immediately under the following conditions<br><br />
42℃ 60min<br />
(6) Inactivate the enzymes by incubation at 95℃ for 5min, followed by cooling on ice. <br />
</dd><br />
</dl><br />
<br />
<dl><br />
<dt><a name="">PCR</a></dt><br />
<dd class="dd_1"><br />
<strong>3-1 Colony PCR</strong><br><br />
<table cellpadding="0" cellspacing="0" border="0" style="color:#676767; font-size:13px; font-family:'Arial';"><br />
<tr><td style="padding-right:10px">(1) System: </td><td style="padding-right:20px">2×PrimeStar</td><td style="padding-right:20px">12.5μL</td></tr><br />
<tr><td></td><td style="padding-right:20px">Forward primer</td><td style="padding-right:20px">1~2.5μL</td></tr><br />
<tr><td></td><td style="padding-right:20px">Reverse primer</td><td style="padding-right:20px">1~2.5μL</td></tr><br />
<tr><td></td><td style="padding-right:20px">Bacteria solution</td><td style="padding-right:20px">1~2μL</td></tr><br />
<tr><td></td><td style="padding-right:20px">dd water</td><td style="padding-right:20px">up to 25μLL</td></tr><br />
</table><br />
<table cellpadding="0" cellspacing="0" border="0" style="color:#676767; font-size:13px; font-family:'Arial';"><br />
<tr><td style="padding-right:10px">(2) Steps:</td><td style="padding-right:20px">Step 1</td><td style="padding-right:20px">94℃</td></tr><br />
<tr><td></td><td style="padding-right:20px">Step 2</td><td style="padding-right:20px">94℃</td></tr><br />
<tr><td></td><td style="padding-right:20px">Step 3</td><td style="padding-right:20px">Annealing temperature</td></tr><br />
<tr><td></td><td style="padding-right:20px">Step 4</td><td style="padding-right:20px">72℃</td></tr><br />
<tr><td></td><td style="padding-right:20px">Step 5</td><td style="padding-right:20px">go to Step 2, 25~35 cycles</td></tr><br />
<tr><td></td><td style="padding-right:20px">Step 6</td><td style="padding-right:20px">72℃</td></tr><br />
<tr><td></td><td style="padding-right:20px">Step 4</td><td style="padding-right:20px">Pause at 4℃</td></tr><br />
</table><br/><br />
<br />
<strong>3-2 PCR with DNA as Template</strong><br> <br />
<table cellpadding="0" cellspacing="0" border="0" style="color:#676767; font-size:13px; font-family:'Arial';"><br />
<tr><td style="padding-right:10px">(1) System:</td><td style="padding-right:20px">2×PrimeStar</td><td style="padding-right:20px">12.5μL</td></tr><br />
<tr><td></td><td style="padding-right:20px">Forward primer</td><td style="padding-right:20px">1μL</td></tr><br />
<tr><td></td><td style="padding-right:20px">Reverse primer</td><td style="padding-right:20px">1μL</td></tr><br />
<tr><td></td><td style="padding-right:20px">Template DNA</td><td style="padding-right:20px">0.5μL</td></tr><br />
<tr><td></td><td style="padding-right:20px">dd water</td><td style="padding-right:20px">up to 25μL</td></tr><br />
</table><br />
<table cellpadding="0" cellspacing="0" border="0" style="color:#676767; font-size:13px; font-family:'Arial';"><br />
<tr><td style="padding-right:10px">(2) Steps: </td><td style="padding-right:20px">Step 1</td><td style="padding-right:20px">94℃</td></tr><br />
<tr><td></td><td style="padding-right:20px">Step 2</td><td style="padding-right:20px">94℃</td></tr><br />
<tr><td></td><td style="padding-right:20px">Step 3</td><td style="padding-right:20px">Annealing temperature</td></tr><br />
<tr><td></td><td style="padding-right:20px">Step 4</td><td style="padding-right:20px">72℃</td></tr><br />
<tr><td></td><td style="padding-right:20px">Step 5</td><td style="padding-right:20px">go to Step 2, 25~35 cycles</td></tr><br />
<tr><td></td><td style="padding-right:20px">Step 6</td><td style="padding-right:20px">72 ℃</td></tr><br />
<tr><td></td><td style="padding-right:20px">Step 7</td><td style="padding-right:20px">Pause at 4℃</td></tr><br />
</table> <br />
</dd><br />
</dl><br />
<dl><br />
<dt><a href="###">Double Digestion (40μL System)</a></dt><br />
<dd class="dd_1"> <br />
(1) Mix the following in a PCR tube:<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Enzyme A&nbsp;&nbsp;&nbsp;&nbsp;2μL<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Enzyme B&nbsp;&nbsp;&nbsp;&nbsp;2μL<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;10Xbuffer&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;2 or 4μL (depend on the buffer used)<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;BSA&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;0 or 4μL (depend on the buffer used)<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;DNA&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;20μL (insert DNA) or 10μl (vector DNA)<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;dd water&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;up to 40μL<br><br />
(2) Incubate at appropriate temperature (30 or 37℃, depend on the enzymes used) for 8~10h (shorter time if the enzymes used have star activity).<br><br />
(3) Pause at 4 ℃.<br><br />
(4) Gel running and purify the digestion product. <br> <br />
</dd><br />
</dl><br />
<br />
<dl><br />
<dt><a href="###">Agarose gel electrophoresis</a></dt><br />
<dd class="dd_1"><br />
(1) Weigh agarose powder according to the volume of the gel wanted (0.1%) and measure the volume of 1×TAE. Add them to a flask.<br><br />
(2) Melt the mixture in a microwave until the solution becomes clear.<br><br />
(3) Let the solution cool to about 50℃. Add corresponding volume of DuRed (1:10000, V/V) and mix thoroughly.<br><br />
