Team:XMU-China/protocols

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     <p align="center"><span class="tit">Protocols</span></p>
     <p align="center"><span class="tit">Protocols</span></p>
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     <p> <strong ><span class="subtitle">General  protocols</span></strong><br />
+
     <p align="left"> <strong class="subtitle"><a name="_Toc01" id="Toc01"></a>1 General  protocols</strong><br />
-
       <strong class="subtitle">Stock solution</strong><br />
+
       <strong class="subtitle"><a name="_Toc02" id="Toc"></a></strong><strong class="subsubtitle">1.1 Stock solution</strong><br />
-
       50mg/mL Kanamycin <br />
+
       50 mg/mL Kanamycin <br />
-
       <strong>-</strong> 0.5g  Kan, 10mL water, filter sterilize with millipore express membrane, freeze in aliquots <br />
+
       <strong>- </strong>0.5 g Kan, 10 mL water, filter sterilize with millipore express membrane, freeze in aliquots <br />
-
       100mg/mL Amp <br />
+
       100 mg/mL Ampicillin <br />
-
       - 1g Amp, 10mL  water, filter sterilize with millipore express membrane, freeze in aliquots. <br />
+
       -1 g Amp, 10 mL water, filter sterilize with millipore  express membrane, freeze in aliquots. <br />
-
       50mmol/L Arabinose<br />
+
       50 mmol/L Arabinose<br />
-
       - 0.1876g Arabinose, 25mL water, filter sterilize  with millipore express membrane.</p>
+
       - 0.1876 g Arabinose, 25 mL water, filter sterilize with millipore express membrane.<br />
-
     <p><strong class="subtitle">Preparation of  Competent BL21 </strong> <br />
+
      0.25 mg/mL Anhydrotetracycline<br />
-
       - Thaw an aliquot of cells (without any  plasmid in them) on ice <br />
+
      -  0.1 mg Anhydrotetracycline, 0.4 mL PBS, filter sterilize with millipore express membrane.</p>
-
       - To 50 mL of sterile LB, add 100&mu;L aliquot of the thawed cells: remember, this LB does not have any  antibiotic in it, so work as aseptically as possible (i.e. autoclave all  solutions and use sterile pipettes).<br />
+
    <p align="left">&nbsp;</p>
-
       - Grow cells in the shaker at 37℃ until they reach an OD = 0.3-0.4 at 600nm (1cm  pathlength cell). This usually takes 1.5-2 hours.<br />
+
     <p><strong class="subtitle"><a name="_Toc03" id="Toc2"></a></strong><strong class="subsubtitle">1.2 Preparation of  Competent BL21 </strong> <br />
-
       - Ice down the LB with growing cells for 10 min.<br />
+
       Thaw an aliquot of cells (without any  plasmid in them) on ice <br />
-
       - Aliquot into sterile 1.5mL tubes and Spin down at 6000rpm for 10 minutes at 4℃; discard supernatant and.<br />
+
       - To 50 mL of sterile LB, add 100&mu;L aliquot of the thawed cells: remember, this LB does not have any  antibiotic in it, so work as aseptically as possible (i.e. autoclave all  solutions and use sterile pipettes).<br />
-
       - Ice down sterile 100mM CaCl<code>2</code> and 100mM MgC<code>2</code>solutions during centrifugation. <br />
+
       - Grow cells in the shaker at 37 ℃ and 200 rpm, until they reach an OD<sub>600</sub>= 0.3-0.4. This usually takes 1.5-2 hours.<br />
-
       - Gently resuspend each pellet in 8mL 0.1M MgCl<code>2</code> and 2mL 0.1 M CaCl<code>2</code> and combine into 2 tubes (this should take 1-2 minutes per tube) <br />
+
       - Ice down the LB with growing cells for 10 min.<br />
-
       - Centrifuge 6000rpm for 10 minutes and discard supernatant. <br />
+
       - Aliquot into sterile 1.5 mL tubes and spin down at 6000rpm for 10 min at 4 ℃; discard supernatant.<br />
-
       - Resuspend each pellet on ice in 100&mu;L 0.1M  of ice cold CaCl<code>2</code> and combine into one tube </p>
+
       - Ice down sterile 100 mM  CaCl<sub>2</sub> and 100mM MgCl<sub>2</sub> solutions during centrifugation. <br />
-
     <p><strong class="subtitle">Transformation </strong> <br />
+
       - Gently resuspend each pellet with 400 &mu;L 0.1 M MgCl<sub>2</sub> and 100 &mu;L 0.1 M CaCl<sub>2</sub>.<br />
-
       - Add 10&mu;L of DNA. Swirl gently with pipette. <br />
+
       - Centrifuge 6000 rpm for 10 min and discard supernatant. <br />
-
       - Incubate tubes on ice for 20 minutes <br />
+
       - Resuspend each pellet on ice in 100 &mu;L 0.1 M ice cold CaCl<sub>2</sub> and combine into one tube </p>
-
       - Heat pulse tubes in 42℃ water bath for 30 seconds. <br />
+
     <p><strong class="subtitle"><a name="_Toc04" id="Toc3"></a></strong><strong class="subsubtitle">1.3 Transformation </strong> <br />
-
       - Incubate on ice for 2 minutes <br />
+
       - Add 10 &mu;L of DNA. Swirl gently with pipette. <br />
-
       - Add 790uL of LB broth to each tube and incubate for an hour at 37℃ with shaking. <br />
+
       - Incubate tubes on ice for 20 min <br />
-
      - Spread 100uL and 50uL of each culture on an  LB agar plate containing the appropriate antibiotics and incubate overnight at 37℃ (spread using beads). </p>
+
       - Heat pulse tubes in 42 ℃ water bath for 30 seconds. <br />
-
     <p><strong class="subtitle">Purity Plasmid Miniprep</strong> <br />
+
       - Incubate on ice for 2 min <br />
-
       <strong>- </strong>Centrifuge sample in Eppendorf tube approximately 1.5 mL at a time, draining off supernatant after each spin and adding more cell solution <br />
+
       - Add 790 &mu;L of LB broth to each tube and incubate for an hour at 37 ℃ with shaking. <br />
-
       <strong>- </strong>Resuspened  the pelleted cells in 250 &mu;L of the Resuspension Solution (Mixture with Solution I and RNasa A). The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain.<br />
+
    - Spread 100 &mu;L and 50μL of each culture on an  LB agar plate containing the appropriate antibiotics and incubate overnight at 37 ℃ (spread using beads). </p>
-
       <strong>- </strong>Add 250 &mu;L of the Lysis Solution (Solution II) and mix thoroughly and gently by inverting the tube 5-6 times, letting it stand for 1-2minutes at room temperature until the solution becomes viscous and slightly clear.<br />
+
     <p align="left"><strong class="subtitle"><a name="_Toc05" id="Toc4"></a></strong><strong class="subsubtitle">1.4 Plasmid Purification</strong> <br />
-
       <strong>- </strong>Add 350 &mu;L of the Neutralization Solution (Solution III) and mix immediately and thoroughly by inverting the tube 5-6 times.<br />
+
       <strong>- </strong>Centrifuge sample in eppendorf tube approximately 1.5 mL at a time, draining off supernatant after each spin and adding more cell solution <br />
-
       - Centrifuge for 10 min at 12,000rpm to pellet cell debris.<br />
+
       <strong>-</strong>Resuspend the pelleted cells in 250 &mu;L of the resuspension Solution (mixture with Solution I and RNasa A). The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain.<br />
-
       - Apply the supernatant to the supplied spin column by decanting. Avoid disturbing or applying the white precipitate.<br />
+
