Difference between revisions of "Team:Freiburg/Protocols/prepLysate"

Line 12: Line 12:
 
<div class="level1">
 
<div class="level1">
 
<p>
 
<p>
−
<em>Preparation of a Lysate for Cell-free exxpression </em>
+
<em>Preparation of a Lysate for Cell-free expression </em>
 
</p>
 
</p>
 
<p>
 
<p>
−
<a class="media" href="https://static.igem.org/mediawiki/2015/4/4b/Freiburg_wiki-symbols-orga.png" title="wiki:symbols:orga.png"><img alt="" class="media" src="https://static.igem.org/mediawiki/2015/4/4b/Freiburg_wiki-symbols-orga.png"/></a> material: <br/>
+
<a class="media" href="https://static.igem.org/mediawiki/2015/4/4b/Freiburg_wiki-symbols-orga.png" title="wiki:symbols:orga.png"><img alt="" class="media" src="https://static.igem.org/mediawiki/2015/4/4b/Freiburg_wiki-symbols-orga.png"/></a> material:see below <br/>
 
<a class="media" href="https://static.igem.org/mediawiki/2015/b/bb/Freiburg_wiki-symbols-alarm2.png" title="wiki:symbols:alarm2.png"><img alt="" class="media" src="https://static.igem.org/mediawiki/2015/b/bb/Freiburg_wiki-symbols-alarm2.png"/></a> duration: Preparation 1 week <br/>
 
<a class="media" href="https://static.igem.org/mediawiki/2015/b/bb/Freiburg_wiki-symbols-alarm2.png" title="wiki:symbols:alarm2.png"><img alt="" class="media" src="https://static.igem.org/mediawiki/2015/b/bb/Freiburg_wiki-symbols-alarm2.png"/></a> duration: Preparation 1 week <br/>
 
</p>
 
</p>
Line 28: Line 28:
 
</tr>
 
</tr>
 
<tr class="row1">
 
<tr class="row1">
−
<td class="col0"> TRIS-acetate (pH 8 </td><td class="col1"> 10 mM </td>
+
<td class="col0"> TRIS-acetate pH 8 </td><td class="col1"> 10 mM </td>
 
</tr>
 
</tr>
 
<tr class="row2">
 
<tr class="row2">
Line 60: Line 60:
 
</tr>
 
</tr>
 
<tr class="row1">
 
<tr class="row1">
−
<td class="col0"> Tris Ac, pH 7.6</td><td class="col1"> - </td><td class="col2"> 300mM </td>
+
<td class="col0"> Tris Ac, pH 7.6</td><td class="col1"> 1M </td><td class="col2"> 300mM </td>
 
</tr>
 
</tr>
 
<tr class="row2">
 
<tr class="row2">
−
<td class="col0"> Mg(OAc)2 </td><td class="col1"> - </td><td class="col2"> 10 mM </td>
+
<td class="col0"> Mg(OAc)<sub>2</sub> </td><td class="col1"> 1M </td><td class="col2"> 10 mM </td>
 
</tr>
 
</tr>
 
<tr class="row3">
 
<tr class="row3">
−
<td class="col0"> ATP </td><td class="col1"> at least 217 ng/µl </td><td class="col2"> 10mM </td>
+
<td class="col0"> ATP </td><td class="col1"> 100mM </td><td class="col2"> 10mM </td>
 
</tr>
 
</tr>
 
<tr class="row4">
 
<tr class="row4">
−
<td class="col0"> phosphoenol pyruvate (PEP) </td><td class="col1"> - </td><td class="col2"> 80mM </td>
+
<td class="col0"> phosphoenol pyruvate (PEP) </td><td class="col1"> 1M </td><td class="col2"> 80mM </td>
 
</tr>
 
</tr>
 
<tr class="row2">
 
<tr class="row2">
−
<td class="col0"> Amino Acid mix </td><td class="col1"> - </td><td class="col2"> 5mM </td>
+
<td class="col0"> Amino Acid mix </td><td class="col1"> 5mM </td><td class="col2"> 40µM </td>
 
</tr>
 
</tr>
 
<tr class="row3">
 
<tr class="row3">
−
<td class="col0"> pyruvate kinase</td><td class="col1"> at least 217 ng/µl </td><td class="col2"> 3250 - 5000 ng </td>
+
<td class="col0"> pyruvate kinase</td><td class="col1"> 2U/µl </td><td class="col2"> 8U/µl</td>
 
</tr>
 
</tr>
 
<tr class="row4">
 
<tr class="row4">
−
<td class="col0"> DEPC-H2O </td><td class="col1"> - </td><td class="col2"> up to 50 µl </td>
+
<td class="col0"> DEPC-H2O </td><td class="col1"> - </td><td class="col2"> ad 750µl </td>
 