(4) Pour the solution into the gel casting tray with appropriate comb.<br><br />
(5) Let the gel cool until it is solid.<br><br />
(6) Pull out the comb and place the gel in the electrophoresis chamber carefully.<br><br />
(7) Add enough 1×TAE to ensure that there is a thin layer of buffer above the surface of the gel.<br><br />
(8) Pipette DNA samples mixed with appropriate amount of loading buffer and DNA marker into wells on the gel.<br><br />
(9) Run the gel at 135V for about 20min.<br />
<br />
</dd><br />
</dl><br />
<br />
<dl><br />
<dt><a href="###">Gel purification with Axygen Gel Extraction Kit</a></dt><br />
<dd class="dd_1"><br />
<br />
(1) Excise the DNA fragment from the agarose gel with a clean shaver blade.<br><br />
(2) Weigh the gel slice in a 1.5mL microcentrifuge tube and add 3 volumes of Buffer DE-A to 1 volume of gel.<br><br />
(3) Incubate at 75°C for 10min (or until the gel slice has completely dissolved). To help dissolve gel, mix by inverting the tube several times every 2~3 min during the incubation.<br><br />
(4) Add a half volume of Buffer DE-B to 1volume of Buffer DE-A.<br><br />
(5) Add a volume of isopropanol to 1 volume of the gel if the DNA fragment is shorter than 400bp.<br><br />
(6) Apply the solution from the last step to the column.<br><br />
(7) Centrifuge at 12000rpm for 1min and discard the flow-through.<br><br />
(8) Wash the column by adding 500μL Buffer W1 and centrifuge at 12000rpm for 30sec. Discard the flow-through.<br><br />
(9) Wash the column by adding 700μL Buffer W2 and centrifuge at 12000 rpm for 30sec. Discard the flow-through.<br><br />
(10) Wash the column by adding 700μL Buffer W2 and centrifuge at 12000 rpm for 1min. Discard the flow-through.<br><br />
(11) Centrifuge at 12000rpm for 1min to remove the residual wash buffer.<br><br />
(12) Place the column in a new clean 1.5mL microcentrifuge tube and add 30μL ddH2O to the center of the column. Stand for 1min (or longer for better elution).<br><br />
(13) Centrifuge at 12000rpm for 1min (or longer for better elution).<br><br />
(14) Apply the flow-through from the last step to the center of the column. Stand for 1min (or longer for better elution).<br><br />
(15) Centrifuge at 12000rpm for 1min (or longer for better elution) and discard the column.<br><br />
<br />
</dd><br />
</dl><br />
<br />
<dl><br />
<dt><a href="###">Ligation</a></dt><br />
<dd class="dd_1"><br />
(1) Test the concentration of the DNA samples.<br><br />
(2) Pipet the following into a PCR tube:<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Linearized vector DNA <br><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Insert DNA<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Solution I<br><br />
(3) Mix thoroughly and incubate the mixture at 16℃ for 2~3h.<br><br />
(4) Pause at 4℃. <br> <br />
</dd><br />
</dl> <br />
<br />
<dl><br />
<dt><a href="###">Tranformatiom</a></dt><br />
<dd class="dd_1"><br />
(1) Get the competent cell from -80℃ and wait for its fusion on ice.<br><br />
(2) Add the entire ligation product or 1μL plasmid into the tube.<br><br />
(3) Mix and incubate on ice for 30min.<br><br />
(4) Heat pulse at 42℃ for 90sec.<br><br />
(5) Put back the tube on ice and incubate for 5min.<br><br />
(6) Add 1mL LB and incubate at 37℃ with vigorous shaking for 1h.<br><br />
(7) Centrifuge at 4000rpm for 5min and remove most of the supernatant with about 100μL left.<br><br />
(8) Resuspend the compact cells and plate the culture on LB plate containing corresponding antibiotics. <br><br />
</dd><br />
</dl> <br />
<br />
<dl><br />
<dt><a href="###">Mini-prep with Axygen Mini-prep Kit</a></dt><br />
<dd class="dd_1"><br />
(1) Pick a single colony from a selective plate and inoculate a culture of 5mL LB medium containing the appropriate antibiotic. Incubate for proper time (depending on strains and antibiotic) at 37℃ with vigorous shaking.<br><br />
(2) Harvest the bacteria cells by centrifuge at 12000 rpm in 1.5mL microcentrifuge for 1min at room temperature and remove all traces of supernatant.<br><br />
(3) Repeat the last step.<br><br />
(4) Resuspend compact cells in 250μL buffer S1.<br><br />
(5) Add 250μL buffer S2 and mix thoroughly by inverting the tubes 4~6 times.<br><br />
(6) Add 350μL buffer S3 and mix thoroughly by inverting the tubes 6~8 times.<br><br />
(7) Centrifuge at 12000rpm for 10min.<br><br />
(8) Apply the supernatant from the last step to the column.<br><br />
(9) Centrifuge at 12000rpm for 1min and discard the flow-through.<br><br />
(10) Wash the column by adding 500μL buffer W1 and centrifuge at 12000rpm for 1min. Discard the flow-through.<br><br />
(11) Wash the column by adding 700μL buffer W2 and centrifuge at 12000rpm for 1min. Discard the flow-through.<br><br />
(12) Repeat the last step.<br><br />
(13) Centrifuge at 12000rpm for 1min to remove the residual wash buffer.<br><br />
(14) Place the column in a new clean 1.5mL microcentrifuge tube and add 30μL ddH2O to the center of the column. Stand for 1min (or longer for better elution).<br><br />
(15) Centrifuge at 12000rpm for 1min (or longer for better elution).<br><br />
(16) Apply the flow-through from the last step to the center of the column. Stand for 1min (or longer for better elution).<br><br />
(17) Centrifuge at 12000rpm for 1min (or longer for better elution) and discard the column.<br> <br />
</dd><br />