       <strong>- </strong>Add 250 &mu;L of the Lysis Solution (Solution II) and mix thoroughly and gently by inverting the tube 5-6 times, letting it stand for 1-2 min at room temperature until the solution becomes viscous and slightly clear.<br />
-
       - Centrifuge for 1 min at 12,000rpm. Discard  flow-through and place the column back into the same collection tube.<br />
+
       <strong>- </strong>Add 350 &mu;L of the Neutralization Solution (Solution III) and mix immediately and thoroughly by inverting the tube 5-6 times.<br />
-
       - Add 500 &mu;L of the Wash Buffer PB to the spin column. Centrifuge for 1minute at 12,000rpm and discard  flow-through. Place the column back into the same collection tube.<br />
+
       - Centrifuge for 10 min at 12,000 rpm to pellet cell debris.<br />
-
       - Add 500 &mu;L of the Wash Buffer W to the spin column. Centrifuge for 1minute at 12,000rpm and discard the flow-through. Place the column back into the same collection tube.<br />
+
       - Apply the supernatant to the supplied spin column by decanting. Avoid disturbing or applying the white precipitate.<br />
-
       - Repeat the step 9 again.<br />
+
       - Centrifuge for 1 min at 12,000 rpm. Discard  flow-through and place the column back into the same collection tube.<br />
 +
       - Add 500 &mu;L of the Wash Buffer PB to the spin column. Centrifuge for 1min at 12,000 rpm and discard  flow-through. Place the column back into the same collection tube.<br />
 +
       - Add 500 &mu;L of the Wash Buffer W to the spin column. Centrifuge for 1min at 12,000 rpm and discard  the flow-through. Place the column back into the same collection tube.<br />
 +
       - Repeat the step 9 again.<br />
       - Discard flow-through  and centrifuge for an additional 3 min to remove residual Wash Solution. <br />
       - Discard flow-through  and centrifuge for an additional 3 min to remove residual Wash Solution. <br />
-
       - Place the spin column in a clean 1.5 mL centrifuge tube, and pipet 20 &mu;L Elution Buffer TE (prewarm to 60℃) directly to the center of the column without touching the membrane. Let it stand for 2 min at room temperature and centrifuge for 1 min at 12,000rpm.<br />
+
       - Place the spin column in a clean 1.5 mL centrifuge tube, and pipet 20 &mu;L Elution Buffer TE (prewarm to 60 ℃) directly to the center of the column without touching the membrane. Let it stand for 2 min at room temperature and centrifuge for 1 min at 12,000 rpm.<br />
-
       - Discard the column and  store the purified plasmid DNA at -20&deg;C.</p>
+
       - Discard the column and  store the purified plasmid DNA at -20 &deg;C.</p>
-
     <p class="subtitle"><strong>Reaction system of restriction endonuclease</strong></p>
+
     <p align="left" class="subsubtitle"><strong class="subtitle"><a name="_Toc06" id="Toc5"></a></strong><strong>1.5 Reaction system of restriction endonuclease</strong></p>
-
     <p align="left"><img width="553" height="297" src="http://2012.igem.org/wiki/images/1/1f/The_digestion_system.png" /><img width="900" height="1" src="image003.jpg" />- System1、2、3 and 4 are used for Standard BioBrick Assembly .<br />
+
     <p align="left"><img src="http://2012.igem.org/wiki/images/7/72/The_digestion_system.jpg" alt="" width="800" /><img src="image005.jpg" alt="" width="900" height="1" />- System1、2、3 and 4 are used for Standard BioBrick Assembly .<br />
-
       - System 5 and 6 are used for Restriction analysis. Digestion of sample: at least 500 ng DNA / 10 &micro;L volume. Digest for h at 37 &deg;C, afterwards inactivated by adding 10x loading buffer and standing for 10 min at room temperature. </p>
+
       - System 5 and 6 are used for restriction analysis. Digestion of sample: at least 500 ng DNA / 10 &mu;L volume. Digest for 1 h at 37 &deg;C, afterwards inactivated by adding 10<strong>&times;</strong> loading buffer and standing for 10 min at room temperature. </p>
-
     <p><strong class="subtitle">Standard BioBrick Assembly</strong><br />
+
     <p align="left"><strong class="subtitle"><a name="_Toc07" id="Toc6"></a></strong><strong class="subsubtitle">1.6 Standard BioBrick Assembly</strong><br />
-
       - Digestion of insert: 2 &mu;g~5 &mu;g DNA / 100 &micro;L volume, 10x H buffer, EcoRI, SpeI. Digestion and inactivation. Clean up the insert via gel electrophoresis. When cutting the insert out of the gel, try avoiding staining or exposure to ultraviolet light of the insert. <br />
+
       - Digestion of insert: 2 &mu;g~5 &mu;g DNA / 100 &mu;L volume, 10<strong>&times;</strong> H buffer, <em>Eco</em>R I, <em>Spe </em>I. Digestion and inactivation. Clean up the insert via gel electrophoresis. When cutting the insert out of the gel, try avoiding staining or exposure to ultraviolet light of the insert. <br />
-
       - Digestion of vector: 2 &mu;g~5 &mu;g DNA / 100 &micro;L volume, 10 x M buffer, EcoRI, XbaI. Digestion and inactivation. Clean up the insert via gel electrophoresis. When cutting the insert out of the gel, try to avoid staining or exposure to ultraviolet light of the insert.</p>
+
       - Digestion of vector: 2 &mu;g~5 &mu;g DNA / 100 &mu;L volume, 10<strong>&times;</strong> M buffer, <em>Eco</em>R I, <em>Xba </em>I. Digestion and inactivation. Clean up the insert via gel electrophoresis. When cutting the insert out of the gel, try to avoid staining or exposure to ultraviolet light of the insert.</p>
-
     <p><strong class="subtitle">Suffix Insertion </strong><br />
+
     <p align="left"><strong class="subtitle"><a name="_Toc08" id="Toc7"></a></strong><strong class="subsubtitle">1.7 Suffix Insertion </strong><br />
-
       - Digestion of insert: 2 &mu;g~5 &mu;g DNA / 100 &micro;L volume, 10x M buffer, XbaI, PstI. Digestion and inactivation. Clean up the insert.<br />
+
       - Digestion of insert: 2 &mu;g~5 &mu;g DNA / 100 &mu;L volume, 10<strong>&times;</strong> M buffer, <em>Xba </em>I, <em>Pst </em>I. Digestion and inactivation. Clean up the insert.<br />
-
       - Digestion of vector : 2 &mu;g~5 &mu;g DNA / 100 &micro;L volume, 10x H buffer, SpeI, PstI. Digestion and inactivation. Clean up the vector.</p>
+
       - Digestion of vector : 2 &mu;g~5 &mu;g DNA / 100 &mu;L volume, 10<strong>&times;</strong> H buffer, <em>Spe </em>I,<em> Pst </em>I. Digestion and inactivation. Clean up the vector.</p>
-
     <p><strong class="subtitle">Ligation </strong><br />
+
     <p align="left"><strong class="subtitle"><a name="_Toc09" id="Toc8"></a></strong><strong class="subsubtitle">1.8 Ligation </strong><br />
-
       - After digestion and clean-up, the next step is ligation. ligation at 16℃ for 4h or at 4℃ for 16h. Table 2 is the system of ligation. <br />
+
       - After digestion and clean-up, the next step is ligation. ligation at 16 ℃  for 4 h or at 4 ℃  for 16 h. Table 2 is the system of ligation. <br />
       Table 2&nbsp; Ligation system</p>
       Table 2&nbsp; Ligation system</p>
     <div align="center">
     <div align="center">
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       <table border="1" cellspacing="0" cellpadding="0">
+
   
