Difference between revisions of "Team:Jilin China/Protocol"

 
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{{Jilin_China}}
 
{{Jilin_China}}
 
<html>
 
<html>
<div class="banner"><img src="https://static.igem.org/mediawiki/2017/b/b1/T--Jilin_China--_sec_bg_t.jpg"></div>
+
<div class="banner"><div class="menu">Protocol</div><img src="https://static.igem.org/mediawiki/2017/b/b1/T--Jilin_China--_sec_bg_t.jpg"></div>
  
 
<div class="thr_box">
 
<div class="thr_box">
 +
<div class="h1_title" style="font-size:24px">Cloning Protocol</div>
 
<p>
 
<p>
<strong style="color: #229d73;">1.PCR</strong> <br />
+
<strong style="color: #229d73;">1. PCR</strong> <br />
 
From plasmid DNA template<br />
 
From plasmid DNA template<br />
 
<strong>Materials:</strong><br />
 
<strong>Materials:</strong><br />
TransTaq○R HiFi DNA Polymerase<br />
+
<i>TransTaq</i><sup>&#174;</sup> HiFi DNA Polymerase<br />
10×TransTaq HiFi Buffer II<br />
+
10×<i>TransTaq</i><sup>&#174;</sup> HiFi Buffer II<br />
2.5mM dNTPs<br />
+
2.5 mM dNTPs<br />
6×DNA Loading Buffer <br />
+
6× DNA Loading Buffer <br />
10 μM forward Primer <br />
+
10 μM Forward Primer <br />
10 μM reverse Primer <br />
+
10 μM Reverse Primer <br />
 
Plasmid DNA<br />
 
Plasmid DNA<br />
ddH2O <br />
+
ddH<sub>2</sub>O <br />
 
PCR tube<br /><br />
 
PCR tube<br /><br />
 
 
 
<strong>Methods:</strong><br />
 
<strong>Methods:</strong><br />
For a 50μL reaction:<br />
+
For a 50 μL reaction:<br />
 
<table cellpadding="0" cellspacing="0">
 
<table cellpadding="0" cellspacing="0">
 
<tr><th>Component</th><th>Volume</th><th>Final Concentration</th></tr>
 
<tr><th>Component</th><th>Volume</th><th>Final Concentration</th></tr>
Line 27: Line 28:
 
Forward Primer (10 μM)<br />
 
Forward Primer (10 μM)<br />
 
Reverse Primer (10 μM)<br />
 
Reverse Primer (10 μM)<br />
10×TransTaq○R HiFi Buffer II<br />
+
10×<i>TransTaq</i><sup>&#174;</sup> HiFi Buffer II<br />
 
2.5 mM dNTPs<br />
 
2.5 mM dNTPs<br />
TransTaq○R HiFi DNA Polymerase<br />
+
<i>TransTaq</i><sup>&#174;</sup> HiFi DNA Polymerase<br />
ddH2O<br />
+
ddH<sub>2</sub>O<br />
 
</td>
 
</td>
 
<td align="right">
 
<td align="right">
Line 57: Line 58:
 
</p>
 
</p>
 
 
<p>Combine all the component together into a PCR tube. Spin down briefly to collect the liquid to the bottom of the PCR tube. Put the PCR tube into a PCR machine.</p>
+
<p>Combine all the components together into a PCR tube. Spin down briefly to collect the liquid to the bottom of the PCR tube. Put the PCR tube into a PCR machine.</p>
<p>NOTE: The TransTaq○R HiFi DNA Polymerase should be added last.</p>
+
<p><i>NOTE: The TransTaq<sup>&#174;</sup> HiFi DNA Polymerase should be added last.</i></p>
 
<p>Settings of PCR machine:<br />
 
<p>Settings of PCR machine:<br />
 
<table cellpadding="0" cellspacing="0">
 
<table cellpadding="0" cellspacing="0">
 
<tr><th>Step</th><th>Temperature(℃)</th><th>Time</th></tr>
 
<tr><th>Step</th><th>Temperature(℃)</th><th>Time</th></tr>
<tr><td>Initial denaturation</td><td align="right">94</td><td align="right">5 min</td></tr>
+
<tr><td>Initial denaturation</td><td align="right">94</td><td align="right">5 mins</td></tr>
 
<tr>
 
<tr>
 
<td>35 cycles</td>
 
<td>35 cycles</td>
Line 68: Line 69:
 
<td align="right">30 sec<br />30 sec<br />1-2 kb/min</td>
 
<td align="right">30 sec<br />30 sec<br />1-2 kb/min</td>
 
</tr>
 
</tr>
<tr><td>Final extension</td><td align="right">72</td><td align="right">5 min</td></tr>
+
<tr><td>Final extension</td><td align="right">72</td><td align="right">5 mins</td></tr>
 
<tr><td>Hold</td><td align="right">4</td><td align="right">∞</td></tr>
 
<tr><td>Hold</td><td align="right">4</td><td align="right">∞</td></tr>
 
</table>
 
</table>
 
</p>
 
</p>
 
<p>
 
<p>
<strong style="color: #229d73;">2.Agarose gel electrophoresis</strong> <br />
+
<strong style="color: #229d73;">2. Agarose gel electrophoresis</strong> <br />
 
<strong>Materials:</strong><br />
 
<strong>Materials:</strong><br />
 
Agarose powder<br />
 
Agarose powder<br />
Line 84: Line 85:
 
6× DNA loading dye<br />
 
6× DNA loading dye<br />
 
<strong>Methods:</strong><br />
 
<strong>Methods:</strong><br />
1.Prepare 1% w/v solution agarose powder in 1×TAE buffer(e.g. 1g agarose powder in 100mL 1×TAE buffer).<br />
+
1.Prepare 1% w/v agarose solution with 1×TAE buffer(e.g. 1 g agarose powder in 100 mL 1×TAE buffer).<br />
 
2.Heat the mixture in microwave oven until agarose is completely dissolved.<br />
 
2.Heat the mixture in microwave oven until agarose is completely dissolved.<br />
3.Cooling the solution till around 50℃ , add Ethidium bromide to 0.1‰ v/v final concentration.<br />
+
3.Cooling the solution till around 50℃ , add ethidium bromide to 0.1‰ v/v final concentration.<br />
 
4.Pour the solution into a gel mould.<br />
 
4.Pour the solution into a gel mould.<br />
 
5.Put the solution at room temperature till the gel is set.<br />
 
5.Put the solution at room temperature till the gel is set.<br />
 
6.Remove the comb and transfer the agarose gel to a gel tank.<br />
 
6.Remove the comb and transfer the agarose gel to a gel tank.<br />
7.Mix DNA samples with proper amount of 6× DNA loading buffer and load the samples and DNA marker into the gel.<br />
+
7.Mix DNA samples with proper amounts of 6× DNA loading buffer and load the samples and DNA marker into the gel.<br />
8.Run the gel for 30 minutes of 5V/cm. <br />
+
8.Run the gel for 30 minutes of 5 V/cm. <br />
 
</p>
 
</p>
 
<p>
 
<p>
<strong style="color: #229d73;">3.Gel extraction</strong> <br />
+
<strong style="color: #229d73;">3. Gel extraction</strong> <br />
 
Gel extraction was performed according to the TIANgel Midi Purification Kit
 
Gel extraction was performed according to the TIANgel Midi Purification Kit
 
</p>
 
</p>
 
<p>
 
<p>
<strong style="color: #229d73;">4.Digestion</strong> <br />
+
<strong style="color: #229d73;">4. Digestion</strong> <br />
 
<strong>Materials:</strong><br />
 
<strong>Materials:</strong><br />
 
Restriction Enzyme (NEB)<br />
 
Restriction Enzyme (NEB)<br />
 
10× cutsmart buffer (NEB)<br />
 
10× cutsmart buffer (NEB)<br />
 
Plasmid DNA or DNA fragment<br />
 
Plasmid DNA or DNA fragment<br />
ddH2O<br />
+
ddH<sub>2</sub>O<br />
 
<strong>Methods:</strong><br />
 
<strong>Methods:</strong><br />
 
<table cellpadding="0" cellspacing="0">
 
<table cellpadding="0" cellspacing="0">
Line 109: Line 110:
 
<tr>
 
<tr>
 
<td>
 
<td>
Restriction enzyme<br />
+
Restriction Enzyme<br />
10× cutsmart buffer<br />
+
10× cutsmart <br/>buffer<br />
Plasmid DNA or DNA fragment<br />
+
Plasmid DNA or <br/>DNA fragment<br />
ddH2O<br />
+
ddH<sub>2</sub>O<br />
 
</td>
 
</td>
 
<td align="right">
 
<td align="right">
 
0.5 μL + 0.5 μL<br />
 
0.5 μL + 0.5 μL<br />
1 μL<br />
+
1 μL<br/><br />
Variable (~ 500 ng)<br />
+
Variable (~ 500 ng)<br /><br />
 
Add to 10 μL<br />
 
Add to 10 μL<br />
 
</td>
 
</td>
 
<td align="right">
 
<td align="right">
 
3 μL + 3 μL<br />
 
3 μL + 3 μL<br />
5 μL<br />
+
5 μL<br/><br />
Variable (~ 3 μg)<br />
+
Variable (~ 3 μg)<br /><br />
 
Add to 50 μL<br />
 
Add to 50 μL<br />
 
</td>
 
</td>
 
</tr>
 
</tr>
</table>
+
</table><br/>
 +
1.Mix all the components following the table above.<br/>
 +
2.Incubate the digestion mixture at 37℃ for 1~2 hrs.
 