</dl><br />
<br />
<dl><br />
<dt><a href="###">Preparation of competent cells</a></dt><br />
<dd class="dd_1"><br />
(1) Pick a single colony from a selective plate and inoculate a culture of 20mL LB medium. Incubate overnight at 37℃ with vigorous shaking.<br><br />
(2) Add 2mL of the incubated bacteria solution into 50mL LB medium. Incubate for about 1.5h by shaking at 220rpm until OD600 grows to between 0.5 and 0.6.<br><br />
(3) Incubate on ice for 15min. Harvest the bacteria cells by centrifuge at 6000rpm for 5min at 4℃.<br><br />
(4) Remove all traces of supernatant and add 10 ml precooled buffer I on ice. Shake slightly to resuspend the cells.<br><br />
(5) Put the tube on ice for 2h. Harvest the bacteria cells by centrifuge at 6000rpm for 5min at 4℃.<br><br />
(6) Remove all traces of supernatant and resuspend the cells with 3.5mL precooled buffer II.<br><br />
(7) For every 100μL, separate the solution into precooled sterile Eppendorf tube.<br><br />
(8) Store at -80℃ after liquid nitrogen freezing.<br><br />
<table cellpadding="0" cellspacing="0" border="0" style="color:#676767; font-size:13px; font-family:'Arial';"><br />
<tr><td style="padding-right:10px">Buffer I:</td><td style="padding-right:20px">100mM RbCl </td><td style="padding-right:20px">1.2g/100mL</td></tr><br />
<tr><td></td><td style="padding-right:20px">45mM MnCl2·4H2O</td><td style="padding-right:20px">0.891g/100mL</td></tr><br />
<tr><td></td><td style="padding-right:20px">30mM KAc(pH 7.5) </td><td style="padding-right:20px">0.294g/100mL</td></tr><br />
<tr><td></td><td style="padding-right:20px">10mM CaCl2·2H2O</td><td style="padding-right:20px">0.147g/100mL</td></tr><br />
<tr><td></td><td style="padding-right:20px">glycerol (pH 6.8)</td><td style="padding-right:20px">15% (w/v)</td></tr><br />
</table><br />
<table cellpadding="0" cellspacing="0" border="0" style="color:#676767; font-size:13px; font-family:'Arial';"><br />
<tr><td style="padding-right:10px">Buffer II: </td><td style="padding-right:20px">10mM MOPS </td><td style="padding-right:20px">0.209g/100mL</td></tr><br />
<tr><td></td><td style="padding-right:20px">10mM RbCl </td><td style="padding-right:20px">0.12g/100mL</td></tr><br />
<tr><td></td><td style="padding-right:20px">75mM CaCl2·2H2O</td><td style="padding-right:20px">1.1025g/100mL</td></tr><br />
<tr><td></td><td style="padding-right:20px">glycerol (pH 6.8)</td><td style="padding-right:20px">15% (w/v)</td></tr><br />
</table><br />
</pre> <br />
</dd><br />
</dl><br />
<br />
<dl><br />
<dt><a name="Observation">Measuring GFP expression with ELISA</a></dt><br />
<dd class="dd_1"><br />
(1) Pick a single colony with required plasmids from a selective plate and inoculate a culture of 5mL LB medium containing the appropriate antibiotic.<br><br />
(2) Incubate at 37℃ for 20h with vigorous shaking.<br><br />
(3) Sample 1mL of the bacteria solution every 2h until 10h and sample 1mL of the bacteria solution after 20h since the start of the incubation.<br><br />
(4) Measure the fluorescence (excitation: 485nm, detection: 535nm) <br> <br />
</dd><br />
</dl><br />
<br />
<dl><br />
<dt><a href="###">Observation GFP expression with Confocal Microscope</a></dt><br />
<dd class="dd_1"><br />
(1) Pick a single colony with required plasmids from a selective plate and inoculate a culture of 5mL LB medium containing the appropriate antibiotic.<br><br />
(2) Incubate at 37℃ for 20h with vigorous shaking.<br><br />
(3) Sample 1mL of the bacteria solution every 2h until 10h and sample 1mL of the bacteria solution after 20h since the start of the incubation.<br><br />
(4) Observation GFP expression with Confocal Microscope. <br> <br />
</dd><br />
</dl><br />
<br />
<dl><br />
<dt><a name="Drawing">Drawing growth curve</a></dt><br />
<dd class="dd_1"><br />
<br />
(1) Prepare the mother solution of atrazine (200mmol/L) with ethanol.<br><br />
(2) Dilute the mother solution with LB into four different concentrations of atrazine: 200μmol/L; 500μmol/L; 1000μmol/L.<br><br />
(3) Take 52 15mL tubes, divide them into four groups: Group 0, Group 200, Group 500 and Group 1000, 14 tubes each. <br><br />
(4) Label number 0-13 on each tube in each group. (Each group has tubes labeled 1-13.)<br><br />
(5) Add 10mL diluted atrazine solutions into Group 0, 200, 500 and 1000. 0μmol/L atrazine solution for Group 0, 200μmol/L atrazine solution for Group 200, 500μmol/L atrazine solution for Group 500 and 1000μmol/L atrazine solution for Group 1000.<br><br />
(6) Add 1μL Bacteria culture (K12) into each tube, and put them all together into the shaker.<br><br />
(7) Set the speed of the shaker to 200rpm.<br><br />
(8) Take corresponding tubes out of the shaker every 1h. For example, tubes which are labeled 4 should be taken out after four hours, tubes which are labeled 13 should be taken out after 13 hours.<br><br />
(9) Test the OD of the solution in the tubes immediately. Each tube has to be tested for three times. (Use the bacteria-free atrazine solution with corresponding concentration as control.)<br><br />
(10) Record the data and analyze them. <br> <br />
</dd><br />
</dl> <br />
<br />
<dl><br />
<dt><a name="HPLC">HPLC</a></dt><br />
<dd class="dd_1"><br />
(1) Prepare the mother solution of atrazine (100mmol/L) with ethanol.<br><br />
(2) Dilute the mother solution with LB into 500μmol/L.<br><br />