 +
       <table >
         <tr>
         <tr>
           <td width="284" valign="top"><br />
           <td width="284" valign="top"><br />
             <strong>Components</strong></td>
             <strong>Components</strong></td>
-
           <td width="284" valign="top"><p align="center">Volume/&micro;L </p></td>
+
           <td width="284" valign="top"><p align="left">Volume/&mu;L </p></td>
         </tr>
         </tr>
         <tr>
         <tr>
-
           <td width="284" valign="top"><p align="center">Digested vector </p></td>
+
           <td width="284" valign="top"><p align="left">Digested vector </p></td>
-
           <td width="284" valign="top"><p align="center">7 </p></td>
+
           <td width="284" valign="top"><p align="left">7 </p></td>
         </tr>
         </tr>
         <tr>
         <tr>
-
           <td width="284" valign="top"><p align="center">Digested insert </p></td>
+
           <td width="284" valign="top"><p align="left">Digested insert </p></td>
-
           <td width="284" valign="top"><p align="center">1 </p></td>
+
           <td width="284" valign="top"><p align="left">1 </p></td>
         </tr>
         </tr>
         <tr>
         <tr>
-
           <td width="284" valign="top"><p align="center">10<strong>&times; T4 ligation buffer</strong></p></td>
+
           <td width="284" valign="top"><p align="left">10&times; T4 ligation buffer </p></td>
-
           <td width="284" valign="top"><p align="center">1 </p></td>
+
           <td width="284" valign="top"><p align="left">1 </p></td>
         </tr>
         </tr>
         <tr>
         <tr>
-
           <td width="284" valign="top"><p align="center">T4 ligase </p></td>
+
           <td width="284" valign="top"><p align="left">T4 ligase </p></td>
-
           <td width="284" valign="top"><p align="center">1 </p></td>
+
           <td width="284" valign="top"><p align="left">1 </p></td>
         </tr>
         </tr>
         <tr>
         <tr>
-
           <td width="284" valign="top"><p align="center">Total </p></td>
+
           <td width="284" valign="top"><p align="left">Total </p></td>
-
           <td width="284" valign="top"><p align="center">10 </p></td>
+
           <td width="284" valign="top"><p align="left">10 </p></td>
         </tr>
         </tr>
       </table>
       </table>
     </div>
     </div>
-
     <p>&nbsp;</p>
+
     <p align="left">&nbsp;</p>
-
     <p><strong class="subtitle">Restriction analysis</strong><br />
+
     <p align="left"><strong class="subtitle"><a name="_Toc10" id="Toc9"></a></strong><strong class="subsubtitle">1.9 Restriction analysis</strong><br />
-
       - Pick one colony with a sterile tip and cultivation in 20mL LB for overnight at 37 ℃<br />
+
       - Pick one colony with a sterile tip and cultivation in 20 mL LB for overnight at 37 ℃<br />
       - Isolation of Plasmid<br />
       - Isolation of Plasmid<br />
-
       - Digest BioBrick,the system of Restriction analysis refer to table1<br />
+
       - Digest BioBrick,the system of Restriction analysis refer to table1<br />
-
       - Gel electrophoresis:add 2.2 &micro;L loading buffer to digestion mixture. An agarose concentration is 1 %.</p>
+
       - Gel electrophoresis:add 2.2 &mu;L loading buffer to digestion mixture. An agarose concentration is 1 %.</p>
-
     <p><strong class="subtitle">Gel Extraction</strong><br />
+
     <p align="left"><strong class="subtitle"><a name="_Toc11" id="Toc10"></a></strong><strong class="subsubtitle">1.10 Gel Extraction</strong><br />
-
       - Weigh a 1.5mL centrifuge tube  for each DNA fragment to be isolated and record the weight.<br />
+
       - Weigh a 1.5 mL centrifuge tube  for each DNA fragment to be isolated and record the weight.<br />
-
       - Excise gel slice containing the  DNA fragment using a clean scalpel or razor blade. Cut as close to the DNA as  possible to minimize the gel volume. Place the gel slice into a pre-weighed 1.5  Ml tube and weigh. Record the weight of the gel slice.<br />
+
       - Excise gel slice containing the  DNA fragment using a clean scalpel or razor blade. Cut as close as possible to the DNA to minimize the gel volume. Place the gel slice into a pre-weighed 1.5 mL tube and weigh. Record the weight of the gel slice.<br />
-
       - Add Bing Buffer BD at a ratio  of 100&mu;L of solution per 100mg of agarose gel slices.<br />
+
       - Add Bing Buffer BD at a ratio  of 100 &mu;L of solution per 100 mg of agarose gel slices.<br />
-
       - &nbsp;Incubate the gel mixture at 55-65℃ for 7-10 min or until the gel slice is completely dissolved. Mix the tube by inversion every few minutes to facilitate the melting process. Ensure that the gel is completely dissolved.<br />
+
       - Incubate the gel mixture at 55-65 ℃ for 7-10 min or until the gel slice is completely dissolved. Mix the tube by inversion every few minutes to facilitate the melting process. Ensure that the gel is completely dissolved.<br />
-
       - &nbsp;After the dissolved gel mixture cool down, transfer it to the Spin Columns assembly and incubate for 2 minute at  room temperature.<br />
+
       - After the dissolved gel mixture cool down, transfer it to the Spin Columns assembly and incubate for 2 min at  room temperature.<br />
-
       - Centrifuge the Spin Columns  assembly in a microcentrifuge at 12,000 rpm for 1 minute, then discard the  flow-through.<br />
+
       - Centrifuge the Spin Columns  assembly in a microcentrifuge at 12,000 rpm for 1 min, and discard the  flow-through.<br />
-
       - &nbsp;Wash the columns by adding 500 &mu;L of Wash Buffer PE to the Columns. Centrifuge the columns assembly for 1 minute at 12,000 rpm, then discard the flow-through.<br />
+
       - Wash the columns by adding 500 &mu;L of Wash Buffer PE to the Columns. Centrifuge the columns assembly for 1 min  at 12,000 rpm, and discard the flow-through.<br />
-
       - &nbsp;Repeat step 7 again.<br />
+
       - Repeat step 7 again.<br />
       - Centrifuge the Columns for an  additional 3 min to completely remove residual wash buffer.<br />
       - Centrifuge the Columns for an  additional 3 min to completely remove residual wash buffer.<br />
-
       - &nbsp;Empty the Collection Tube and  recentrifuge the column assembly for 1 minute with the microcentrifuge lid open  (or off) to allow evaporation of any residual ethanol.<br />
+
       - Empty the Collection Tube and  recentrifuge the column assembly for 1 min with the microcentrifuge lid open  (or off) to allow evaporation of any residual ethanol.<br />
-
       - Place the spin column in a  clean 1.5 mL microcentrifuge tube, and pipet 20 &mu;L deionized water (pH is  8.0-8.5 and prewarm to 60℃)directly to the center of the column without touching the membrane. Incubate at room temperature for 2 min.<br />
+
       - Place the spin column in a  clean 1.5 mL microcentrifuge tube, and pipet 20 &mu;L deionized water (pH is  8.0-8.5 and prewarm to 60 ℃)directly to the center of the column without touching the membrane. Incubate at room temperature for 2 min.<br />
-
       - Centrifuge for 1 minute at 12,000 rpm. Discard the columns and store the microcentrifuge tube containing the eluted DNA at &ndash;20℃.</p>
+
       - Centrifuge for 1 min at 12,000 rpm. Discard the columns and store the microcentrifuge tube containing the eluted DNA at&ndash;20 ℃ .</p><hr>
-
     <p><span class="subtitle"><strong>Characterization</strong><br />
+
     <p align="left"><strong class="subtitle"><a name="_Toc12" id="Toc11"></a>2 Characterization</strong><br />
-
       <strong>Fluorescence Measurements</strong></span><br />
+
       <strong class="subtitle"><a name="_Toc01" id="Toc13"></a></strong><strong class="subsubtitle">2.1 Fluorescence Measurements</strong><br />
-
       - The samples to be tested are cultured from plates in 20 ml of the Basal Minimal Medium with appropriate antibiotics and incubated overnight at 37 &deg;C at 200 rpm. <br />
+
       - The samples to be tested are cultured from plates in 20 mL of the Basal Minimal Medium with  appropriate antibiotics and incubated overnight at 37 at 200 rpm. <br />
-
       - The culture is checked for OD600  next day and then subculture by the same medium with antibiotics at 37 &deg;C shaking for 2 hours.&nbsp; <br />
+
       - The culture is checked for OD<sub>600</sub> next day and then subculture by the same medium with antibiotics at 37 ℃ shaking for 2 hours. <br />
-
       - Add corresponding inducer at concentration gradients into the above-mentioned culture and keep on incubating. During the time incubating, every 15 min, take 1 mL bacteria liquid, then centrifuge the cells( 6000 rpm, 10 min ) and resuspend them in 1 mL PBS. At last, pipette to a 96 well plate.<br />
+
       - Add corresponding inducer at concentration gradients into the above-mentioned culture and keep on incubating. During the time incubating, every 15 min, take 1 mL bacteria liquid, then centrifuge the cells( 6000 rpm, 10 min ) and resuspend them in 1 mL PBS. At last, pipette to a 96 well plate.<br />
-
       - The plate reader made by Molecular Device then read. <br />
+
       - The plate reader made by Molecular Device then read. <br />
-
       - The program does the following: <br />
+
       - The program does the following: <br />
-
       - In Endpoint Reads, following measurements are taken in a time interval of 15 minutes: absorbance (600 nm filter) and fluorescence (485nm and 520nm for GFP). <br />
+
       - In endpoint readsfollowing measurements are taken in a time interval of 15 min: absorbance (600 nm filter) and fluorescence (485 nm and 520 nm for GFP). <br />