</tr>
 
</tr>
 
</table></div>
 
</table></div>
−
<!-- EDIT3 TABLE [946-1151] -->
+
 
−
</div>
+
−
<h4>Implementation</h4>
+
−
<div class="level4">
+
 
<ul>
 
<ul>
−
<li class="level1"><div class="li"> incubate 2-4 hrs at 37 °C in autoclaved tubes or 96 well plate (low bind)</div>
+
<li class="level1"><div class="li"> 750µl of preincubation buffer are necessary for 1 l of culture</div>
 
</li>
 
</li>
 
</ul>
 
</ul>
−
</div>
+
<h3>Initial remarks to the used Bacteria</h3>
−
<h4>Growth of Bacteria</h4>
+
 
<div class="level4">
 
<div class="level4">
 +
<ul>
 +
<li class="level1"><div class="li"> To achieve high levels of protein production, the BL21(DE3) strain  was chosen, which carries the gene coding for a T7 RNA polymerase as a lambda prophage within its chromosome, which is inducible by addition of IPTG</div>
 +
</li>
 +
<li class="level1"><div class="li"> As the system is later to be used for expression of synthetic gene constructs of varying natural hosts, the bacteria need to be able to express so-called “rare co-dons”: AGA, AGG, AUA, CUA, GGA, CCC, and CGG are used less frequently in E. coli as they are e.g in human cells</div>
 +
</li>
 +
<li class="level1"><div class="li"> cells were transformed with the pRARE2 plasmid, taken from Rosetta2  competent cells. The pRARE2 plasmid encodes for tRNAs</div>
 +
</li>
 +
</ul>
  
−
<h4>Harvesting, washing and lysis</h4>
 
−
<div class="level4">
 
  
 +
 +
<h4>Growth of Bacteria</h4>
 +
<div class="level4">
 
<ul>
 
<ul>
−
<li class="level1"><div class="li"> cells were grown in a 2 litre chicanery flask containing 1 litre of Terrific Broth medium until reaching OD600 = 0,8</div>
+
<li class="level1"><div class="li">Prepare an overnight preculture in 10 ml medium in a 100 ml Erlenmeyer-flask @ 37°C, 250 rpm</div>
 +
</li>
 +
<li class="level1"><div class="li"> inoculated in 1000 ml Terrific Broth in a 2000 ml chicanery-flask at 37°C, 150-180 rpm</div>
 +
</li>
 +
<li class="level1"><div class="li"> cells were grown until reaching OD600 = 0,8</div>
 
</li>
 
</li>
 
<li class="level1"><div class="li"> The expression of T7 RNA polymerase then was induced by adding 0.5 ml of 1 M IPTG to the medium</div>
 
<li class="level1"><div class="li"> The expression of T7 RNA polymerase then was induced by adding 0.5 ml of 1 M IPTG to the medium</div>
 
</li>
 
</li>
−
<li class="level1"><div class="li"> further growth until OD600 = 6.5, then cells were put on ice</div>
+
<li class="level1"><div class="li"> further growth until OD600 = 2.5, then cells were put on ice</div>
 
</li>
 
</li>
 
<li class="level1"><div class="li">  cells were pelleted at 6000 g at 4°C for 30 minutes in a fixed-angle rotor</div>
 
<li class="level1"><div class="li">  cells were pelleted at 6000 g at 4°C for 30 minutes in a fixed-angle rotor</div>
Line 107: Line 116:
 
<li class="level1"><div class="li"> supernatant was decanted and the cells were washed two times in buffer A</div>
 
<li class="level1"><div class="li"> supernatant was decanted and the cells were washed two times in buffer A</div>
 
</li>
 
</li>
−
<li class="level1"><div class="li"> Again, the cells were pelleted at 5000 g for 20 min at 4°C and after-wards resuspended in 1.3 ml of buffer B</div>
+
<li class="level1"><div class="li"> Again, the cells were pelleted at 5000 g for 20 min at 4°C and afterwards resuspended in 1.3 ml of buffer B</div>
 
</li>
 
</li>
−
<li class="level1"><div class="li"> feed: Using a french press with a 1“ diameter piston, the cells were lysed at 1000 psi (≈ 6.9 MPa)</div>
+
<li class="level1"><div class="li">Using a french press with a 1“ diameter piston, the cells were lysed at 1000 psi (≈ 6.9 MPa)</div>
 
</li>
 
</li>
 
<li class="level1"><div class="li">About 7.5 ml of lysate was obtained after pressing.</div>
 
<li class="level1"><div class="li">About 7.5 ml of lysate was obtained after pressing.</div>
Line 115: Line 124:
 