</p>
 
</p>
 
<p>
 
<p>
<strong style="color: #229d73;">5.DNA purification</strong> <br />
+
<strong style="color: #229d73;">5. DNA purification</strong> <br />
 
PCR purification was performed according to the TIANquick Midi Purification Kit
 
PCR purification was performed according to the TIANquick Midi Purification Kit
 
</p>
 
</p>
 
<p>
 
<p>
<strong style="color: #229d73;">6.Ligation</strong> <br />
+
<strong style="color: #229d73;">6. Ligation</strong> <br />
 
 
 
T4 DNA Ligase<br />
 
T4 DNA Ligase<br />
Line 141: Line 144:
 
Insert DNA<br />
 
Insert DNA<br />
 
Microcentrifuge tube<br />
 
Microcentrifuge tube<br />
ddH2O<br />
+
ddH<sub>2</sub>O<br />
 
<strong>Methods:</strong><br />
 
<strong>Methods:</strong><br />
 
<table cellpadding="0" cellspacing="0">
 
<table cellpadding="0" cellspacing="0">
Line 151: Line 154:
 
10× T4 DNA ligase buffer<br />
 
10× T4 DNA ligase buffer<br />
 
T4 DNA Ligase<br />
 
T4 DNA Ligase<br />
ddH2O<br />
+
ddH<sub>2</sub>O<br />
 
</td>
 
</td>
 
<td align="right">
 
<td align="right">
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</tr>
 
</tr>
 
</table><br />
 
</table><br />
1.Add all the components list in the table above to a microcentrifuge tube.<br />
+
1.Add all the components listed in the table above to a microcentrifuge tube.<br />
2.Incubate the ligation mixture at 16℃ over night.
+
2.Incubate the ligation mixture at 16℃ overnight.
 
</p>
 
</p>
 
<p>
 
<p>
<strong style="color: #229d73;">7.Chemical Transformation</strong> <br />
+
<strong style="color: #229d73;">7. Chemical Transformation</strong> <br />
 
<strong>Materials:</strong><br />
 
<strong>Materials:</strong><br />
 
Ice<br />
 
Ice<br />
 
SOC medium<br />
 
SOC medium<br />
 
Selection plates<br />
 
Selection plates<br />
Chemically competent cell<br />
+
Chemical competent cell<br />
 
Plasmid DNA or ligation reation mix<br />
 
Plasmid DNA or ligation reation mix<br />
<strong>Methods:</strong>
+
<strong>Methods:</strong><br/>
 
1. Put 50 μL chemical competent cells on ice for 10 minutes.<br />
 
1. Put 50 μL chemical competent cells on ice for 10 minutes.<br />
2. Add 100ng DNA of known plasmid or 10 μL ligation reaction mix into the competent cells .  <br />
+
2. Add 100 ng DNA of known plasmid or 10 μL ligation reaction mix into the competent cells .  <br />
 
3. Softly flick the tube 4-5 times to mix cells and DNA.<br />
 
3. Softly flick the tube 4-5 times to mix cells and DNA.<br />
4. Put the tube on ice for 30 minutes.<br />
+
4. Put the tube on ice for 30 mins.<br />
5. Heat shock at exactly 42℃ for exactly 45 seconds.<br />
+
5. Heat shock at exactly 42℃ for exactly 45 secs.<br />
6. Put the tube on ice for 2 minutes.<br />
+
6. Put the tube on ice for 2 mins.<br />
 
7. Add 950 μL SOC medium into the tube.<br />
 
7. Add 950 μL SOC medium into the tube.<br />
8. Incubate at 37℃ ,200rpm for 60 minutes.<br />
+
8. Incubate at 37℃ ,200 rpm for 60 mins.<br />
 
9. Spread:<br />
 
9. Spread:<br />
 
&nbsp;&nbsp;
 
&nbsp;&nbsp;
Line 190: Line 193:
 
For ligation reaction mix:<br />
 
For ligation reaction mix:<br />
 
&nbsp;&nbsp;&nbsp;&nbsp;
 
&nbsp;&nbsp;&nbsp;&nbsp;
Centrifuge the whole 1000 μL transformation cell mixture at 6000rpm for 2 minutes.<br />
+
Centrifuge the whole 1000 μL transformation cell mixture at 6000 rpm for 2 mins.  
&nbsp;&nbsp;&nbsp;&nbsp;
+
Remove 900 μL supernatant and re-suspended cells with 100 μL supernatant left. Transfer the 100 μL resuspended cells onto a selection plate. <br />
Remove 900 μL supernatant and re-suspend cells with 100 μL supernatant left. Transfer the 100 μL resuspended cells onto a selection plate. <br />
+
 
10.  Incubate overnight at 37℃ with plates upside down.<br />
 
10.  Incubate overnight at 37℃ with plates upside down.<br />
 
</p>
 
</p>
 
<p>
 
<p>
<strong style="color: #229d73;">8.Plasmid DNA extraction</strong> <br />
+
<strong style="color: #229d73;">8. Plasmid DNA extraction</strong> <br />
 
Plasmid DNA extraction was carried out according to the TIANprep Rapid Mini Plasmid Kit<br />
 
Plasmid DNA extraction was carried out according to the TIANprep Rapid Mini Plasmid Kit<br />
<strong>EGFP Measurement</strong><br />
+
</p>
 +
</div>
 +
<div class="thr_box">
 +
<div class="h1_title" style="font-size:24px">EGFP Measurement Protocol</div>
 +
<p>
 
<strong>Materials:</strong><br />
 
<strong>Materials:</strong><br />
 
Liquid LB medium <br />
 
Liquid LB medium <br />
100mg/mL Kanamycin<br />
+
100 mg/mL Kanamycin<br />
 
BL21 competent cells<br />
 
BL21 competent cells<br />
Plasmids: pET28a vector, pET28a-J23114-Dmpr-GFP-Rluc, pET28a-J23101-Dmpr-GFP-Rluc, pET28a-J23107-Dmpr-GFP-Rluc, pET28a-Pr-Dmpr-GFP-Rluc, pET28a-Pr-PEKDmpr-GFP-Rluc,<br />
+
Plasmids: pET28a vector, pET28a-J23114-DmpR-GFP-Rluc, pET28a-J23101-DmpR-GFP-Rluc, pET28a-J23107-DmpR-GFP-Rluc, pET28a-Pr-DmpR-GFP-Rluc, pET28a-Pr-PEKDmpR-GFP-Rluc,<br />
 +
transparent flat bottom 96-well plates (for Absorbance 600 measurement),<br/>
 +
white flat bottom 96-well plates (for EGFP measurement)<br/>
 
<strong>Methods:</strong><br />
 
<strong>Methods:</strong><br />
 
1. Transform plasmids into BL21 competent cells. Incubate the plates at 37°C overnight.<br />
 
1. Transform plasmids into BL21 competent cells. Incubate the plates at 37°C overnight.<br />
3. Pick a single colony and inoculate into liqud LB medium containing kanamycin. Overnight culture.<br />
+
2. Pick one monoclonal colony and inoculate into 5 mL liqud LB medium containing 50 μg/mL kanamycin. Overnight culture.<br />
4.Seal the tubes and incubate overnight at 37°C shaking at 200-250 rpm<br />
+
3. Transfer 1 mL overnight-cultured bacteria into 15 mL LB medium containing 50 μg/mL kanamycin.<br/>
 +
4. Take 100 μL samples from the 15 mL growing bacteria at 0, 2, 4, 6, 8, 12 and 24  hours and transfer into transparent flat bottom 96-well plates for Absorbance 600 measurement or white flat bottom 96-well plates for EGFP detection.<br />
 +
5. Measure Absorbance 600 or EGFP by Biotek microplate reader at indicated time.<br/>
 