       - The results then  transfer to excel sheet and interpret.</p>
       - The results then  transfer to excel sheet and interpret.</p>
-
     <p><span class="subtitle"><strong>Immobilization</strong><br />
+
     <p align="left">&nbsp;</p>
-
       <strong>Prepare Sodiumcellu-losesulfate (NaCS)</strong></span><br />
+
    <p align="left"><strong class="subtitle"><a name="_Toc14" id="Toc13"></a></strong><strong class="subsubtitle">2.2 Protein electrophoresis</strong><br />
-
       - Deepfreeze H2SO4 and absolute alcohol at -20℃ for at least 2 hours;<br />
+
       <strong class="subtitle"><a name="_Toc15" id="Toc14"></a></strong><strong class="sub3title">2.2.1 Preparation of Samples</strong><br />
-
       - Prepare a sulphuric acid and ethanol solution at the proportion of 1.51:1(120mL H2SO4 and 80mL alcohol), maintaining it at -18℃ for at least 2 hours;<br />
+
      - The samples to be  tested are cultured in the Basal Medium with appropriate antibiotics, and take  200 &mu;L bacteria liquid to determine  its OD<sub>600</sub> at appropriate time.<br />
-
       - Put the sulphuric acid and ethanol solution and 500mL industrial alcohol in an ice box, maintaining them at 0℃ for at least 1 hour;<br />
+
      - Dilute or  concentrate the next 200 &mu;L bacteria liquid in  order to let the OD<sub>600</sub> equals to 4.0 while the computational formula  is the actual OD<sub>600</sub> * 200=2.0 * X, and X presents the total volume  of the bacteria liquid after being diluted or concentrated while its unit is &mu;L  as well. <br />
-
       - Immerse 4g dry absorbent cotton in the solution in ice-bath for 66 min. Then squeezed out the solution and rinsed the reacted linters with 0℃ industrial alcohol in draught cupboard;<br />
+
      - Add 30 &mu;L  diluted or concentrated liquid into corresponding 1.5 mL centrifugal tubes,  then mix up them with 10 &mu;L loading buffer. <br />
-
       - Squeeze out the alcohol, then put the linters in 400mL deionized water and regulated pH to about 3. Stir and dissolve it for 10 min, then filtrate it;<br />
+
      - Put these  centrifugal tubes into metal bath and heat them in 100 ℃ in around 5 to 8 min,  then centrifuge them at the speed of 13000 rpm for 5 min, the supernatant is what we need. </p>
-
       - Collected the filtrate and regulated pH to 9.3 accurately;<br />
+
    <p align="left"><strong class="subtitle"><a name="_Toc16" id="Toc15"></a></strong><strong class="subsubtitle"><span class="sub3title">2.2.2 Manufacture Albumen Gel</span></strong><br />
-
       - Add industrial alcohol gradually to the solution until there appear the largest volume of white retiary floccule on the top of the solution;<br />
+
      - Prepare a clear  centrifuge tube in the capacity of 50 mL, and make running gel, high  concentration one, following the formula below. Then mix up them and pour the  mixture into a glass pane. </p>
-
       - Centrifuge the floccule for 5 min at 5,000rpm and collect it;<br />
+
    <div align="center">
-
       - 65℃ drying for at least 24 hours until it is completely dry, then collect the final production.</p>
+
      <table border="1" cellspacing="0" cellpadding="0">
-
     <p class="subtitle"><strong>Prepare microcapsules</strong></p>
+
        <tr>
-
     <p>- Centrifuge 10mL bacteria sample for 3 min at 6000rpm and collect the deposit;<br />
+
          <td width="444" colspan="2" valign="top"><p align="left">Running Gel</p></td>
-
       - Add 10mL NaCS solution and mix it throughly with the cells;<br />
+
        </tr>
-
       - Put a 6% PDMDAAC solution on a magnetic stirrer and stir it at a certain speed, maintaining a small eddy in the center of liquid surface;<br />
+
        <tr>
-
       - Drop the mixture into the fringe of the eddy by a 1mL injector until it form an spheroidic membrane. It takes 10 min to react completely and form microcapsules;<br />
+
          <td width="444" colspan="2" valign="top"><p align="left">Final Gel    Concentration (5 mL; 1 ea ;1.0    mm thick; 10%)</p></td>
-
       - Tip all microcapsules to a strainer and rinse it with sterile water. Then transfer all microcapsules&nbsp; into&nbsp; LB medium with Ampicillin, 37℃ shaker incubate at 100rpm.</p>
+
        </tr>
-
     <p>&nbsp;</p>
+
        <tr>
 +
          <td width="341" valign="top"><p align="left">ddH<sub>2</sub>O</p></td>
 +
          <td width="103" valign="top"><p align="left">1.18 mL</p></td>
 +
        </tr>
 +
        <tr>
 +
          <td width="341" valign="top"><p align="left">4 <strong>&times;</strong> Running Gel Buffer (pH 8.8,1.5 M    Tris-HCl)</p></td>
 +
          <td width="103" valign="top"><p align="left">1.25 mL</p></td>
 +
        </tr>
 +
        <tr>
 +
          <td width="341" valign="top"><p align="left">Monomer Solution</p></td>
 +
          <td width="103" valign="top"><p align="left">2.48 mL</p></td>
 +
        </tr>
 +
        <tr>
 +
          <td width="341" valign="top"><p align="left">10% SDS</p></td>
 +
          <td width="103" valign="top"><p align="left">50 mL</p></td>
 +
        </tr>
 +
        <tr>
 +
          <td width="341" valign="top"><p align="left">10% Ammonium    Persulfate</p></td>
 +
          <td width="103" valign="top"><p align="left">50 mL</p></td>
 +
        </tr>
 +
        <tr>
 +
          <td width="341" valign="top"><p align="left">TEMED</p></td>
 +
          <td width="103" valign="top"><p align="left">5 mL</p></td>
 +
        </tr>
 +
      </table>
 +
    </div>
 +
    <p align="left">- Add some absolute alcohol to planish the top of gel. There will be approximately 60 min for its  solidification. <br />
 +
      - After  solidification, pour out the alcohol and make stacking gel following the  formula below. Then mix them up, add the solution onto the running gel in the  glass panes until it being filled up with the gel. Insert a clean comb into  stacking gel. Wait for about 40 min for stacking gel solidification.</p>
 +
    <div align="center">
 +
      <table border="1" cellspacing="0" cellpadding="0">
 +
        <tr>
 +
          <td width="444" colspan="2" valign="top"><p align="left">Stacking Gel</p></td>
 +
        </tr>
 +
        <tr>
 +
          <td width="444" colspan="2" valign="top"><p align="left">Stacking Gel    Concentration (1 ea ;1.0 mm    thick; 10%)</p></td>
 +
        </tr>
 +
        <tr>
 +
          <td width="341" valign="top"><p align="left">ddH<sub>2</sub>O</p></td>
 +
          <td width="103" valign="top"><p align="right">1.35 mL</p></td>
 +
        </tr>
 +
        <tr>
 +
          <td width="341" valign="top"><p align="left">4 <strong>&times;</strong> Running Gel Buffer (pH 6.8,1.5    M Tris-HCl)</p></td>
 +
          <td width="103" valign="top"><p align="right">0.58 mL</p></td>
 +
        </tr>
 +
        <tr>
 +
          <td width="341" valign="top"><p align="left">Monomer Solution</p></td>
 +
          <td width="103" valign="top"><p align="right">0.3 mL</p></td>
 +
        </tr>
 +
        <tr>
 +
          <td width="341" valign="top"><p align="left">10% SDS</p></td>
 +
          <td width="103" valign="top"><p align="right">25 mL</p></td>
 +
        </tr>
 +
        <tr>
 +
          <td width="341" valign="top"><p align="left">10% Ammonium    Persulfate</p></td>
 +
          <td width="103" valign="top"><p align="right">25 mL</p></td>
 +
        </tr>
 +
        <tr>
 +
          <td width="341" valign="top"><p align="left">TEMED</p></td>
 +
          <td width="103" valign="top"><p align="right">5 mL</p></td>
 +
        </tr>
 +
      </table>
 +
    </div>
 +
    <p align="left"><strong class="subtitle"><a name="_Toc17" id="Toc16"></a></strong><strong class="sub3title">2.2.3 Electrophoresis </strong><br />
 +
      - Take out the glass  pane with finished gel and then fasten it in an electrophoresis tank. Add some  1<strong>&times; </strong>Tank Buffer to detect  whether liquid leak or not.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; <br />
 +
      - Take out the comb  slowly and use pipette to add approximately 10 to 20 &mu;L  processed samples into the wells in stacking gel. <br />
 +
      - Add 1<strong>&times; </strong>Tank Buffer until the liquid  level is above the platinum line in the electrophoresis tank. <br />
 +
      - Cover up the  electrophoresis tank and connect it with the electrophoresis device. Set the program  120 V- 60 min and start it up. <br />
 +
      - When the green  marker band run to the bottom of running gel, stop the device. </p>
 +
    <p align="left"><strong class="subtitle"><a name="_Toc18" id="Toc17"></a></strong><strong class="subsubtitle"><span class="sub3title">2.2.4 Dyeing (Colloidal Coomassie Brilliant Blue)</span></strong><br />
 +
       - Take out the gel and put it into a clean petri dish. And add appropriate Commassie Blue Staining Solution.  Please make sure that the solution can cover all the gel. <br />
 +
      - Put the petri dish  onto the orbital shaker and dye for approximate 30 min.<br />
 +
      - Pour out the  staining solution then add enough destaining solution. Destain about 30 min. <br />
 +
      - Renew the destaining  solution for about 2 or 3 times until the blue background of gel being taken  off. <br />
 +
      - Pour out the  destaining solution and add appropriate water to clear it. </p>
 +
    <p align="left"><strong class="subtitle"><a name="_Toc19" id="Toc18"></a></strong><strong class="subsubtitle"><span class="sub3title">2.2.5 Scanning</span></strong><br />
 +
      Scan the processed gel  and save the picture for analysis. </p><hr>
 +
     