</ul>
 
</ul>
  
−
<h4>Harvesting, washing and lysis</h4>
+
<h4>Washing and lysis</h4>
 
<div class="level4">
 
<div class="level4">
  
 
<ul>
 
<ul>
 
<li class="level1"><div class="li"> obtained lysate is pelleted via ultracentrifugation at 30,000 g for 30 min at 4°C</div>
 
<li class="level1"><div class="li"> obtained lysate is pelleted via ultracentrifugation at 30,000 g for 30 min at 4°C</div>
−
</li>
 
−
<li class="level1"><div class="li"> The expression of T7 RNA polymerase then was induced by adding 0.5 ml of 1 M IPTG to the medium</div>
 
 
</li>
 
</li>
 
<li class="level1"><div class="li"> The resulting extract is mixed with the preincubation buffer in a ratio of 10:1 (lysate:buffer)</div>
 
<li class="level1"><div class="li"> The resulting extract is mixed with the preincubation buffer in a ratio of 10:1 (lysate:buffer)</div>

Revision as of 21:22, 14 September 2015

""

Protocol Cell-free expression

Preparation of a Lysate for Cell-free expression

material:see below
duration: Preparation 1 week

Buffer A:

Component final concentration
TRIS-acetate pH 8 10 mM
Potassium acetate 60 mM
Magnesium acetate 14 mM
DTT 1 mM
ß-mercaptoethanol 7 mM
  • Buffer B is the same as A, but without the ß-mercaptoethanol
  • Buffer C is the same as B, but without DTT

Preincubation Buffer

Component stock concentration endconcentration [µl]
Tris Ac, pH 7.6 1M 300mM
Mg(OAc)2 1M 10 mM
ATP 100mM 10mM
phosphoenol pyruvate (PEP) 1M 80mM
Amino Acid mix 5mM 40µM
pyruvate kinase 2U/µl 8U/µl
DEPC-H2O - ad 750µl
  • 750µl of preincubation buffer are necessary for 1 l of culture

Initial remarks to the used Bacteria

  • To achieve high levels of protein production, the BL21(DE3) strain was chosen, which carries the gene coding for a T7 RNA polymerase as a lambda prophage within its chromosome, which is inducible by addition of IPTG
  • As the system is later to be used for expression of synthetic gene constructs of varying natural hosts, the bacteria need to be able to express so-called “rare co-dons”: AGA, AGG, AUA, CUA, GGA, CCC, and CGG are used less frequently in E. coli as they are e.g in human cells
  • cells were transformed with the pRARE2 plasmid, taken from Rosetta2 competent cells. The pRARE2 plasmid encodes for tRNAs

Growth of Bacteria

  • Prepare an overnight preculture in 10 ml medium in a 100 ml Erlenmeyer-flask @ 37°C, 250 rpm
  • inoculated in 1000 ml Terrific Broth in a 2000 ml chicanery-flask at 37°C, 150-180 rpm
  • cells were grown until reaching OD600 = 0,8
  • The expression of T7 RNA polymerase then was induced by adding 0.5 ml of 1 M IPTG to the medium
  • further growth until OD600 = 2.5, then cells were put on ice
  • cells were pelleted at 6000 g at 4°C for 30 minutes in a fixed-angle rotor
  • supernatant was decanted and the cells were washed two times in buffer A
  • Again, the cells were pelleted at 5000 g for 20 min at 4°C and afterwards resuspended in 1.3 ml of buffer B
  • Using a french press with a 1“ diameter piston, the cells were lysed at 1000 psi (≈ 6.9 MPa)
  • About 7.5 ml of lysate was obtained after pressing.

Washing and lysis

  • obtained lysate is pelleted via ultracentrifugation at 30,000 g for 30 min at 4°C
  • The resulting extract is mixed with the preincubation buffer in a ratio of 10:1 (lysate:buffer)
  • preincubated lysate is then transferred to a 3.5 kDa dialysis tubing and desalted by dialysing it against buffer C at 4°C for 90 minutes
  • after renewing the buffer, once more over night at slow stirring
  • the lysate is ready for use and, after shock-freezing in liquid N2, can be stored in small ali-quots at - 80°C.

Remarks

  • The Preincubation step takes care of what is called the „run off“ step, a step imple-mented in order to eliminate endogenous cellular mRNA. The removal of endog-enous mRNA bound to ribosomes/polysomes is accomplished by finishing their synthesis via the added PEP, pyruvate kinase and ATP at 37°C. The residual endogenous mRNA will then subsequently become degraded due to the high RNase content of the extract. This step is precautionally performed in the dark, because some components are slightly light sensitive.
  • The lysate can be frozen again after use, but looses effectivity

Reaction is adapted from the European Molecular Biology Laboratory (EMBL), Heidelberg, Germany