(3) Take 24 15mL tubes, divide them into 8 groups: 0, K12, GFP, TRM, TrzN-, TrzN+, TRM+TrzN-, TRM+TrzN+, 3 tubes each.<br><br />
(4) Add 10mL 500μmol/L atrazine solution into each tube.<br><br />
(5) Do nothing to Group 0; add 1μL bacteria culture with corresponding features into each group of tubes.<br> <br />
(6) Put all the tubes into the shaker, set the speed to 200rpm.<br><br />
(7) Add 12μL solution IPTG into Group TrzN+ and Group TRM+TrzN+ to induce the expression of TrzN after 5 hours of culture.<br><br />
(8) After 24h of culture, take all the tubes out and put them on the ice.<br><br />
(9) Treat all the tubes with centrifuge for 10000rpm, 2min.<br><br />
(10) Filtrate the 1mL supernate in each tube with 0.22μm-filter membrane into EP tubes.<br><br />
(11) Analyze the samples with HPLC. <br> <br />
</dd><br />
</dl> <br />
<br />
<dl><br />
<dt><a name="Verification">Verification of the motility from phenotype</a></dt><br />
<dd class="dd_1"><br />
(1) Pick a single colony with required plasmids from a selective plate and inoculate a culture of 5mL LB medium containing the appropriate antibiotic.<br><br />
(2) Incubate at 37℃ for appropriate time with vigorous shaking until the bacteria reach their plateau phase.<br><br />
(3) Add 5μL bacteria solution from the last step into 5mL LB medium and incubate at 37℃ for 3h with vigorous shaking.<br><br />
(4) Inoculate 2μL suspension from the last step on semisolid (the concentration of agar or agarose depends) LB plate.<br><br />
(5) Incubate at 37℃ for 20h.<br><br />
(6) Take photos with Gel Analysis System.<br><br />
</pre> <br />
</dd><br />
</dl> <br />
<br />
</div><br />
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<div class="team_bt"><a name="member">Team Members</a></div><br />
<dl class="member"><br />
<dt><img src="https://static.igem.org/mediawiki/2013/d/db/M1.jpg" width="116" height="129"></dt><br />
<dd class="dd_1">Mao Yafei</dd><br />
<dd class="dd_2">He is the team leader of Nanjing-China. He is in charge of arranging all team work, making sure that all team members collaborate well with each other. He also participated in all data analysis.</dd><br />
</dl><br />
<dl class="member"><br />
<dt><img src="https://static.igem.org/mediawiki/2013/c/cd/M2.jpg" width="126" height="129"></dt><br />
<dd class="dd_1">Lv Kun</dd><br />
<dd class="dd_2">He is one of the core members of Nanjing-China. His work mainly lies in designing experiments to test the functions of our parts, TrzN and TRM. And this responsible guy also helped team leader complete all the forms we should submit to the HQ.</dd><br />
</dl><br />
<dl class="member"><br />
<dt><img src="https://static.igem.org/mediawiki/2013/7/77/M3.jpg" width="126" height="129"></dt><br />
<dd class="dd_1">Yang Jianchen</dd><br />
<dd class="dd_2">He is one of the core members of Nanjing-China.The main idea of this program mainly came from this intellectual guy. He is in charge of constructing our gene circuits and testing the functions of the systems.</dd><br />
</dl><br />
<dl class="member"><br />
<dt><img src="https://static.igem.org/mediawiki/2013/3/3a/CaoZhipeng.jpg" width="116" height="129"></dt><br />
<dd class="dd_1">Cao Zhipeng</dd><br />
<dd class="dd_2">He is one of the core members of Nanjing-China. He is a good experimenter whose work mainly lies in constructing our gene circuits. This funnny guy always creates miracles for our team work.</dd><br />
</dl><br />
<dl class="member"><br />
<dt><img src="https://static.igem.org/mediawiki/2013/4/49/M5.jpg" width="116" height="129"></dt><br />
<dd class="dd_1">Zhang Juyan</dd><br />
<dd class="dd_2">He is one of the core members of Nanjing-China. He spent a lot of time helping others, including experiments and website design. This diligent guy contributed a lot to our team. <br />
</dd><br />
</dl><br />
<dl class="member"><br />
<dt><img src="https://static.igem.org/mediawiki/2013/9/9e/6_a.jpg" width="116" height="129"></dt><br />
<dd class="dd_1">Pu Ruokun</dd><br />
<dd class="dd_2">He is the group leader of the modeling part of Nanjing-China. He completed the main model with help from Tsinghua-A. And he also helped us to collect the basic information of atrazine.</dd><br />
</dl><br />
<dl class="member"><br />
<dt><img src="https://static.igem.org/mediawiki/2013/6/6c/M7.jpg" width="116" height="129"></dt><br />
<dd class="dd_1">Gao Jian</dd><br />
<dd class="dd_2">He is the group leader of the website part of Nanjing-China.He is in charge of working out the idea about website design. And this shutterbug prepared all the photos for our team.</dd><br />
</dl><br />
<dl class="member"><br />
<dt><img src="https://static.igem.org/mediawiki/2013/0/07/M8.jpg" width="116" height="129"></dt><br />
<dd class="dd_1">Wang Yuanyuan</dd><br />
<dd class="dd_2">She is one of the main members of Nanjing-China. She assists others on the experiments, and she independently completed the growth curve drawing.</dd><br />
</dl><br />
<dl class="member"><br />
<dt><img src="https://static.igem.org/mediawiki/2013/8/8d/M9.jpg" width="116" height="129"></dt><br />
<dd class="dd_1">Gao Jiajia</dd><br />
<dd class="dd_2">She is one of the core members of the website part of Nanjing-China. She helped us to complete the wiki and worked out the elegant video for our team.</dd><br />
</dl><br />
<dl class="member"><br />
<dt><img src="https://static.igem.org/mediawiki/2013/6/61/M10.jpg" width="116" height="129"></dt><br />