 +
    <p align="left"><strong class="subtitle"><a name="_Toc20" id="Toc19"></a>3 Immobilization</strong><br />
 +
      <strong class="subtitle"><a name="_Toc21" id="Toc20"></a></strong><strong class="subsubtitle">3.1 Prepare Sodiumcellu-losesulfate (NaCS)</strong><br />
 +
      - Deep freeze H<sub>2</sub>SO<sub>4</sub> and absolute ethanol at -20 ℃ for at least 2 hours;<br />
 +
       - Prepare a sulphuric acid and ethanol solution at the proportion of 1.51:1(120 mL H<sub>2</sub>SO<sub>4</sub>  and 80 mL alcohol), maintaining it at -18 ℃ for at least 2 hours;<br />
 +
       - Put the sulphuric acid and ethanol solution and 500mL industrial alcohol in an ice box, maintaining them at 0 ℃ for at least 1 hour;<br />
 +
       - Immerse 4 g dry absorbent cotton in the solution in ice-bath for 66 min. Then squeezed out the solution and rinsed the reacted linters with 0℃ industrial alcohol in draught cupboard;<br />
 +
       - Squeeze out the alcohol, then put the linters in 400 mL deionized water and regulated pH to about 3. Stir and dissolve it for 10 min, then filtrate it;<br />
 +
       - Collected the filtrate and regulated pH to 9.3 accurately;<br />
 +
       - Add industrial alcohol gradually to the solution until there appears the largest volume of white retiary floccule on the top of the solution;<br />
 +
       - Centrifuge the floccule for 5 min at 5,000 rpm and collect it;<br />
 +
       - 65 ℃ drying for at least 24 hours until it is completely dry, then collect the final production.</p>
 +
     <p align="left" class="subsubtitle"><strong class="subtitle"><a name="_Toc22" id="Toc21"></a></strong><strong>3.2 Prepare microcapsules</strong></p>
 +
     <p align="left">- Centrifuge 10 mL bacteria sample for 3 min at 6000 rpm and collect the deposit;<br />
 +
       - Add 10 mL NaCS solution and mix it completely with the cells;<br />
 +
       - Put a 6% PDMDAAC solution on a magnetic stirrer and stir it at a certain speed, maintaining a small eddy in the center of liquid surface;<br />
 +
       - Drop the mixture into the fringe of the eddy by a 1 mL injector until it form a spheroidic membrane. It takes 10 min to react completely and form microcapsules;<br />
 +
       - Tip all microcapsules to a strainer and rinse it with sterile water. Then transfer all microcapsules into LB medium with Ampicillin, 37 ℃ shaker incubate at 100 rpm.</p>
 +
     <p align="left" class="subsubtitle"><strong class="subtitle"><a name="_Toc23" id="Toc22"></a></strong><strong>3.3 Immobilize cells into calcium alginate beads:</strong></p>
 +
    <table border="1" cellspacing="0" cellpadding="0">
 +
      <tr>
 +
        <td width="262" valign="top"><br />
 +
          Solutions </td>
 +
        <td width="306" valign="top"><p align="left">Preparation</p></td>
 +
      </tr>
 +
      <tr>
 +
        <td width="262" valign="top"><p align="left">3% w/v sodium alginate solution</p></td>
 +
        <td width="306" valign="top"><p align="left">3 g sodium alginate+100 mL    deionized water </p></td>
 +
      </tr>
 +
      <tr>
 +
        <td width="262" valign="top"><p align="left">0.05 mol/L CaCl<sub>2</sub> solution</p></td>
 +
        <td width="306" valign="top"><p align="left">5.55 g CaCl<sub>2</sub>+1000 mL    deionized water(121 ℃ autoclaving for 20 min) </p></td>
 +
      </tr>
 +
    </table>
 +
    <p align="left">- Centrifuge 10 mL  sample at a time for 1 min at 6,000 rpm, drain off supernatant after each  spin. Repeat the procedure until 0.2~1.0 g deposits are collected.<br />
 +
      - Resuspend the  deposits with sterile water and centrifuge for 1min at 6000 rpm,drain off  supernatant.<br />
 +
      - Mix the deposits  with sterile water in a mass ratio of 1:5.<br />
 +
      - Add equivalent  volume of sodium alginate solution, mixing thoroughly.<br />
 +
      - Draw the mixture in  a 1 mL injector and then drip it one by one through the pinhead into 100 mL  stirring CaCl<sub>2</sub> solution. Set it aside and let the beads fully harden  for 2 hours.<br />
 +
      - Drain off the CaCl<sub>2</sub>  solution and rinsed the calcium alginate beads with sterile water for 1~2  times. After that, add 100mL CaCl<sub>2</sub> solution to the beads and set it  overnight.<br />
 +
      - Separate the calcium  alginate beads and CaCl<sub>2</sub> solution by a colander. The immobilization  of cell sample is finished.</p>
 +
    <p align="left" class="subsubtitle"><strong class="subtitle"><a name="_Toc24" id="Toc23"></a></strong><strong>3.4 Immobilize cells into intra-hollow Ca-alginate capsules:</strong></p>
 +
    <table border="1" cellspacing="0" cellpadding="0">
 +
      <tr>
 +
        <td width="262" valign="top"><br />
 +
          Solutions </td>
 +
        <td width="306" valign="top"><p align="left">Preparation</p></td>
 +
      </tr>
 +
      <tr>
 +
        <td width="262" valign="top"><p align="left">1% w/v sodium    alginate solution </p></td>
 +
        <td width="306" valign="top"><p align="left">1.5 g sodium alginate    powder+ 100 mL sterile distilled water(121 ℃ autoclaving for 15 min) </p></td>
 +
      </tr>
 +
      <tr>
 +
        <td width="262" valign="top"><p align="left">Cationic Mixture</p></td>
 +
        <td width="306" valign="top"><p>1.2 g CMC powder(121 ℃ autoclaving for 15 min) <br />
 +
          50 mL 4% w/v CaCl<sub>2</sub> solution(121 ℃ autoclaving for 15 min) <br />
 +
          Under sterile    condition, add 50mL of sterile distilled water into the CMC powder. Dissolve    the CMC and mix it with the 50 mL 4% w/v CaCl<sub>2</sub> solution. </p></td>
 +
      </tr>
 +
      <tr>
 +
        <td width="262" valign="top"><p align="left">3% CaCl2 Solution</p></td>
 +
        <td width="306" valign="top"><p>12 g CaCl<sub>2</sub> powder+ 400 mL distilled water. (121 ℃ autoclaving for 20 min) </p></td>
 +
      </tr>
 +
    </table>
 +
    <p align="left">- Place 3 mL cationic  mixture with 0.6mL cell suspension into a 4 mL centrifuge tube, mixing  thoroughly.<br />
 +
      - Put a magnetic  stirrer at the bottom of a beaker, agitate a 100 mL sodium alginate solution at  a constant rate, which maintains a small eddy in the center of liquid surface.<br />
 +
      - Draw the mixture  (CaCl<sub>2</sub>/CMC/<em>E</em>. coli) with a  1 mL syringe, and then drip it one by one from a height of 10 cm into the  sodium alginate solution. Keep stirring for 5 min.<br />
 +
      - Before sifting out  the capsules,&nbsp;dilute the gelation reaction system by adding 100 mL aseptic  distilled water. Then rinse the capsules with aseptic distilled water on a  colander.<br />
 +
      - Transfer the  capsules to a 3% w/v CaCl<sub>2</sub> solution and stir for 20 min. <br />
 +
      - Finally, rinse the  capsules with distilled water to remove excess CaCl<sub>2</sub>.<br />
 +
      - The immobilization  of cell sample is finished. </p>
 +
    <p align="left"><span class="subsubtitle"><strong class="subtitle"><a name="_Toc25" id="Toc24"></a></strong>3.5 Immobilize cells  into NaCS-PDMDAAC microcapsules</span><br />
 +
      Solutions:<br />
 +
      200 mL sulfuric acid  and ethanol solution(1.51:1) maintained at 0 ℃  ;<br />
 +
      1000 mL industrial  alcohol maintained at 0 ℃; <br />
 +
      Deionized water; <br />
 +
      NaOH solution.</p>
 +
    <p id="fin">All of the above procedures are carried out at room temperature  and all steps are performed under aseptic condition.</p>
 +
    <p align="left">&nbsp;</p>
   </div>
   </div>
-
  <p>&nbsp;</p>
 