</p>
 
</p>
 +
</div>
 +
<div class="thr_box">
 +
<div class="h1_title" style="font-size:24px">Renilla Luciferase Assay</div>
 
<p>
 
<p>
<strong style="color: #229d73;">4.Bacterial Glycerol Stocks:</strong> <br />
+
<strong>Materials:</strong><br/>
<strong>Materials:</strong><br />
+
600 μM Promega ViviRen Renila Luciferase substrate <br/>
0.2mL sterile 40% glycerol solution<br />
+
100 mM phenol solution <br/>
0.2mL bacteria liquid<br />
+
100 mM 2-chlorophenol solution <br/>
 +
100 mM 4-chlorophenol solution <br/>
 +
100 mM 2, 4-dichlorophenol solution <br/>
 +
100 mM pyrocatechol solution <br/>
 +
Liquid LB medium <br/>
 +
100 mg/mL Kanamycin <br/>
 +
BL21 competent cells <br/>
 +
Plasmids: pET28a vector, pET28a-J23114-DmpR-GFP-Rluc, pET28a-J23101-DmpR-GFP-Rluc, pET28a-J23107-DmpR-GFP-Rluc, pET28a-Pr-DmpR-GFP-Rluc, pET28a-Pr-PEKDmpR-GFP-Rluc<br/>
 
<strong>Methods:</strong><br />
 
<strong>Methods:</strong><br />
1.Put 0.2ml bacterial culture in a sterile eppendorf tube<br />
+
1. Transform plasmids into BL21 competent cells. Incubate the plates at 37°C overnight.<br/>
2.Add 0.2ml sterile 80% glycerol solution and mix well<br />
+
2. Pick one monoclonal colony and inoculate into 50 mL LB medium containing 50 μg/mL kanamycin. Overnight culture.<br/>
3.Seal the eppendorf tube and store at -80°C<br />
+
3. Transfer 594 μL of 50 mL overnight bacteria into 1.5 mL eppendorf tubes.<br/>
 +
4. Add 6 μL phenols into the aliquot 594 μL bacteria in the eppendorf tubes.<br/>
 +
5. Seal the tubes and incubate at 37°C, 250 rpm, 3 hrs for vector, J23114, J23101 and J23107 groups. Incubate at 37°C, 250 rpm, 12 hrs for vector, Pr-DmpR and Pr-PEKDmpR groups.<br/>
 +
6. Take 45 μL bacteria containing phenols mixture from tubes and put it into a new 1.5 mL eppendorf tube. Transfer corresponding 100 μL mixture to the transparent flat bottom 96-well plates as well.<br/>
 +
7. Add 5 μL 600 μM renilla luciferase substrate into the 45 μL mixture in 1.5 mL eppendorf tube.<br/>
 +
8. Vortex 7 seconds and measure the luciferase reading.<br/>
 +
9. Measure the Absorbance 600 of the corresponding 100 μL mixture in the transparent flat  96-well plates.<br/>
 
</p>
 
</p>
 +
</div>
 +
<div class="thr_box">
 +
<div class="h1_title" style="font-size:24px">Poisoning Protocol</div>
 
<p>
 
<p>
<strong style="color: #229d73;">5.Bacterial amplification:</strong> <br />
+
<strong>Materials:</strong><br/>
<strong>Materials:</strong><br />
+
10% L-arabinose<br/>
15mL LB medium<br />
+
LB medium<br/>
7.5 L 100mg/mL Kanamycin<br />
+
100 mg/mL Kanamycin<br/>
50mL centrifugal tubes<br />
+
<strong>Methods</strong><br/>
<strong>Methods: </strong><br />
+
1. Inoculate 7.5 mL overnight cultured bacteria into 800 mL LB medium with 50 μg/mL kanamycin. Make sure the initial reading of Absorbance 600 is around 0.015.<br/>
1.Add 15 ml liquid LB medium into 50mL centrifugal tubes<br />
+
2. Sub-pack the bacteria into 4 conical flasks, 150 mL each at Absorbance 600 around 0.15-0.2.<br/>
2.Add 7.5 L 100mg/mL Kanamycin into the medium<br />
+
3. Add L-arabinose to the 4 conical flasks for 0%, 0.1%, 0.2%, 0.6% final concentration respectively.<br/>
3.Add 1mL bacteria liquid<br />
+
4. Measure Absorbance 600 at once after inoculation. Every hour in the beginning 6 hrs and every 2 hrs in the next 6 hrs respectively.<br/>
4.Seal the centrifugal tubes and incubate at 37°C shaking at 200-250 rpm<br />
+
 
</p>
 
</p>
 +
</div>
 +
<div class="thr_box">
 +
<div class="h1_title" style="font-size:24px">IPTG Adding Protocol</div>
 
<p>
 
<p>
<strong style="color: #229d73;">6.Detection of EGFP:</strong> <br />
+
<strong>Materials:</strong><br/>
<strong>Materials:</strong><br />
+
100 mM IPTG<br/>
96-well plates<br />
+
LB medium<br/>
<strong>Methods: </strong><br />
+
100 mg/mL Kanamycin<br/>
1.Take 100 L samples of the 50mL centrifugal tubes into the 96-well plates<br />
+
<strong>Methods</strong><br/>
2.Use Microplate reader to shake the 96-well plates and measure the samples (OD600 and EGFP) at 0, 2, 4, 6, 8, 10, 12 and 24 hours of amplification<br />
+
1. Inoculate 7.5 mL overnight cultured bacteria into 800 mL LB medium with 50 μg/mL kanamycin. Make sure the initial reading of Absorbance 600 is around 0.015.<br/>
<strong>Renilla Luciferase Assay </strong><br />
+
2. Sub-pack the bacteria into 4 conical flasks, 150 mL each at Absorbance 600 around 0.15-0.2.<br/>
<strong>Materials: </strong><br />
+
3. Add IPTG to the 4 conical flasks for 0, 0.1, 0.2, 0.5 mM final concentration respectively.<br/>
60 mM Promega ViviRen Renila Luciferase substrate <br />
+
4. Measure Absorbance 600 at once after inoculation. Every hour in the beginning 6 hrs and every 2 hrs in the next 6 hrs respectively.<br/>
1 M Phenol solution<br />
+
1 M 2-Chlorophenol solution<br />
+
1 M 4-Chlorophenol solution<br />
+
1 M 2,4-Dichlorophenol solution<br />
+
1 M Catechol solution<br />
+
<strong>Methods: </strong><br />
+
1.Take 595μlbacteria liquid from 15mL culture tube and add into 1.5mL eppendorf tube<br />
+
2.Mix 6 L Phenols into 595 L bacteria liquid in the 1.5mL eppendorf tube<br />
+
3.For J23114, J23101, J23107: Seal the centrifugal tubes and incubate at 37°C shaking at 200-250 rpm for 3h<br />
+
4.For wt-pr, wt-prpk: Seal the centrifugal tubes and incubate at 37°C shaking at 200-250 rpm for 12h<br />
+
5.Take 45 L mixture from 1.5mL eppendorf tube and add into new 1.5mL eppendorf tube<br />
+
6.Mix 5 L 0.6mmol/mL Luciferase substrate Renila into the mixture in 1.5mL eppendorf tube<br />
+
7.Vortex 7 seconds and detect the Luciferase<br />
+
+
 
</p>
 
</p>
 +
</div>
 +
<div class="thr_box">
 +
<div class="h1_title" style="font-size:24px">Detoxification Protocol</div>
 