<dd class="dd_1">Xiao Shuke</dd><br />
<dd class="dd_2">He is one of the main members of Nanjing-China. He always helps us to complete the basic experiments. This cute guy is good at technology using. He also did some human practices with other team members.</dd><br />
</dl><br />
<dl class="member"><br />
<dt><img src="https://static.igem.org/mediawiki/2013/5/57/11_a.jpg" width="116" height="129"></dt><br />
<dd class="dd_1">Li Yangyang</dd><br />
<dd class="dd_2">He is in charge of the part of human practice. And this clever guy majors in business so that he helped us to collect much information about atrazine, and calculated the value of our program in market.</dd><br />
</dl><br />
<dl class="member"><br />
<dt><img src="https://static.igem.org/mediawiki/2013/b/b0/12_a.jpg" width="116" height="129"></dt><br />
<dd class="dd_1">Li Yezheng</dd><br />
<dd class="dd_2">He is one of the main members of the model part of Nanjing-China. He helped others model from the primary idea.</dd><br />
</dl><br />
<dl class="member"><br />
<dt><img src="https://static.igem.org/mediawiki/2013/5/5f/13_a.jpg" width="116" height="129"></dt><br />
<dd class="dd_1">Zeng Mingzhi</dd><br />
<dd class="dd_2">He is one of the main members of the wiki design part of Nanjing-China. He worked out the perfect wiki with his brilliant expertise.</dd><br />
</dl><br />
<br />
<br />
<div class="team_bt"><a name="instructors">Instructors</a></div><br />
<dl class="instructors"><br />
<dt><a href="http://114.212.232.25/staff/?p=176"><img src="https://static.igem.org/mediawiki/2013/1/19/Inst_1.jpg" width="140" height="150"></a></dt><br />
<dd class="dd_1">Lan WenZhi</dd><br />
<dd class="dd_2">Professor<br/>Research: the function of iron channel; the mechanism of plant cell signaling</dd><br />
</dl><br />
<dl class="instructors"><br />
<dt><img src="https://static.igem.org/mediawiki/2013/0/0f/Inst_2.jpg" width="140" height="150"></dt><br />
<dd class="dd_1">Wang Bin</dd><br />
<dd class="dd_2">Associated professor <br/>Research: molecular genetics and evolution of mycorrhizae</dd><br />
</dl><br />
<dl class="instructors"><br />
<dt><a href="http://114.212.232.25/staff/?p=333"><img src="https://static.igem.org/mediawiki/2013/4/45/Inst_3.jpg" width="140" height="150"></a></dt><br />
<dd class="dd_1">Zheng WeiJuan</dd><br />
<dd class="dd_2">Professor<br>Research: the separation and purification of proteins; genetic engineering of peptide drug</dd><br />
</dl><br />
<br />
<br />
<div class="team_bt" style=" margin-top:20px;"><a name="acknowledgement">Acknowledgements</a></div><br />
<dl class="instructors"><br />
<dt><a href="http://114.212.232.25/staff/?p=170" target="_blank"><img src="https://static.igem.org/mediawiki/2013/f/f3/1.jpg" width="140" height="150"></a></dt><br />
<dd class="dd_1">Kong Lingdong</dd><br />
</dl><br />
<dl class="instructors"><br />
<dt><a href="http://gattaca.nju.edu.cn/sihai.html" target="_blank"><img src="https://static.igem.org/mediawiki/2013/b/bd/YangSihai.jpg" width="140" height="150"></a></dt><br />
<dd class="dd_1">Yang Sihai</dd><br />
</dl><br />
<dl class="instructors"><br />
<dt><img src="https://static.igem.org/mediawiki/2013/7/79/3.jpg" width="140" height="150"></dt><br />
<dd class="dd_1">Zeng Shuiyun</dd><br />
</dl><br />
<dl class="instructors"><br />
<dt><a href="https://2013.igem.org/Team:Tsinghua-A" target="_blank"><img src="https://static.igem.org/mediawiki/2013/f/f4/Tsinghua-A.jpg" width="140" height="150"></a></dt><br />
<dd class="dd_1">Tsinghua-A</dd><br />
</dl><br />
<dl class="instructors"><br />
<dt><a href="http://www.biochem.hku.hk/huanglab/Huang_Lab_at_HKU" target="_blank"><img src="https://static.igem.org/mediawiki/2013/b/b9/5_c.jpg" width="140" height="150"></a></dt><br />
<dd class="dd_1">Huang Lab</dd><br />
</dl><br />
<div class="miaoshu">Here we express our gratitude to the great support from professor Kong Lingdong and professor Yang Sihai from the School of Life Sciences, Nanjing University, and technical support from technologist Zeng Shuiyun. And especially, we express our heartfelt thanks to Tsinghua-A, another 2013 iGEM team, for their help in our basic model building. Finally, we want to express great gratitude to Dr. Huang Jiandong's lab, University of Hong Kong which provided us with some important plasmids and strains.</div><br />
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</html></div>Nidhogghttp://2013.igem.org/Team:Nanjing-China/teamTeam:Nanjing-China/team2013-09-27T15:08:00Z<p>Nidhogg: </p>
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<div class="team_fl"><br />
<div class="team_bt"><a name="member">Team Members</a></div><br />
<dl class="member"><br />
<dt><img src="https://static.igem.org/mediawiki/2013/d/db/M1.jpg" width="116" height="129"></dt><br />
<dd class="dd_1">Mao Yafei</dd><br />
<dd class="dd_2">He is the team leader of Nanjing-China. He is in charge of arranging all team work, making sure that all team members collaborate well with each other. He also participated in all data analysis.</dd><br />
</dl><br />
<dl class="member"><br />
<dt><img src="https://static.igem.org/mediawiki/2013/c/cd/M2.jpg" width="126" height="129"></dt><br />
<dd class="dd_1">Lv Kun</dd><br />
<dd class="dd_2">He is one of the core members of Nanjing-China. His work mainly lies in designing experiments to test the functions of our parts, TrzN and TRM. And this responsible guy also helped team leader complete all the forms we should submit to the HQ.</dd><br />