-
</div>
 
-
</body>
 
 +
</body>
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Latest revision as of 19:36, 26 September 2012

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Contents[hide] [show]
  • 1 General protocols
  •        1.1 Stock solution
  •        1.2 Preparation of Competent BL21
  •        1.3 Transformation
  •        1.4 Plasmid Purification
  •        1.5 Reaction system of restriction endonuclease
  •        1.6 Standard BioBrick Assembly
  •        1.7 Suffix Insertion
  •        1.8 Ligation
  •        1.9 Restriction analysis
  •        1.10 Gel Extraction
  • 2 Characterization
  •        2.1 Fluorescence Measurements
  •        2.2 Protein electrophoresis
  •               2.2.1 Preparation of Samples
  •               2.2.2 Manufacture Albumen Gel
  •               2.2.3 Electrophoresis
  •               2.2.4 Dyeing (Colloidal Coomassie Brilliant Blue)
  •               2.2.5 Scanning
  • 3 Immobilization
  •        3.1 Prepare Sodiumcellu-losesulfate (NaCS)
  •        3.2 Prepare microcapsules
  •        3.3 Immobilize cells into calcium alginate beads
  •        3.4 Immobilize cells into intra-hollow Ca-alginate capsules
  •        3.5 Immobilize cells into NaCS-PDMDAAC microcapsules
  • protocols

    Protocols

    1 General protocols
    1.1 Stock solution
    50 mg/mL Kanamycin
    - 0.5 g Kan, 10 mL water, filter sterilize with millipore express membrane, freeze in aliquots
    100 mg/mL Ampicillin
    -1 g Amp, 10 mL water, filter sterilize with millipore express membrane, freeze in aliquots.
    50 mmol/L Arabinose
    - 0.1876 g Arabinose, 25 mL water, filter sterilize with millipore express membrane.
    0.25 mg/mL Anhydrotetracycline
    - 0.1 mg Anhydrotetracycline, 0.4 mL PBS, filter sterilize with millipore express membrane.

     

    1.2 Preparation of Competent BL21
    Thaw an aliquot of cells (without any plasmid in them) on ice
    - To 50 mL of sterile LB, add 100μL aliquot of the thawed cells: remember, this LB does not have any antibiotic in it, so work as aseptically as possible (i.e. autoclave all solutions and use sterile pipettes).
    - Grow cells in the shaker at 37 ℃ and 200 rpm, until they reach an OD600= 0.3-0.4. This usually takes 1.5-2 hours.
    - Ice down the LB with growing cells for 10 min.
    - Aliquot into sterile 1.5 mL tubes and spin down at 6000rpm for 10 min at 4 ℃; discard supernatant.
    - Ice down sterile 100 mM CaCl2 and 100mM MgCl2 solutions during centrifugation.
    - Gently resuspend each pellet with 400 μL 0.1 M MgCl2 and 100 μL 0.1 M CaCl2.
    - Centrifuge 6000 rpm for 10 min and discard supernatant.
    - Resuspend each pellet on ice in 100 μL 0.1 M ice cold CaCl2 and combine into one tube

    1.3 Transformation
    - Add 10 μL of DNA. Swirl gently with pipette.
    - Incubate tubes on ice for 20 min
    - Heat pulse tubes in 42 ℃ water bath for 30 seconds.
    - Incubate on ice for 2 min
    - Add 790 μL of LB broth to each tube and incubate for an hour at 37 ℃ with shaking.
    - Spread 100 μL and 50μL of each culture on an LB agar plate containing the appropriate antibiotics and incubate overnight at 37 ℃ (spread using beads).