<p>
 
<p>
<strong style="color: #229d73;">1.Detoxification curve</strong> <br />
+
<strong>Materials:</strong><br/>
<strong>materials:</strong><br />
+
10% L-arabinose<br/>
arabinose;<br />
+
100 mM IPTG<br/>
IPTG<br />
+
LB medium<br/>
LB culture medium (with 50μg/mL kanamycin;)<br />
+
100 mg/mL Kanamycin<br/>
Microplate reader<br />
+
Plasmids: pET28a vector, T-A (pET28a-totion-antitoxin)<br/>
<strong>Methods:</strong><br />
+
<strong>Methods:</strong><br/>
1.Inoculated 7.5 mL overnight cultured bacteria into 800mL LB media added kanamycin, making its initial value of Abs600 around 0.015.<br />
+
1. Inoculate 7.5 mL overnight cultured bacteria into 800 mL LB medium with 50 μg/mL kanamycin. Make sure the initial reading of Absorbance 600 is around 0.015.<br/>
2.When value of Abs600 reached 0.15-0.2, sub-packed 150 mL medium into a conical flask. Add 0.2% arabinose into the rest 650mL medium.<br />
+
2. Sub-pack 150 mL bacteria into a new conical flask as the no IPTG and no L- arabinose control at Absorbance 600 around 0.15-0.2.<br/>
3.When OD600 was steady, added IPTG(0.1mM) into the medium.<br />
+
3. Add 13 ml L-arabinose into the rest 650 mL bacteria (0.2% final concentration).<br/>
4.Abs600 measurement began at inoculation, every 1h in the beginning 6hours and every     2hours in the next 6hours, respectively.<br />
+
4. Take 150 mL bacteria into a new conical flask as the no IPTG control when Absorbance 600 does not change any more.<br/>
 +
5. Sub-pack 150 mL bacteria into a new conical flask and add 150 μl IPTG into the 150 mL bacteria (0.1 mM final concentration) when Absorbance 600 does not change any more.<br/>
 +
6. Measure the Absorbance 600 at inoculation beginning. Every hour in the beginning 6 hrs and every two hours in the next 6 hrs respectively.<br/>
 
</p>
 
</p>
 +
</div>
 +
<div class="thr_box">
 +
<div class="h1_title" style="font-size:24px">Enzyme tfdB-JLU Induction and Purification Protocol</div>
 
<p>
 
<p>
<strong style="color: #229d73;">2.poisoning curve</strong> <br />
+
<strong>Materials</strong><br/>
<strong>materials:</strong><br />
+
IPTG<br/>
arabinose;<br />
+
LB medium<br/>
LB culture medium (50μg/ml kanamycin;)<br />
+
100 mg/ml Kanamycin<br/>
Microplate reader<br />
+
Ni-NTA His Bind column<br/>
<strong>Methods:</strong><br />
+
lysis buffer: 200 mM NaCl, 20% glycerol, 20 mM Tris–HCl, and pH 7.5<br/>
Inoculated 7.5 mL overnight cultured bacteria into 800mL LB media added kanamycin, making its initial value of Abs600 around 0.015. measured Abs600 every hour. When value of Abs600 reached 0.15-0.2, sub-packed medium into 4 conical flasks, 150mL each. Then added 0%, 0.1%, 0.2%, 0.6% arabinose respectively, measured Abs600 every 1h in the beginning 6hours and every 2hours in the next 6hours, respectively.<br />
+
buffer A: 20 mM Tris-HCl buffer, pH 7.5, containing 20% (v/v) glycerol, 200 mM NaCl<br/>
 +
buffer B: 500 mM imidazole,20 mM Tris-HCl buffer, pH 7.5, containing 20% (v/ v) glycerol, 200 mM NaCl<br/>
 +
<strong>Methods:</strong><br/>
 +
<i>Day 1</i><br/>
 +
1. Inoculate <i>E. coli</i> harboring tfdB-JLU in LB medium supplemented with 50 μg/mL kanamycin at 37℃, 220 rpm, overnight culture.<br/>
 +
<i>Day 2</i><br/>
 +
2. Transfer 1 mL overnight cultured cells into 200 mL LB.<br/>
 +
3. Add IPTG to final concentration 0.3 mM at OD600 around 0.4 of the grown bacteria.<br/>
 +
4. Incubate the cells at 18℃ for 16 hrs by shaking at 180 rpm to induce tfdB-JLU enzyme.<br/>
 +
<i>Day 3</i><br/>
 +
5. Harvest overnight-inducted cells. Centrifuge at 4000 rpm for 30 mins at 4℃. Discard the supernatant.<br/>
 +
6. Resuspend with 50 mL lysis buffer (200 mM NaCl, 20% glycerol, 20 mM Tris–HCl, and pH 7.5).<br/>
 +
7. Sonicate 5 s with 5 s interval each time at 20 kHz for 1 hr on ice.<br/>
 +
8. Remove cell debris by centrifugation at 12000 rpm for 30 mins.<br/>
 +
9. Purify the His-tagged recombinant enzyme by passing the supernatant through a Ni-NTA His Bind column.<br/>
 +
10. Reload the supernatant twice before washing.<br/>
 +
11. Use 8 mL volumes of buffer A wash the column.<br/>
 +
12. Wash the column with 10 mL buffer A containing 20 mM imidazole (using buffer A dilute buffer B).<br/>
 +
13. Collect samples, 1 mL/tube.<br/>
 +
14. Wash the column with 10 mL buffer A containing 50 mM imidazole, collect samples, 1 mL/tube.<br/>
 +
15. Wash the column with 6 mL buffer A containing 100 mM imidazole, collect samples, 1 mL/tube.<br/>
 +
16. Wash the column with 10 mL buffer A containing 250 mM imidazole, collect samples, 1 mL/tube.<br/>
 +
17. Wash the column with 40 mL buffer B.<br/>
 +
18. Wash the column with 40 mL buffer A.<br/>
 +
19. Wash the column with 40 mL H<sub>2</sub>O.<br/>
 
</p>
 
</p>
 +
</div>
 +
<div class="thr_box">
 +
<div class="h1_title" style="font-size:24px">Enzyme tfdB-JLU Characterization Protocol</div>
 
<p>
 
<p>
<strong style="color: #229d73;">3.IPTG curve</strong> <br />
+
<strong>Materials:</strong><br/>
<strong>materials:</strong><br />
+
nicotinamide adenine dinucleotide phosphate (NADPH)<br/>
IPTG<br />
+
FAD (flavin adenine dinucleotide)<br/>
LB culture medium(kanamycin;)<br />
+
sodium phosphate buffer (pH 7.5)<br/>
Microplate reader<br />
+
phenol substrate:2,4-DCP, 3-CP, 4-CP, phenol, 2,3-DCP, 2,5-DCP, 2,6-DCP, 3,4-DCP, 3,5-DCP, 2,4,5-TCP<br/>
+
<strong>Methods:</strong><br/>
+
Determine the activities of chlorophenol hydroxylases by monitoring the decrease in absorbance at 340 nm (e340 = 6,220 M-1 cm-1) following the substrate-dependent oxidation of NADPH. Define one unit of activity as the amount of enzyme required to consume 1 μmol NADPH per min at 25℃.<br/>
<strong>Methods:</strong><br />
+
1. Incubate 12 μg/mL tfdB-JLU with 0.1 mM substracts, 5 μM FAD and 0.2 mM NADPH in 50 mM sodium phosphate buffer (pH 7.5) at 25℃ in 1 mL mixture system.<br/>
1.the bacterio-liquid(7.5ml)shaken overnight at 37℃ were transferred into LB culture medium(800ml) with kanamycin(1b).<br />
+
2. Monitor Absorbance 340 for 1 hr.
2.Measure its abs600 once an hour when abs600 reaches 0.015.<br />
+
3.Sub-pack in four Conical flasks with 150ml adding different concentrations of IPTG(0.1mM 0.2mM 0.5mM),when abs600 reaches 0.150-0.200.<br />
+
4.Since transferring ,measure abs600 once an hour in the first six hour ,and then once two hour.<br />
+
<br />
+
Inoculated 7.5 mL overnight cultured bacteria into 800mL LB media added kanamycin, making its initial value of Abs600 around 0.015. measured Abs600 every hour. When value of Abs600 reached 0.15-0.2, sub-packed medium into 4 conical flasks, 150mL each. Then added 0.1mM, 0.2mM, 0.5mM IPTG respectively, measured Abs600 every 1h in the beginning 6hours and every 2hours in the next 6hours, respectively.<br />
+
 
</p>
 
</p>
 +
</div>
 +
<div class="thr_box">
 +
<div class="h1_title" style="font-size:24px">Phenol Degradation Protocol (4-AAP Assay)</div>
 +
<p>
 +
<strong>Principles:</strong><br/>
 +
Phenol can react with 4-AAP in alkaline medium (pH =10.0±0.2), with the oxidizer K<sub>3</sub>Fe(CN)<sub>6</sub>. Reaction product antipyrine dye appears orange, whose absorption peak is at 510nm.<br/>
 +
</p>
 +
<div align="center"><img src="https://static.igem.org/mediawiki/2017/e/e5/T--Jilin_China--protocol_phenol_test_15E3A9.jpg"/></div>
 