</dl><br />
<dl class="member"><br />
<dt><img src="https://static.igem.org/mediawiki/2013/7/77/M3.jpg" width="126" height="129"></dt><br />
<dd class="dd_1">Yang Jianchen</dd><br />
<dd class="dd_2">He is one of the core members of Nanjing-China.The main idea of this program mainly came from this intellectual guy. He is in charge of constructing our gene circuits and testing the functions of the systems we constructed.</dd><br />
</dl><br />
<dl class="member"><br />
<dt><img src="https://static.igem.org/mediawiki/2013/3/3a/CaoZhipeng.jpg" width="116" height="129"></dt><br />
<dd class="dd_1">Cao Zhipeng</dd><br />
<dd class="dd_2">He is one of the core members of Nanjing-China. He is a good experimenter whose work mainly lies in constructing our gene circuits. This funnny guy always creates miracles for our team work.</dd><br />
</dl><br />
<dl class="member"><br />
<dt><img src="https://static.igem.org/mediawiki/2013/4/49/M5.jpg" width="116" height="129"></dt><br />
<dd class="dd_1">Zhang Juyan</dd><br />
<dd class="dd_2">He is one of the core members of Nanjing-China. He spent a lot of time helping others, including experiments and website design. This diligent guy contributed a lot to our team. <br />
</dd><br />
</dl><br />
<dl class="member"><br />
<dt><img src="https://static.igem.org/mediawiki/2013/9/9e/6_a.jpg" width="116" height="129"></dt><br />
<dd class="dd_1">Pu Ruokun</dd><br />
<dd class="dd_2">He is the group leader of the modeling part of Nanjing-China. He completed the main model with help from Tsinghua-A. And he also helped us to collect the basic information of atrazine.</dd><br />
</dl><br />
<dl class="member"><br />
<dt><img src="https://static.igem.org/mediawiki/2013/6/6c/M7.jpg" width="116" height="129"></dt><br />
<dd class="dd_1">Gao Jian</dd><br />
<dd class="dd_2">He is the group leader of the website part of Nanjing-China.He is in charge of working out the idea about website design. And this shutterbug prepared all the photos for our team.</dd><br />
</dl><br />
<dl class="member"><br />
<dt><img src="https://static.igem.org/mediawiki/2013/0/07/M8.jpg" width="116" height="129"></dt><br />
<dd class="dd_1">Wang Yuanyuan</dd><br />
<dd class="dd_2">She is one of the main members of Nanjing-China. She assists others on the experiments, and she independently completed the growth curve drawing.</dd><br />
</dl><br />
<dl class="member"><br />
<dt><img src="https://static.igem.org/mediawiki/2013/8/8d/M9.jpg" width="116" height="129"></dt><br />
<dd class="dd_1">Gao Jiajia</dd><br />
<dd class="dd_2">She is one of the core members of the website part of Nanjing-China. She helped us to complete the wiki and worked out the elegant video for our team.</dd><br />
</dl><br />
<dl class="member"><br />
<dt><img src="https://static.igem.org/mediawiki/2013/6/61/M10.jpg" width="116" height="129"></dt><br />
<dd class="dd_1">Xiao Shuke</dd><br />
<dd class="dd_2">He is one of the main members of Nanjing-China. He always helps us to complete the basic experiments. This cute guy is good at technology using. He also did some human practices with other team members.</dd><br />
</dl><br />
<dl class="member"><br />
<dt><img src="https://static.igem.org/mediawiki/2013/5/57/11_a.jpg" width="116" height="129"></dt><br />
<dd class="dd_1">Li Yangyang</dd><br />
<dd class="dd_2">He is in charge of the part of human practice. And this clever guy majors in business so that he helped us to collect much information about atrazine, and calculated the value of our program in market.</dd><br />
</dl><br />
<dl class="member"><br />
<dt><img src="https://static.igem.org/mediawiki/2013/b/b0/12_a.jpg" width="116" height="129"></dt><br />
<dd class="dd_1">Li Yezheng</dd><br />
<dd class="dd_2">He is one of the main members of the model part of Nanjing-China. He helped others model from the primary idea.</dd><br />
</dl><br />
<dl class="member"><br />
<dt><img src="https://static.igem.org/mediawiki/2013/5/5f/13_a.jpg" width="116" height="129"></dt><br />
<dd class="dd_1">Zeng Mingzhi</dd><br />
<dd class="dd_2">He is one of the main members of the wiki design part of Nanjing-China. He worked out the perfect wiki with his brilliant expertise.</dd><br />
</dl><br />
<br />
<br />
<div class="team_bt"><a name="instructors">Instructors</a></div><br />
<dl class="instructors"><br />
<dt><a href="http://114.212.232.25/staff/?p=176"><img src="https://static.igem.org/mediawiki/2013/1/19/Inst_1.jpg" width="140" height="150"></a></dt><br />
<dd class="dd_1">Lan WenZhi</dd><br />
<dd class="dd_2">Professor<br/>Research: the function of iron channel; the mechanism of plant cell signaling</dd><br />
</dl><br />
<dl class="instructors"><br />
<dt><img src="https://static.igem.org/mediawiki/2013/0/0f/Inst_2.jpg" width="140" height="150"></dt><br />
<dd class="dd_1">Wang Bin</dd><br />
<dd class="dd_2">Associated professor <br/>Research: molecular genetics and evolution of mycorrhizae</dd><br />
</dl><br />
<dl class="instructors"><br />
<dt><a href="http://114.212.232.25/staff/?p=333"><img src="https://static.igem.org/mediawiki/2013/4/45/Inst_3.jpg" width="140" height="150"></a></dt><br />
<dd class="dd_1">Zheng WeiJuan</dd><br />
<dd class="dd_2">Professor<br>Research: the separation and purification of proteins; genetic engineering of peptide drug</dd><br />