    1.4 Plasmid Purification
    - Centrifuge sample in eppendorf tube approximately 1.5 mL at a time, draining off supernatant after each spin and adding more cell solution
    -Resuspend the pelleted cells in 250 μL of the resuspension Solution (mixture with Solution I and RNasa A). The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain.
    - Add 250 μL of the Lysis Solution (Solution II) and mix thoroughly and gently by inverting the tube 5-6 times, letting it stand for 1-2 min at room temperature until the solution becomes viscous and slightly clear.
    - Add 350 μL of the Neutralization Solution (Solution III) and mix immediately and thoroughly by inverting the tube 5-6 times.
    - Centrifuge for 10 min at 12,000 rpm to pellet cell debris.
    - Apply the supernatant to the supplied spin column by decanting. Avoid disturbing or applying the white precipitate.
    - Centrifuge for 1 min at 12,000 rpm. Discard flow-through and place the column back into the same collection tube.
    - Add 500 μL of the Wash Buffer PB to the spin column. Centrifuge for 1min at 12,000 rpm and discard flow-through. Place the column back into the same collection tube.
    - Add 500 μL of the Wash Buffer W to the spin column. Centrifuge for 1min at 12,000 rpm and discard the flow-through. Place the column back into the same collection tube.
    - Repeat the step 9 again.
    - Discard flow-through and centrifuge for an additional 3 min to remove residual Wash Solution.
    - Place the spin column in a clean 1.5 mL centrifuge tube, and pipet 20 μL Elution Buffer TE (prewarm to 60 ℃) directly to the center of the column without touching the membrane. Let it stand for 2 min at room temperature and centrifuge for 1 min at 12,000 rpm.
    - Discard the column and store the purified plasmid DNA at -20 °C.

    1.5 Reaction system of restriction endonuclease

    - System1、2、3 and 4 are used for Standard BioBrick Assembly .
    - System 5 and 6 are used for restriction analysis. Digestion of sample: at least 500 ng DNA / 10 μL volume. Digest for 1 h at 37 °C, afterwards inactivated by adding 10× loading buffer and standing for 10 min at room temperature.

    1.6 Standard BioBrick Assembly
    - Digestion of insert: 2 μg~5 μg DNA / 100 μL volume, 10× H buffer, EcoR I, Spe I. Digestion and inactivation. Clean up the insert via gel electrophoresis. When cutting the insert out of the gel, try avoiding staining or exposure to ultraviolet light of the insert.
    - Digestion of vector: 2 μg~5 μg DNA / 100 μL volume, 10× M buffer, EcoR I, Xba I. Digestion and inactivation. Clean up the insert via gel electrophoresis. When cutting the insert out of the gel, try to avoid staining or exposure to ultraviolet light of the insert.

    1.7 Suffix Insertion
    - Digestion of insert: 2 μg~5 μg DNA / 100 μL volume, 10× M buffer, Xba I, Pst I. Digestion and inactivation. Clean up the insert.
    - Digestion of vector : 2 μg~5 μg DNA / 100 μL volume, 10× H buffer, Spe I, Pst I. Digestion and inactivation. Clean up the vector.

    1.8 Ligation
    - After digestion and clean-up, the next step is ligation. ligation at 16 ℃ for 4 h or at 4 ℃ for 16 h. Table 2 is the system of ligation.
    Table 2  Ligation system


    Components

    Volume/μL

    Digested vector

    7

    Digested insert

    1

    10× T4 ligation buffer

    1

    T4 ligase

    1

    Total

    10

     

    1.9 Restriction analysis
    - Pick one colony with a sterile tip and cultivation in 20 mL LB for overnight at 37 ℃
    - Isolation of Plasmid
    - Digest BioBrick,the system of Restriction analysis refer to table1
    - Gel electrophoresis:add 2.2 μL loading buffer to digestion mixture. An agarose concentration is 1 %.

    1.10 Gel Extraction
    - Weigh a 1.5 mL centrifuge tube for each DNA fragment to be isolated and record the weight.
    - Excise gel slice containing the DNA fragment using a clean scalpel or razor blade. Cut as close as possible to the DNA to minimize the gel volume. Place the gel slice into a pre-weighed 1.5 mL tube and weigh. Record the weight of the gel slice.
    - Add Bing Buffer BD at a ratio of 100 μL of solution per 100 mg of agarose gel slices.
    - Incubate the gel mixture at 55-65 ℃ for 7-10 min or until the gel slice is completely dissolved. Mix the tube by inversion every few minutes to facilitate the melting process. Ensure that the gel is completely dissolved.
    - After the dissolved gel mixture cool down, transfer it to the Spin Columns assembly and incubate for 2 min at room temperature.
    - Centrifuge the Spin Columns assembly in a microcentrifuge at 12,000 rpm for 1 min, and discard the flow-through.
    - Wash the columns by adding 500 μL of Wash Buffer PE to the Columns. Centrifuge the columns assembly for 1 min at 12,000 rpm, and discard the flow-through.
    - Repeat step 7 again.
    - Centrifuge the Columns for an additional 3 min to completely remove residual wash buffer.
    - Empty the Collection Tube and recentrifuge the column assembly for 1 min with the microcentrifuge lid open (or off) to allow evaporation of any residual ethanol.
    - Place the spin column in a clean 1.5 mL microcentrifuge tube, and pipet 20 μL deionized water (pH is 8.0-8.5 and prewarm to 60 ℃)directly to the center of the column without touching the membrane. Incubate at room temperature for 2 min.
    - Centrifuge for 1 min at 12,000 rpm. Discard the columns and store the microcentrifuge tube containing the eluted DNA at–20 ℃ .


    2 Characterization
    2.1 Fluorescence Measurements
    - The samples to be tested are cultured from plates in 20 mL of the Basal Minimal Medium with appropriate antibiotics and incubated overnight at 37 ℃ at 200 rpm.
    - The culture is checked for OD600 next day and then subculture by the same medium with antibiotics at 37 ℃ shaking for 2 hours.
    - Add corresponding inducer at concentration gradients into the above-mentioned culture and keep on incubating. During the time incubating, every 15 min, take 1 mL bacteria liquid, then centrifuge the cells( 6000 rpm, 10 min ) and resuspend them in 1 mL PBS. At last, pipette to a 96 well plate.
    - The plate reader made by Molecular Device then read.
    - The program does the following:
    - In endpoint reads, following measurements are taken in a time interval of 15 min: absorbance (600 nm filter) and fluorescence (485 nm and 520 nm for GFP).
    - The results then transfer to excel sheet and interpret.

     

    2.2 Protein electrophoresis
    2.2.1 Preparation of Samples
    - The samples to be tested are cultured in the Basal Medium with appropriate antibiotics, and take 200 μL bacteria liquid to determine its OD600 at appropriate time.
    - Dilute or concentrate the next 200 μL bacteria liquid in order to let the OD600 equals to 4.0 while the computational formula is the actual OD600 * 200=2.0 * X, and X presents the total volume of the bacteria liquid after being diluted or concentrated while its unit is μL as well.
    - Add 30 μL diluted or concentrated liquid into corresponding 1.5 mL centrifugal tubes, then mix up them with 10 μL loading buffer.
    - Put these centrifugal tubes into metal bath and heat them in 100 ℃ in around 5 to 8 min, then centrifuge them at the speed of 13000 rpm for 5 min, the supernatant is what we need.