<p>
 
<p>
<strong>1.Phenol degradation experiments</strong><br />
+
<strong>Materials:</strong><br/>
Materials and methods<br />
+
2% (m/v) 4-amino anti pyrine (4-AAP)<br/>
2%(m/v) 4-amino anti pyrine (4-AAP), 8%(m/v) potassium ferricyanide K3Fe(CN)6, 1mM glucose, 0.9% NaCl, buffer solution: 10g NH4Cl in 50mL ammonium hydroxide. <br />
+
8% (m/v) potassium ferricyanide K3Fe(CN)6<br/>
Phenol can react with 4-AAP in alkaline medium (pH =10.0±0.2), with the oxidizer  K3Fe(CN)6. Reaction product antipyrine dye appears orange, whose absorption peak is at 510nm. <br />
+
1 mM glucose<br/>
<img src="https://static.igem.org/mediawiki/2017/a/a2/T--Jilin_China--protocol01.png" /><br />
+
0.9% NaCl<br/>
4-AAP assay<br />
+
buffer: 10g NH4Cl in 50 mL ammonium hydroxide.<br/>
For a 14.5ml reaction system:<br />
+
<strong>Methods:</strong><br/>
 +
1. Inoculate E. coli containing tfdB-JLU or mock in LB with 50 μg/mL kanamycin at 37℃, 220 rpm, overnight culture.<br/>
 +
2. Transfer 1 mL overnight cultured cells into 200 mL LB.<br/>
 +
3. Add IPTG to final concentration 0.3 mM at OD600 around 0.4 of the grown bacteria.<br/>
 +
4. Incubate at 18℃ for 16 hrs by shaking at 180 rpm to induce tfdB-JLU enzyme.<br/>
 +
5. Take 500 μL induced bacteria samples for measuring Absorbance 600.<br/>
 +
6. Take out 20 mL corresponding bacteria into 50 mL centrifuge tube.<br/>
 +
7. Centrifuge at 3000 rpm for 5 minw, discard supernatant, and suspend with 10 mL solution (1 mM glucose in 0.9% NaCl).<br/>
 +
8. Centrifuge in 3000 rpm for 5 mins again. Discard supernatant.<br/>
 +
9. Re-suspend with 10 mL 0, 1, 2, or 5 mM phenol with 1 mM glucose in 0.9% NaCl solution.<br/>
 +
10. Incubate at 25℃ for 12 hrs.<br/>
 +
11. Centrifuge samples immediately at 12000 rpm for 5 mins.<br/>
 +
12. Take out 0.5 mL supernatant into assay system. Mix a 14.5 mL reaction system as below. Incubate for 10 mins.<br/>
 
<table cellpadding="0" cellspacing="0">
 
<table cellpadding="0" cellspacing="0">
 
<tr><th width="150">Component</th><th width="100">Volume</th></tr>
 
<tr><th width="150">Component</th><th width="100">Volume</th></tr>
 
<tr>
 
<tr>
<td>
+
<td align="center">
 
samples<br />
 
samples<br />
H2O <br />
+
H<sub>2</sub>O <br />
 
buffer<br />
 
buffer<br />
 
2% 4-AAP<br />
 
2% 4-AAP<br />
8% K3Fe(CN)6<br />
+
8% K<sub>3</sub>Fe(CN)<sub>6</sub><br />
 
</td>
 
</td>
<td>
+
<td align="center">
0.5ml<br />
+
0.5 mL<br />
9.5 ml<br />
+
9.5 mL<br />
0.9 ml<br />
+
0.9 mL<br />
1.8ml<br />
+
1.8 mL<br />
1.8ml<br />
+
1.8 mL<br />
 
</td>
 
</td>
 
</tr>
 
</tr>
 
<tr>
 
<tr>
<td>Total volume</td>
+
<td align="center">Total volume</td>
<td>14.5ml</td>
+
<td align="center">14.5 mL</td>
 
</tr>
 
</tr>
</table>
+
</table><br/>
 +
13. Measure Absorbance 510 for all the samples.<br/>
 
</p>
 
</p>
 +
</div>
 +
<div class="thr_box">
 +
<div class="h1_title" style="font-size:24px">Phenol Degradation Protocol (HPLC Assay)</div>
 
<p>
 
<p>
+
<strong>Materials:</strong><br/>
<strong>Experimental group:</strong><br />
+
ZORBAX Eclipse XDB C-18 column<br/>
1mM phenol, 1mM glucose, 0.9%NaCI solution with tfdB-JLU bacteria 0h <br />
+
methanol, acetic acid, phenol,<br/>
2mM phenol, 1mM glucose, 0.9%NaCI solution with tfdB-JLU bacteria 0h<br />
+
sodium phosphate buffer (pH 7.5)<br/>
5mM phenol, 1mM glucose, 0.9%NaCI solution with tfdB-JLU bacteria 0h<br />
+
LB medium with kanamycin<br/>
1mM phenol, 1mM glucose, 0.9%NaCI solution with tfdB-JLU bacteria 20h    <br />  
+
IPTG<br/>
2mM phenol, 1mM glucose, 0.9%NaCI solution with tfdB-JLU bacteria 20h    <br />
+
<strong>HPLC condition:</strong><br/>
5mM phenol, 1mM glucose, 0.9%NaCI solution with tfdB-JLU bacteria 20h<br />
+
ZORBAX Eclipse XDB C-18 column<br/>
+
mobile phase:methanol : 0.2% acetic acid (30:70)<br/>
<strong>Control group:</strong><br />
+
rate: 1 mL/min<br/>
2mM phenol, 1mM glucose, 0.9%NaCI solution with empty vector bacteria 20h<br />
+
injection volume: 10 μL<br/>
+
wavelength: 275 nm<br/>
<strong>Blank:</strong><br />
+
temperature: 25°C<br/>
0mM phenol, 1mM glucose, 0.9%NaCI solution with supernatant of tfdB-JLU bacteria <br />
+
<strong>Methods:</strong><br/>
 
+
1. Inoculate E. coli containing tfdB-JLU or mock in LB medium with 50 μg/mL kanamycin at 37℃, 220 rpm, overnight culture.<br/>
 +
2. Transfer 250 μL overnight cultured cells into 50 mL LB.<br/>
 +
3. Add IPTG to final concentration 0.3 mM at OD600 around 0.4 of the grown bacteria.<br/>
 +
4. Incubate at 18℃ for 16 hrs by shaking at 180 rpm to induce tfdB-JLU enzyme.<br/>
 +
5. Take 1 mL induced bacteria samples for measuring Absorbance 600.<br/>
 +
6. Transfer 1 mL corresponding induced bacteria samples to the tubes. Centrifuge 4000 rpm for 10 mins. Discard the supernatant.<br/>
 +
7. Resuspend the precipitate with 0, 1, 2, or 5 mM phenol in PBS respectively. Incubate at 25℃, shaking for 4 hrs.<br/>
 +
8. Centrifuge 4000 rpm for 10 mins. Keep the supernatant and pass the 0.22 μm filter.<br/>
 +
9. Measure with HPLC.
 
</p>
 
</p>
 +
</div>
 +
<div class="thr_box">
 +
<div class="h1_title" style="font-size:24px">Circuit Protocol</div>
 