</dl><br />
<br />
<br />
<div class="team_bt" style=" margin-top:20px;"><a name="acknowledgement">Acknowledgements</a></div><br />
<dl class="instructors"><br />
<dt><a href="http://114.212.232.25/staff/?p=170" target="_blank"><img src="https://static.igem.org/mediawiki/2013/f/f3/1.jpg" width="140" height="150"></a></dt><br />
<dd class="dd_1">Kong Lingdong</dd><br />
</dl><br />
<dl class="instructors"><br />
<dt><a href="http://gattaca.nju.edu.cn/sihai.html" target="_blank"><img src="https://static.igem.org/mediawiki/2013/b/bd/YangSihai.jpg" width="140" height="150"></a></dt><br />
<dd class="dd_1">Yang Sihai</dd><br />
</dl><br />
<dl class="instructors"><br />
<dt><img src="https://static.igem.org/mediawiki/2013/7/79/3.jpg" width="140" height="150"></dt><br />
<dd class="dd_1">Zeng Shuiyun</dd><br />
</dl><br />
<dl class="instructors"><br />
<dt><a href="https://2013.igem.org/Team:Tsinghua-A" target="_blank"><img src="https://static.igem.org/mediawiki/2013/f/f4/Tsinghua-A.jpg" width="140" height="150"></a></dt><br />
<dd class="dd_1">Tsinghua-A</dd><br />
</dl><br />
<dl class="instructors"><br />
<dt><a href="http://www.biochem.hku.hk/huanglab/Huang_Lab_at_HKU" target="_blank"><img src="https://static.igem.org/mediawiki/2013/b/b9/5_c.jpg" width="140" height="150"></a></dt><br />
<dd class="dd_1">Huang Lab</dd><br />
</dl><br />
<div class="miaoshu">Here we express our gratitude to the great support from professor Kong Lingdong and professor Yang Sihai from the School of Life Sciences, Nanjing University, and technical support from technologist Zeng Shuiyun. And especially, we express our heartfelt thanks to Tsinghua-A, another 2013 iGEM team, for their help in our basic model building. Finally, we want to express great gratitude to Dr. Huang Jiandong's lab, University of Hong Kong which provided us with some important plasmids and strains.</div><br />
</div><br />
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</html></div>Nidhogghttp://2013.igem.org/Team:Nanjing-China/teamTeam:Nanjing-China/team2013-09-27T14:57:01Z<p>Nidhogg: </p>
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<li class=trunk onmouseover=listTrigger(0);><a href="https://2013.igem.org/Team:Nanjing-China/team#member">Team Members</a></li><br />
<li class=trunk onmouseover=listTrigger(0);><a href="https://2013.igem.org/Team:Nanjing-China/team#instructors">Instructors</a></li><br />
<li class=trunk onmouseover=listTrigger(0);><a href="https://2013.igem.org/Team:Nanjing-China/team#acknowledgement">Acknowledgements</a></li><br />
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<div class="team_bt"><a name="member">Team Members</a></div><br />
<dl class="member"><br />
<dt><img src="https://static.igem.org/mediawiki/2013/d/db/M1.jpg" width="116" height="129"></dt><br />
<dd class="dd_1">Mao Yafei</dd><br />
<dd class="dd_2">He is the team leader of Nanjing-China. He is in charge of arranging all team work, making sure that all team members collaborate well with each other. He also participated in all data analysis.</dd><br />
</dl><br />
<dl class="member"><br />
<dt><img src="https://static.igem.org/mediawiki/2013/c/cd/M2.jpg" width="126" height="129"></dt><br />
<dd class="dd_1">Lv Kun</dd><br />
<dd class="dd_2">He is one of the core members of Nanjing-China. His work mainly lies in designing experiments to test the functions of our parts, TrzN and TRM. And this responsible guy also helped team leader complete all the forms we should submit to the HQ.</dd><br />
</dl><br />
<dl class="member"><br />
<dt><img src="https://static.igem.org/mediawiki/2013/7/77/M3.jpg" width="126" height="129"></dt><br />
<dd class="dd_1">Yang Jianchen</dd><br />
<dd class="dd_2">He is one of the core members of Nanjing-China.The main idea of this program mainly came from this intellectual guy. He is in charge of constructing our gene circuits and testing the functions of the systems we constructed.</dd><br />
</dl><br />
<dl class="member"><br />
<dt><img src="https://static.igem.org/mediawiki/2013/3/3a/CaoZhipeng.jpg" width="116" height="129"></dt><br />
<dd class="dd_1">Cao Zhipeng</dd><br />
<dd class="dd_2">He is one of the core members of Nanjing-China. He is a good experimenter whose work mainly lies in constructing our gene circuits. This funnny guy always creates miracles for our team work.</dd><br />
</dl><br />
<dl class="member"><br />
<dt><img src="https://static.igem.org/mediawiki/2013/4/49/M5.jpg" width="116" height="129"></dt><br />
<dd class="dd_1">Zhang Juyan</dd><br />
<dd class="dd_2">He is one of the core members of Nanjing-China. He spent a lot of time helping others, including experiments and website design. This diligent guy contributed a lot to our team. <br />
</dd><br />
</dl><br />
<dl class="member"><br />
<dt><img src="https://static.igem.org/mediawiki/2013/9/9e/6_a.jpg" width="116" height="129"></dt><br />
<dd class="dd_1">Pu Ruokun</dd><br />
<dd class="dd_2">He is the group leader of the modeling part of Nanjing-China. He completed the main model with help from Tsinghua-A. And he also helped us to collect the basic information of atrazine.</dd><br />
</dl><br />
<dl class="member"><br />
<dt><img src="https://static.igem.org/mediawiki/2013/6/6c/M7.jpg" width="116" height="129"></dt><br />
<dd class="dd_1">Gao Jian</dd><br />