    2.2.2 Manufacture Albumen Gel
    - Prepare a clear centrifuge tube in the capacity of 50 mL, and make running gel, high concentration one, following the formula below. Then mix up them and pour the mixture into a glass pane.

    Running Gel

    Final Gel Concentration (5 mL; 1 ea ;1.0 mm thick; 10%)

    ddH2O

    1.18 mL

    4 × Running Gel Buffer (pH 8.8,1.5 M Tris-HCl)

    1.25 mL

    Monomer Solution

    2.48 mL

    10% SDS

    50 mL

    10% Ammonium Persulfate

    50 mL

    TEMED

    5 mL

    - Add some absolute alcohol to planish the top of gel. There will be approximately 60 min for its solidification.
    - After solidification, pour out the alcohol and make stacking gel following the formula below. Then mix them up, add the solution onto the running gel in the glass panes until it being filled up with the gel. Insert a clean comb into stacking gel. Wait for about 40 min for stacking gel solidification.

    Stacking Gel

    Stacking Gel Concentration (1 ea ;1.0 mm thick; 10%)

    ddH2O

    1.35 mL

    4 × Running Gel Buffer (pH 6.8,1.5 M Tris-HCl)

    0.58 mL

    Monomer Solution

    0.3 mL

    10% SDS

    25 mL

    10% Ammonium Persulfate

    25 mL

    TEMED

    5 mL

    2.2.3 Electrophoresis
    - Take out the glass pane with finished gel and then fasten it in an electrophoresis tank. Add some 1× Tank Buffer to detect whether liquid leak or not.     
    - Take out the comb slowly and use pipette to add approximately 10 to 20 μL processed samples into the wells in stacking gel.
    - Add 1× Tank Buffer until the liquid level is above the platinum line in the electrophoresis tank.
    - Cover up the electrophoresis tank and connect it with the electrophoresis device. Set the program 120 V- 60 min and start it up.
    - When the green marker band run to the bottom of running gel, stop the device.

    2.2.4 Dyeing (Colloidal Coomassie Brilliant Blue)
    - Take out the gel and put it into a clean petri dish. And add appropriate Commassie Blue Staining Solution. Please make sure that the solution can cover all the gel.
    - Put the petri dish onto the orbital shaker and dye for approximate 30 min.
    - Pour out the staining solution then add enough destaining solution. Destain about 30 min.
    - Renew the destaining solution for about 2 or 3 times until the blue background of gel being taken off.
    - Pour out the destaining solution and add appropriate water to clear it.

    2.2.5 Scanning
    Scan the processed gel and save the picture for analysis.


    3 Immobilization
    3.1 Prepare Sodiumcellu-losesulfate (NaCS)
    - Deep freeze H2SO4 and absolute ethanol at -20 ℃ for at least 2 hours;
    - Prepare a sulphuric acid and ethanol solution at the proportion of 1.51:1(120 mL H2SO4 and 80 mL alcohol), maintaining it at -18 ℃ for at least 2 hours;
    - Put the sulphuric acid and ethanol solution and 500mL industrial alcohol in an ice box, maintaining them at 0 ℃ for at least 1 hour;
    - Immerse 4 g dry absorbent cotton in the solution in ice-bath for 66 min. Then squeezed out the solution and rinsed the reacted linters with 0℃ industrial alcohol in draught cupboard;
    - Squeeze out the alcohol, then put the linters in 400 mL deionized water and regulated pH to about 3. Stir and dissolve it for 10 min, then filtrate it;
    - Collected the filtrate and regulated pH to 9.3 accurately;
    - Add industrial alcohol gradually to the solution until there appears the largest volume of white retiary floccule on the top of the solution;
    - Centrifuge the floccule for 5 min at 5,000 rpm and collect it;
    - 65 ℃ drying for at least 24 hours until it is completely dry, then collect the final production.

    3.2 Prepare microcapsules

    - Centrifuge 10 mL bacteria sample for 3 min at 6000 rpm and collect the deposit;
    - Add 10 mL NaCS solution and mix it completely with the cells;
    - Put a 6% PDMDAAC solution on a magnetic stirrer and stir it at a certain speed, maintaining a small eddy in the center of liquid surface;
    - Drop the mixture into the fringe of the eddy by a 1 mL injector until it form a spheroidic membrane. It takes 10 min to react completely and form microcapsules;
    - Tip all microcapsules to a strainer and rinse it with sterile water. Then transfer all microcapsules into LB medium with Ampicillin, 37 ℃ shaker incubate at 100 rpm.

    3.3 Immobilize cells into calcium alginate beads:


    Solutions

    Preparation

    3% w/v sodium alginate solution

    3 g sodium alginate+100 mL deionized water

    0.05 mol/L CaCl2 solution

    5.55 g CaCl2+1000 mL deionized water(121 ℃ autoclaving for 20 min)

    - Centrifuge 10 mL sample at a time for 1 min at 6,000 rpm, drain off supernatant after each spin. Repeat the procedure until 0.2~1.0 g deposits are collected.
    - Resuspend the deposits with sterile water and centrifuge for 1min at 6000 rpm,drain off supernatant.
    - Mix the deposits with sterile water in a mass ratio of 1:5.
    - Add equivalent volume of sodium alginate solution, mixing thoroughly.
    - Draw the mixture in a 1 mL injector and then drip it one by one through the pinhead into 100 mL stirring CaCl2 solution. Set it aside and let the beads fully harden for 2 hours.
    - Drain off the CaCl2 solution and rinsed the calcium alginate beads with sterile water for 1~2 times. After that, add 100mL CaCl2 solution to the beads and set it overnight.
    - Separate the calcium alginate beads and CaCl2 solution by a colander. The immobilization of cell sample is finished.

    3.4 Immobilize cells into intra-hollow Ca-alginate capsules:


    Solutions

    Preparation

    1% w/v sodium alginate solution

    1.5 g sodium alginate powder+ 100 mL sterile distilled water(121 ℃ autoclaving for 15 min)

    Cationic Mixture

    1.2 g CMC powder(121 ℃ autoclaving for 15 min)
    50 mL 4% w/v CaCl2 solution(121 ℃ autoclaving for 15 min)
    Under sterile condition, add 50mL of sterile distilled water into the CMC powder. Dissolve the CMC and mix it with the 50 mL 4% w/v CaCl2 solution.

    3% CaCl2 Solution

    12 g CaCl2 powder+ 400 mL distilled water. (121 ℃ autoclaving for 20 min)

    - Place 3 mL cationic mixture with 0.6mL cell suspension into a 4 mL centrifuge tube, mixing thoroughly.
    - Put a magnetic stirrer at the bottom of a beaker, agitate a 100 mL sodium alginate solution at a constant rate, which maintains a small eddy in the center of liquid surface.
    - Draw the mixture (CaCl2/CMC/E. coli) with a 1 mL syringe, and then drip it one by one from a height of 10 cm into the sodium alginate solution. Keep stirring for 5 min.
    - Before sifting out the capsules, dilute the gelation reaction system by adding 100 mL aseptic distilled water. Then rinse the capsules with aseptic distilled water on a colander.
    - Transfer the capsules to a 3% w/v CaCl2 solution and stir for 20 min.
    - Finally, rinse the capsules with distilled water to remove excess CaCl2.
    - The immobilization of cell sample is finished.

    3.5 Immobilize cells into NaCS-PDMDAAC microcapsules
    Solutions:
    200 mL sulfuric acid and ethanol solution(1.51:1) maintained at 0 ℃ ;
    1000 mL industrial alcohol maintained at 0 ℃;
    Deionized water;
    NaOH solution.

    All of the above procedures are carried out at room temperature and all steps are performed under aseptic condition.