<p>
 
<p>
<strong>Phenol disposing system with tfdB-JLU bacteria </strong><br />
+
<strong>Materials:</strong><br/>
1. E. coli harboring the cloned pET-28a-tfdB-JLU was incubated in 3 mL LB media supplemented with 30μg/mL of kanamycin at 37℃ on a rotary shaker operating at 220 rpm at 37℃ overnight. <br />
+
phenol<br/>
2. Enlarged overnight cells. The cells were grown until the value of OD600 reached 0.4. Cooled down the medium untill it reached 16℃, 0.2mM IPTG was then added, and the cells were incubated in 16℃ for 12h by shaking at 220rpm to induce tfdB-JLU enzyme. <br />
+
LB with kanamycin<br/>
3. Took out 20ml inducted bacteria into 50mL concentrator bowls, marked 1, 2, 3, then centrifuged in 3000rpm for 5min, discarded supernatant, resuspended with 10mL 1mM glucose and 0.9% NaCl solution, centrifuged in 3000rpm for 5min, took out 1mL supernatant into 1ml EP tube, marked 0-0. Discarded concentrator bowls’ supernatant, then resuspended with 10mL 1, 2, 5mM phenol with 1mM glucose, 0.9% NaCl solution and centrifuged in 3000rpm for 5min. took out 1mL supernatant marked 1-0, 2-0, 3-0. Resuspended again and took 5mL sample for measuring OD600. Put concentrator bowls in 25℃ for further disposing.<br />
+
HPLC assay materials and condition<br/>
+
4-AAP assay materials and condition<br/>
<strong>Phenol disposing system with empty vector bacteria</strong><br />
+
<strong>Methods:</strong><br/>
1. E. coli harboring the cloned pET-28a was incubated in 3 mL LB media supplemented with 30μg/mL of kanamycin at 37℃ on a rotary shaker operating at 220 rpm at 37℃ overnight. <br />
+
1. Inoculate E. coli containing J23107-DmpR-tfdB-JLU or mock in LB with 50 μg/mL kanamycin at 37℃, 220 rpm, overnight culture.<br/>
2. Enlarged overnight cells. The cells were grown until the value of OD600 reached 0.4. Cooled down the medium till it reached 16℃, then incubated for 12h by shaking at 220rpm. <br />
+
2. Transfer 500 μL overnight cultured cells into 100 mL LB. Shake at 37 ℃ for 2 hrs.<br/>
3. Took out 20ml inducted bacteria into 50mL concentrator bowls, marked empty vector, then centrifuged in 3000rpm for 5min, discarded supernatant, resuspended with 10mL 1mM glucose and 0.9% NaCl solution, centrifuged in 3000rpm for 5min. Discarded concentrator bowls’ supernatant, then resuspended with 10mL 2mM phenol with 1mM glucose, 0.9% NaCl solution and centrifuged in 3000rpm for 5min. took out 1mL supernatant marked empty vector-0. Resuspended again and put concentrator bowls in 25℃ for further disposing.<br />
+
3. Add 0, 1, 2, or 5 mM phenol into the cultured bacteria.<br/>
4. Centrifuged empty vector-0 in 12000rpm for 5min, took out 0.5mL sample into 10mL concentrator bowl for assay system. Incubated 10min and measured A510. (blanked with water) <br />
+
4. Measure Abs600 right after phenol adding and subsequent every 2 hrs till 24 hrs culture.<br/>
5. The high-performance liquid chromatography (HPLC) column was calibrated initially with the mobile phase (methanol and 0.2% acetic acid 30:70) at a flow rate of 1 mL/min for 20 min. The detection wavelength was at 275 nm.<br />
+
5. Phenol was tested 2, 6, 10 or 24 hrs after its adding with both HPLC method and 4-AAP assay.<br/>
6. Prepared 1mM, 2mM, 5mM phenol PBS solution and 1mM, 2mM catethol PBS solution. Measured with HPLC and recorded retention time and peak area.<br />
+
7. Day 0: inoculated tfdB-JLU and empty vector bacteria strain.<br />
+
8. Day 1: enlarged overnight bacteria, cells were grown until the value of OD600 reached 0.4. 0.3mM (IPTG) was then added, and the cells were incubated in 18℃ for 16-24h by shaking at 220rpm for induction.<br />
+
9. Took 1mL tfdb and 1mL empty vector bacteria into 1mL EP tube, centrifuged in 4000rpm for 10min, discarded supernatant, resuspended with 1mM, 2mM, 5mM phenol respectively, incubated for 1h, 2h, 4h, 20h, then measured with HPLC.<br />
+
10. <br />
+
11. Experimental group<br />
+
12. 1mM phenol disposed with tfdB-JLU bacteria for 0h <br />
+
13. 2mM phenol disposed with tfdB-JLU bacteria for 0h<br />
+
14. 5mM phenol disposed with tfdB-JLU bacteria for 0h<br />
+
15. 1mM phenol disposed with tfdB-JLU bacteria for 1h    <br />
+
16. 2mM phenol disposed with tfdB-JLU bacteria for 1h    <br />
+
17. 5mM phenol disposed with tfdB-JLU bacteria for 1h<br />
+
18. 1mM phenol disposed with tfdB-JLU bacteria for 2h    <br />
+
19. 2mM phenol disposed with tfdB-JLU bacteria for 2h    <br />
+
20. 5mM phenol disposed with tfdB-JLU bacteria for 2h<br />
+
21. 1mM phenol disposed with tfdB-JLU bacteria for 4h    <br />
+
22. 2mM phenol disposed with tfdB-JLU bacteria for 4h    <br />
+
23. 5mM phenol disposed with tfdB-JLU bacteria for 4h<br />
+
24. 1mM phenol disposed with tfdB-JLU bacteria for 20h    <br />
+
25. 2mM phenol disposed with tfdB-JLU bacteria for 20h    <br />
+
26. 5mM phenol disposed with tfdB-JLU bacteria for 20h<br />
+
27. <br />
+
28. Control group:<br />
+
29. 1mM phenol disposed with empty vector bacteria for 0h <br />
+
30. 2mM phenol disposed with empty vector bacteria for 0h<br />
+
31. 5mM phenol disposed with empty vector bacteria for 0h<br />
+
32. 1mM phenol disposed with empty vector bacteria for 1h    <br />
+
33. 2mM phenol disposed with empty vector bacteria for 1h    <br />
+
34. 5mM phenol disposed with empty vector bacteria for 1h<br />
+
35. 1mM phenol disposed with empty vector bacteria for 2h    <br />
+
36. 2mM phenol disposed with empty vector bacteria for 2h    <br />
+
37. 5mM phenol disposed with empty vector bacteria for 2h<br />
+
38. 1mM phenol disposed with empty vector bacteria for 4h    <br />
+
39. 2mM phenol disposed with empty vector bacteria for 4h    <br />
+
40. 5mM phenol disposed with empty vector bacteria for 4h<br />
+
41. 1mM phenol disposed with empty vector bacteria for 20h    <br />
+
42. 2mM phenol disposed with empty vector bacteria for 20h    <br />
+
43. 5mM phenol disposed with empty vector bacteria fo<br />
+
 
+
</p>
+
<p>
+
<strong style="color: #229d73;">Overexpression and purification of target enzyme</strong> <br />
+
<strong>Day 1</strong><br />
+
E. coli harboring the cloned pET-28a was incubated in LB media supplemented with 50μg/mL of kanamycin at 37℃ on a rotary shaker operating at 220 rpm at 37℃ overnight. <br />
+
<strong>Day2</strong><br />
+
Suspend overnight cells. The cells were grown until the value of OD600 reached 0.4. 0.3mM (IPTG) was then added, and the cells were incubated in 18℃ for 16h by shaking at 180 rpm to induce tfdB-JLU enzyme. <br />
+
<strong>Day3</strong><br />
+
Harvest overnight-inducted cells. The harvested cells were centrifuged at 4000rpm for 30 min in 4℃, and supernatant was then discarded. The cells were resuspended in 50 mL of lysis buffer (200mM NaCl, 20% glycerol, 20mM Tris–HCl, and pH 7.5) and sonicated 5s with 5s interval each time at 20 kHz for 1h on ice. The cell debris was removed by centrifugation at 12000rpm for 30 min. The His-tagged recombinant enzyme was purified by passing the supernatant through a Ni-NTA His Bind column. The column was washed with wash buffer (20 mM Tris-HCl buffer, pH 7.5, containing 20% (v/ v) glycerol, 200mM NaCl and 20mM, 50mM, 100mM imidazole grads) and eluted with elution buffer ((20 mM Tris-HCl buffer, pH 7.5, containing 20% (v/ v) glycerol, 200mM NaCl and 250mM imidazole).<br />
+
Enzyme assay and characterisation<br />
+
The activities of chlorophenol hydroxylases were determined by monitoring the decrease in absorbance at 340 nm (e340 = 6,220 M-1 cm-1) following the substrate-dependent oxidation of NADPH. Unless otherwise indicated, standard enzyme activity assays were performed by incubating the purified enzyme with 0.1 mM 2,4-DCP and 0.2 mM NADPH in 50 mM sodium phosphate buffer (pH 7.5) at 25℃ in 1 ml. One unit of activity was defined as the amount of enzyme required to consume 1 μmol NADPH per min at 25℃.<br />
+
 
+
 
</p>
 
</p>
 
</div>
 
</div>
 
</html>
 
</html>
 +
{{Jilin_footer}}

Latest revision as of 14:04, 1 November 2017

Cloning Protocol

1. PCR
From plasmid DNA template
Materials:
TransTaq® HiFi DNA Polymerase
10×TransTaq® HiFi Buffer II
2.5 mM dNTPs
6× DNA Loading Buffer
10 μM Forward Primer
10 μM Reverse Primer
Plasmid DNA
ddH2O
PCR tube

Methods:
For a 50 μL reaction:

ComponentVolumeFinal Concentration
Template
Forward Primer (10 μM)
Reverse Primer (10 μM)
10×TransTaq® HiFi Buffer II
2.5 mM dNTPs
TransTaq® HiFi DNA Polymerase
ddH2O
Variable(usually 50 ng)
1 μL
1 μL
5 μL
4 μL
0.5 μL
Add to 50 μL
As required
0.2 μM
0.2 μM

0.2 mM
2.5 units
-
Total volume 50 μL -

Combine all the components together into a PCR tube. Spin down briefly to collect the liquid to the bottom of the PCR tube. Put the PCR tube into a PCR machine.