<dd class="dd_2">He is the group leader of the website part of Nanjing-China.He is in charge of working out the idea about website design. And this shutterbug prepared all the photos for our team.</dd><br />
</dl><br />
<dl class="member"><br />
<dt><img src="https://static.igem.org/mediawiki/2013/0/07/M8.jpg" width="116" height="129"></dt><br />
<dd class="dd_1">Wang Yuanyuan</dd><br />
<dd class="dd_2">She is one of the main members of Nanjing-China. She assists others on the experiments, and she independently completed the growth curve drawing.</dd><br />
</dl><br />
<dl class="member"><br />
<dt><img src="https://static.igem.org/mediawiki/2013/8/8d/M9.jpg" width="116" height="129"></dt><br />
<dd class="dd_1">Gao Jiajia</dd><br />
<dd class="dd_2">She is one of the core members of the website part of Nanjing-China. She helped us to complete the wiki and worked out the elegant video for our team.</dd><br />
</dl><br />
<dl class="member"><br />
<dt><img src="https://static.igem.org/mediawiki/2013/6/61/M10.jpg" width="116" height="129"></dt><br />
<dd class="dd_1">Xiao Shuke</dd><br />
<dd class="dd_2">He is one of the main members of Nanjing-China. He always helps us to complete the basic experiments. This cute guy is good at technology using. He also did some human practices with other team members.</dd><br />
</dl><br />
<dl class="member"><br />
<dt><img src="https://static.igem.org/mediawiki/2013/5/57/11_a.jpg" width="116" height="129"></dt><br />
<dd class="dd_1">Li Yangyang</dd><br />
<dd class="dd_2">He is in charge of the part of human practice. And this clever guy majors in business so that he helped us to collect much information about atrazine, and calculated the value of our program in market.</dd><br />
</dl><br />
<dl class="member"><br />
<dt><img src="https://static.igem.org/mediawiki/2013/b/b0/12_a.jpg" width="116" height="129"></dt><br />
<dd class="dd_1">Li Yezheng</dd><br />
<dd class="dd_2">He is one of the main members of the model part of Nanjing-China. He helped others model from the primary idea.</dd><br />
</dl><br />
<dl class="member"><br />
<dt><img src="https://static.igem.org/mediawiki/2013/5/5f/13_a.jpg" width="116" height="129"></dt><br />
<dd class="dd_1">Zeng Mingzhi</dd><br />
<dd class="dd_2">He is one of the main members of the wiki design part of Nanjing-China. He worked out the perfect wiki with his brilliant expertise.</dd><br />
</dl><br />
<br />
<br />
<div class="team_bt"><a name="instructors">Instructors</a></div><br />
<dl class="instructors"><br />
<dt><a href="http://114.212.232.25/staff/?p=176"><img src="https://static.igem.org/mediawiki/2013/1/19/Inst_1.jpg" width="140" height="150"></a></dt><br />
<dd class="dd_1">Lan WenZhi</dd><br />
<dd class="dd_2">Professor<br/>Research: the function of iron channel the mechanism of plant cell signaling</dd><br />
</dl><br />
<dl class="instructors"><br />
<dt><img src="https://static.igem.org/mediawiki/2013/0/0f/Inst_2.jpg" width="140" height="150"></dt><br />
<dd class="dd_1">Wang Bin</dd><br />
<dd class="dd_2">Associated professor <br/>Research: molecular genetics and evolution of mycorrhizae</dd><br />
</dl><br />
<dl class="instructors"><br />
<dt><a href="http://114.212.232.25/staff/?p=333"><img src="https://static.igem.org/mediawiki/2013/4/45/Inst_3.jpg" width="140" height="150"></a></dt><br />
<dd class="dd_1">Zheng WeiJuan</dd><br />
<dd class="dd_2">Professor<br>Research: the separation and purification of proteins genetic engineering of peptide drug</dd><br />
</dl><br />
<br />
<br />
<div class="team_bt" style=" margin-top:20px;"><a name="acknowledgement">Acknowledgements</a></div><br />
<dl class="instructors"><br />
<dt><a href="http://114.212.232.25/staff/?p=170" target="_blank"><img src="https://static.igem.org/mediawiki/2013/f/f3/1.jpg" width="140" height="150"></a></dt><br />
<dd class="dd_1">Kong Lingdong</dd><br />
</dl><br />
<dl class="instructors"><br />
<dt><a href="http://gattaca.nju.edu.cn/sihai.html" target="_blank"><img src="https://static.igem.org/mediawiki/2013/b/bd/YangSihai.jpg" width="140" height="150"></a></dt><br />
<dd class="dd_1">Yang Sihai</dd><br />
</dl><br />
<dl class="instructors"><br />
<dt><a href="###"><img src="https://static.igem.org/mediawiki/2013/7/79/3.jpg" width="140" height="150"></a></dt><br />
<dd class="dd_1">Zeng Shuiyun</dd><br />
</dl><br />
<dl class="instructors"><br />
<dt><a href="https://2013.igem.org/Team:Tsinghua-A" target="_blank"><img src="https://static.igem.org/mediawiki/2013/f/f4/Tsinghua-A.jpg" width="140" height="150"></a></dt><br />
<dd class="dd_1">Tsinghua-A</dd><br />
</dl><br />
<dl class="instructors"><br />
<dt><a href="http://www.biochem.hku.hk/huanglab/Huang_Lab_at_HKU" target="_blank"><img src="https://static.igem.org/mediawiki/2013/b/b9/5_c.jpg" width="140" height="150"></a></dt><br />
<dd class="dd_1">Huang Lab</dd><br />
</dl><br />
<div class="miaoshu">Here we express our gratitude to the great support from professor Kong Lingdong and professor Yang Sihai of the School of Life Sciences of Nanjing University, and technical support by technologist Zeng Shuiyun. And especially, we express our heartfelt thanks to Tsinghua-A, another 2013 iGEM team, for their help in our basic model building. Finally, we want express great gratitude to Dr. Huang Jiandong's lab of the University of Hong Kong which provided us with some important plasmids and strains.</div><br />
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