NOTE: The TransTaq® HiFi DNA Polymerase should be added last.

Settings of PCR machine:

StepTemperature(℃)Time
Initial denaturation945 mins
35 cycles 94
50-60
72
30 sec
30 sec
1-2 kb/min
Final extension725 mins
Hold4

2. Agarose gel electrophoresis
Materials:
Agarose powder
TAE buffer
Gel mould
Gel comb
Gel Tank
Ethidium bromide
DNA marker DL10000
6× DNA loading dye
Methods:
1.Prepare 1% w/v agarose solution with 1×TAE buffer(e.g. 1 g agarose powder in 100 mL 1×TAE buffer).
2.Heat the mixture in microwave oven until agarose is completely dissolved.
3.Cooling the solution till around 50℃ , add ethidium bromide to 0.1‰ v/v final concentration.
4.Pour the solution into a gel mould.
5.Put the solution at room temperature till the gel is set.
6.Remove the comb and transfer the agarose gel to a gel tank.
7.Mix DNA samples with proper amounts of 6× DNA loading buffer and load the samples and DNA marker into the gel.
8.Run the gel for 30 minutes of 5 V/cm.

3. Gel extraction
Gel extraction was performed according to the TIANgel Midi Purification Kit

4. Digestion
Materials:
Restriction Enzyme (NEB)
10× cutsmart buffer (NEB)
Plasmid DNA or DNA fragment
ddH2O
Methods:

ComponentTest digestion (double digestion)Assemble digestion(double digestion)
Restriction Enzyme
10× cutsmart
buffer
Plasmid DNA or
DNA fragment
ddH2O
0.5 μL + 0.5 μL
1 μL

Variable (~ 500 ng)

Add to 10 μL
3 μL + 3 μL
5 μL

Variable (~ 3 μg)

Add to 50 μL

1.Mix all the components following the table above.
2.Incubate the digestion mixture at 37℃ for 1~2 hrs.

5. DNA purification
PCR purification was performed according to the TIANquick Midi Purification Kit

6. Ligation
T4 DNA Ligase
10× T4 DNA ligase buffer
Vector DNA
Insert DNA
Microcentrifuge tube
ddH2O
Methods:

ComponentVolume
Vector DNA
Insert DNA
10× T4 DNA ligase buffer
T4 DNA Ligase
ddH2O
Variable (~50 ng)
Variable (molar ratio of vector:insert=1:3)
2 μL
1 μL
Add to 20 μL

1.Add all the components listed in the table above to a microcentrifuge tube.
2.Incubate the ligation mixture at 16℃ overnight.

7. Chemical Transformation
Materials:
Ice
SOC medium
Selection plates
Chemical competent cell
Plasmid DNA or ligation reation mix
Methods:
1. Put 50 μL chemical competent cells on ice for 10 minutes.
2. Add 100 ng DNA of known plasmid or 10 μL ligation reaction mix into the competent cells .
3. Softly flick the tube 4-5 times to mix cells and DNA.
4. Put the tube on ice for 30 mins.
5. Heat shock at exactly 42℃ for exactly 45 secs.
6. Put the tube on ice for 2 mins.
7. Add 950 μL SOC medium into the tube.
8. Incubate at 37℃ ,200 rpm for 60 mins.
9. Spread:
   For known plasmid:
     Transfer 100 μL of transformation cell mixture onto a selection plate.
   For ligation reaction mix:
     Centrifuge the whole 1000 μL transformation cell mixture at 6000 rpm for 2 mins. Remove 900 μL supernatant and re-suspended cells with 100 μL supernatant left. Transfer the 100 μL resuspended cells onto a selection plate.
10. Incubate overnight at 37℃ with plates upside down.

8. Plasmid DNA extraction
Plasmid DNA extraction was carried out according to the TIANprep Rapid Mini Plasmid Kit

EGFP Measurement Protocol

Materials:
Liquid LB medium
100 mg/mL Kanamycin
BL21 competent cells
Plasmids: pET28a vector, pET28a-J23114-DmpR-GFP-Rluc, pET28a-J23101-DmpR-GFP-Rluc, pET28a-J23107-DmpR-GFP-Rluc, pET28a-Pr-DmpR-GFP-Rluc, pET28a-Pr-PEKDmpR-GFP-Rluc,
transparent flat bottom 96-well plates (for Absorbance 600 measurement),
white flat bottom 96-well plates (for EGFP measurement)
Methods:
1. Transform plasmids into BL21 competent cells. Incubate the plates at 37°C overnight.
2. Pick one monoclonal colony and inoculate into 5 mL liqud LB medium containing 50 μg/mL kanamycin. Overnight culture.
3. Transfer 1 mL overnight-cultured bacteria into 15 mL LB medium containing 50 μg/mL kanamycin.
4. Take 100 μL samples from the 15 mL growing bacteria at 0, 2, 4, 6, 8, 12 and 24 hours and transfer into transparent flat bottom 96-well plates for Absorbance 600 measurement or white flat bottom 96-well plates for EGFP detection.
5. Measure Absorbance 600 or EGFP by Biotek microplate reader at indicated time.

Renilla Luciferase Assay

Materials:
600 μM Promega ViviRen Renila Luciferase substrate
100 mM phenol solution
100 mM 2-chlorophenol solution
100 mM 4-chlorophenol solution
100 mM 2, 4-dichlorophenol solution
100 mM pyrocatechol solution
Liquid LB medium
100 mg/mL Kanamycin
BL21 competent cells
Plasmids: pET28a vector, pET28a-J23114-DmpR-GFP-Rluc, pET28a-J23101-DmpR-GFP-Rluc, pET28a-J23107-DmpR-GFP-Rluc, pET28a-Pr-DmpR-GFP-Rluc, pET28a-Pr-PEKDmpR-GFP-Rluc
Methods:
1. Transform plasmids into BL21 competent cells. Incubate the plates at 37°C overnight.
2. Pick one monoclonal colony and inoculate into 50 mL LB medium containing 50 μg/mL kanamycin. Overnight culture.
3. Transfer 594 μL of 50 mL overnight bacteria into 1.5 mL eppendorf tubes.
4. Add 6 μL phenols into the aliquot 594 μL bacteria in the eppendorf tubes.
5. Seal the tubes and incubate at 37°C, 250 rpm, 3 hrs for vector, J23114, J23101 and J23107 groups. Incubate at 37°C, 250 rpm, 12 hrs for vector, Pr-DmpR and Pr-PEKDmpR groups.
6. Take 45 μL bacteria containing phenols mixture from tubes and put it into a new 1.5 mL eppendorf tube. Transfer corresponding 100 μL mixture to the transparent flat bottom 96-well plates as well.
7. Add 5 μL 600 μM renilla luciferase substrate into the 45 μL mixture in 1.5 mL eppendorf tube.
8. Vortex 7 seconds and measure the luciferase reading.
9. Measure the Absorbance 600 of the corresponding 100 μL mixture in the transparent flat 96-well plates.

Poisoning Protocol

Materials:
10% L-arabinose
LB medium
100 mg/mL Kanamycin
Methods
1. Inoculate 7.5 mL overnight cultured bacteria into 800 mL LB medium with 50 μg/mL kanamycin. Make sure the initial reading of Absorbance 600 is around 0.015.
2. Sub-pack the bacteria into 4 conical flasks, 150 mL each at Absorbance 600 around 0.15-0.2.
3. Add L-arabinose to the 4 conical flasks for 0%, 0.1%, 0.2%, 0.6% final concentration respectively.
4. Measure Absorbance 600 at once after inoculation. Every hour in the beginning 6 hrs and every 2 hrs in the next 6 hrs respectively.

IPTG Adding Protocol

Materials:
100 mM IPTG
LB medium
100 mg/mL Kanamycin
Methods
1. Inoculate 7.5 mL overnight cultured bacteria into 800 mL LB medium with 50 μg/mL kanamycin. Make sure the initial reading of Absorbance 600 is around 0.015.
2. Sub-pack the bacteria into 4 conical flasks, 150 mL each at Absorbance 600 around 0.15-0.2.
3. Add IPTG to the 4 conical flasks for 0, 0.1, 0.2, 0.5 mM final concentration respectively.
4. Measure Absorbance 600 at once after inoculation. Every hour in the beginning 6 hrs and every 2 hrs in the next 6 hrs respectively.

Detoxification Protocol

Materials:
10% L-arabinose
100 mM IPTG
LB medium
100 mg/mL Kanamycin
Plasmids: pET28a vector, T-A (pET28a-totion-antitoxin)
Methods:
1. Inoculate 7.5 mL overnight cultured bacteria into 800 mL LB medium with 50 μg/mL kanamycin. Make sure the initial reading of Absorbance 600 is around 0.015.
2. Sub-pack 150 mL bacteria into a new conical flask as the no IPTG and no L- arabinose control at Absorbance 600 around 0.15-0.2.
3. Add 13 ml L-arabinose into the rest 650 mL bacteria (0.2% final concentration).
4. Take 150 mL bacteria into a new conical flask as the no IPTG control when Absorbance 600 does not change any more.
5. Sub-pack 150 mL bacteria into a new conical flask and add 150 μl IPTG into the 150 mL bacteria (0.1 mM final concentration) when Absorbance 600 does not change any more.
6. Measure the Absorbance 600 at inoculation beginning. Every hour in the beginning 6 hrs and every two hours in the next 6 hrs respectively.

Enzyme tfdB-JLU Induction and Purification Protocol

Materials
IPTG
LB medium
100 mg/ml Kanamycin
Ni-NTA His Bind column
lysis buffer: 200 mM NaCl, 20% glycerol, 20 mM Tris–HCl, and pH 7.5
buffer A: 20 mM Tris-HCl buffer, pH 7.5, containing 20% (v/v) glycerol, 200 mM NaCl
buffer B: 500 mM imidazole,20 mM Tris-HCl buffer, pH 7.5, containing 20% (v/ v) glycerol, 200 mM NaCl
Methods:
Day 1
1. Inoculate E. coli harboring tfdB-JLU in LB medium supplemented with 50 μg/mL kanamycin at 37℃, 220 rpm, overnight culture.
Day 2
2. Transfer 1 mL overnight cultured cells into 200 mL LB.
3. Add IPTG to final concentration 0.3 mM at OD600 around 0.4 of the grown bacteria.
4. Incubate the cells at 18℃ for 16 hrs by shaking at 180 rpm to induce tfdB-JLU enzyme.
Day 3
5. Harvest overnight-inducted cells. Centrifuge at 4000 rpm for 30 mins at 4℃. Discard the supernatant.
6. Resuspend with 50 mL lysis buffer (200 mM NaCl, 20% glycerol, 20 mM Tris–HCl, and pH 7.5).
7. Sonicate 5 s with 5 s interval each time at 20 kHz for 1 hr on ice.
8. Remove cell debris by centrifugation at 12000 rpm for 30 mins.
9. Purify the His-tagged recombinant enzyme by passing the supernatant through a Ni-NTA His Bind column.
10. Reload the supernatant twice before washing.
11. Use 8 mL volumes of buffer A wash the column.
12. Wash the column with 10 mL buffer A containing 20 mM imidazole (using buffer A dilute buffer B).
13. Collect samples, 1 mL/tube.
14. Wash the column with 10 mL buffer A containing 50 mM imidazole, collect samples, 1 mL/tube.
15. Wash the column with 6 mL buffer A containing 100 mM imidazole, collect samples, 1 mL/tube.
16. Wash the column with 10 mL buffer A containing 250 mM imidazole, collect samples, 1 mL/tube.
17. Wash the column with 40 mL buffer B.
18. Wash the column with 40 mL buffer A.
19. Wash the column with 40 mL H2O.

Enzyme tfdB-JLU Characterization Protocol

Materials:
nicotinamide adenine dinucleotide phosphate (NADPH)
FAD (flavin adenine dinucleotide)
sodium phosphate buffer (pH 7.5)
phenol substrate:2,4-DCP, 3-CP, 4-CP, phenol, 2,3-DCP, 2,5-DCP, 2,6-DCP, 3,4-DCP, 3,5-DCP, 2,4,5-TCP
Methods:
Determine the activities of chlorophenol hydroxylases by monitoring the decrease in absorbance at 340 nm (e340 = 6,220 M-1 cm-1) following the substrate-dependent oxidation of NADPH. Define one unit of activity as the amount of enzyme required to consume 1 μmol NADPH per min at 25℃.
1. Incubate 12 μg/mL tfdB-JLU with 0.1 mM substracts, 5 μM FAD and 0.2 mM NADPH in 50 mM sodium phosphate buffer (pH 7.5) at 25℃ in 1 mL mixture system.
2. Monitor Absorbance 340 for 1 hr.

Phenol Degradation Protocol (4-AAP Assay)

Principles:
Phenol can react with 4-AAP in alkaline medium (pH =10.0±0.2), with the oxidizer K3Fe(CN)6. Reaction product antipyrine dye appears orange, whose absorption peak is at 510nm.

Materials:
2% (m/v) 4-amino anti pyrine (4-AAP)
8% (m/v) potassium ferricyanide K3Fe(CN)6
1 mM glucose
0.9% NaCl
buffer: 10g NH4Cl in 50 mL ammonium hydroxide.
Methods:
1. Inoculate E. coli containing tfdB-JLU or mock in LB with 50 μg/mL kanamycin at 37℃, 220 rpm, overnight culture.
2. Transfer 1 mL overnight cultured cells into 200 mL LB.
3. Add IPTG to final concentration 0.3 mM at OD600 around 0.4 of the grown bacteria.
4. Incubate at 18℃ for 16 hrs by shaking at 180 rpm to induce tfdB-JLU enzyme.
5. Take 500 μL induced bacteria samples for measuring Absorbance 600.
6. Take out 20 mL corresponding bacteria into 50 mL centrifuge tube.
7. Centrifuge at 3000 rpm for 5 minw, discard supernatant, and suspend with 10 mL solution (1 mM glucose in 0.9% NaCl).
8. Centrifuge in 3000 rpm for 5 mins again. Discard supernatant.
9. Re-suspend with 10 mL 0, 1, 2, or 5 mM phenol with 1 mM glucose in 0.9% NaCl solution.
10. Incubate at 25℃ for 12 hrs.
11. Centrifuge samples immediately at 12000 rpm for 5 mins.
12. Take out 0.5 mL supernatant into assay system. Mix a 14.5 mL reaction system as below. Incubate for 10 mins.

ComponentVolume
samples
H2O
buffer
2% 4-AAP
8% K3Fe(CN)6
0.5 mL
9.5 mL
0.9 mL
1.8 mL
1.8 mL
Total volume 14.5 mL

13. Measure Absorbance 510 for all the samples.

Phenol Degradation Protocol (HPLC Assay)

Materials:
ZORBAX Eclipse XDB C-18 column
methanol, acetic acid, phenol,
sodium phosphate buffer (pH 7.5)
LB medium with kanamycin
IPTG
HPLC condition:
ZORBAX Eclipse XDB C-18 column
mobile phase:methanol : 0.2% acetic acid (30:70)
rate: 1 mL/min
injection volume: 10 μL
wavelength: 275 nm
temperature: 25°C
Methods:
1. Inoculate E. coli containing tfdB-JLU or mock in LB medium with 50 μg/mL kanamycin at 37℃, 220 rpm, overnight culture.
2. Transfer 250 μL overnight cultured cells into 50 mL LB.
3. Add IPTG to final concentration 0.3 mM at OD600 around 0.4 of the grown bacteria.
4. Incubate at 18℃ for 16 hrs by shaking at 180 rpm to induce tfdB-JLU enzyme.
5. Take 1 mL induced bacteria samples for measuring Absorbance 600.
6. Transfer 1 mL corresponding induced bacteria samples to the tubes. Centrifuge 4000 rpm for 10 mins. Discard the supernatant.
7. Resuspend the precipitate with 0, 1, 2, or 5 mM phenol in PBS respectively. Incubate at 25℃, shaking for 4 hrs.
8. Centrifuge 4000 rpm for 10 mins. Keep the supernatant and pass the 0.22 μm filter.
9. Measure with HPLC.

Circuit Protocol

Materials:
phenol
LB with kanamycin
HPLC assay materials and condition
4-AAP assay materials and condition
Methods:
1. Inoculate E. coli containing J23107-DmpR-tfdB-JLU or mock in LB with 50 μg/mL kanamycin at 37℃, 220 rpm, overnight culture.
2. Transfer 500 μL overnight cultured cells into 100 mL LB. Shake at 37 ℃ for 2 hrs.
3. Add 0, 1, 2, or 5 mM phenol into the cultured bacteria.
4. Measure Abs600 right after phenol adding and subsequent every 2 hrs till 24 hrs culture.
5. Phenol was tested 2, 6, 10 or 24 hrs after its adding with both HPLC method and 4-AAP assay.