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

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<p>
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<em>Preparation of a +Lysate for Cell-free exxpression </em>
+
<em>Preparation of a Lysate for Cell-free exxpression </em>
 
</p>
 
</p>
 
<p>
 
<p>
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<hr/>
 
<hr/>
 
</div>
 
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<h4>Mastermix</h4>
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<h4>Step 1:</h4>
 
<div class="level4">
 
<div class="level4">
 
<div class="table sectionedit2"><table class="inline">
 
<div class="table sectionedit2"><table class="inline">
 +
In this study, a prokaryotic system based upon the well-known bacte-rium Escherichia coli was established. To achieve high levels of protein produc-tion, the BL21(DE3) strain  was chosen, which carries the gene coding for a T7 RNA polymerase as a lambda prophage within its chromosome. It will be expressed in the cells once it becomes induced via addition of isopropyl thio-galactoside (IPTG) to the cell’s environment; in this case its growth medium. IPTG is an allolactose analogon that activates the lac operator due to a release of the lac repressor. The T7 RNA polymerase is commonly used in protein overex-pression because of reduced need to rely upon the bacteria’s own transcriptional apparatus. This causes less interference with the cell’s own metabolism.
 +
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 frequent-ly in E. coli as they are e.g in human cells. [ ] Therefore, the cells were trans-formed with the pRARE2 plasmid, taken from Rosetta2  competent cells. The pRARE2 plasmid encodes for tRNAs that are able to translate the 7 rare codons mentioned above.
 +
 
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<th class="col0">Component </th><th class="col1"> stock concentration </th><th class="col2">amount in 50 µl reaction [µl]</th>
 
<th class="col0">Component </th><th class="col1"> stock concentration </th><th class="col2">amount in 50 µl reaction [µl]</th>
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</ul>
 
</ul>
 
</div>
 
</div>
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<h4>Remarks</h4>
+
<h4>Growth of Bacteria</h4>
 
<div class="level4">
 
<div class="level4">
 +
 +
<h4>Harvesting, washing and lysis</h4>
 +
<div class="level4">
 +
 
<ul>
 
<ul>
−
<li class="level1"><div class="li"> add creatine phosphokinase last to mastermix</div>
+
<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>
 
</li>
−
<li class="level1"><div class="li"> work quickly, sterile and as nuclease free as possible (on ice)</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"> add DNA last to start reactions simultaneously</div>
+
<li class="level1"><div class="li"> further growth until OD600 = 6.5, then cells were put on ice</div>
 
</li>
 
</li>
−
<li class="level1"><div class="li"> amount of lysate used can be varied (here 45%)</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>
 
</li>
 
</li>
−
<li class="level1"><div class="li"> the used chemicals should have a high grade of purity</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"> Certain counterions like Chlor and Sodium should be avoided</div>
+
<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>
 +
<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>
 +
<li class="level1"><div class="li">About 7.5 ml of lysate was obtained after pressing.</div>
 +
</li>
 +
</ul>
 +
 
 +
<h4>Harvesting, washing and lysis</h4>
 +
<div class="level4">
 +
 
 +
<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>
 +
<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 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">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</div>
 +
</li>
 +
<li class="level1"><div class="li">after renewing the buffer, once more over night at slow stirring</div>
 +
</li>
 +
<li class="level1"><div class="li">the lysate is ready for use and, after shock-freezing in liquid N2, can be stored in small ali-quots at - 80°C.</div>
 +
</li>
 +
</ul>
 +
 
 +
 
 +
<h4>Remarks</h4>
 +
<div class="level4">
 +
<ul>
 +
<li class="level1"><div class="li"> 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.</div>
 
</li>
 
</li>
−
<li class="level1"><div class="li"> feed: adding 10 mM Mg(OAc) every 20 minutes can increase reaction output profoundly</div>
+
<li class="level1"><div class="li"> The lysate can be frozen again after use, but looses effectivity</div>
 
</li>
 
</li>
 
</ul>
 
</ul>
 +
 
<p>
 
<p>
 
<strong>Reaction is adapted from the European Molecular Biology Laboratory (EMBL), Heidelberg, Germany </strong>
 
<strong>Reaction is adapted from the European Molecular Biology Laboratory (EMBL), Heidelberg, Germany </strong>

Revision as of 14:39, 14 September 2015

""

Protocol Cell-free expression

Preparation of a Lysate for Cell-free exxpression

material: Mastermix, lysate, nuclease free water and DNA template
duration: Preparation 30-45 min + running time

Step 1:

In this study, a prokaryotic system based upon the well-known bacte-rium Escherichia coli was established. To achieve high levels of protein produc-tion, the BL21(DE3) strain was chosen, which carries the gene coding for a T7 RNA polymerase as a lambda prophage within its chromosome. It will be expressed in the cells once it becomes induced via addition of isopropyl thio-galactoside (IPTG) to the cell’s environment; in this case its growth medium. IPTG is an allolactose analogon that activates the lac operator due to a release of the lac repressor. The T7 RNA polymerase is commonly used in protein overex-pression because of reduced need to rely upon the bacteria’s own transcriptional apparatus. This causes less interference with the cell’s own metabolism. 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 frequent-ly in E. coli as they are e.g in human cells. [ ] Therefore, the cells were trans-formed with the pRARE2 plasmid, taken from Rosetta2 competent cells. The pRARE2 plasmid encodes for tRNAs that are able to translate the 7 rare codons mentioned above.
Component stock concentration amount in 50 µl reaction [µl]
Creatine phosphate 80 mM 4
Potassium glutamate 200 mM 3.7
HEPES-KOH pH 7.5 55 mM 2.75
Folinic acid 35 µg/ml 1.75
Amino acids 2mM 1.25
E. coli tRNA 175 µg/ml 1.14
PEG4000 2% 1
DTT 1,7 mM 0.85
ATP 1,2 mM 0.6
MgOAc 11 mM 0.55
CTP 0.8 mM 0.4
GTP 0.8 mM 0.4
UTP 0.8 mM 0.4
cAMP 0.65 mM 0.325
Creatine phosphokinase 80 mM 0.175

Final reaction

Component stock concentration amount in 50 µl reaction [µl]
Master mix - 17.68
Lysate - 22.5
DNA at least 217 ng/µl 3250 - 5000 ng
H2O (nuclease free) - up to 50 µl

Implementation

  • incubate 2-4 hrs at 37 °C in autoclaved tubes or 96 well plate (low bind)

Growth of Bacteria

Harvesting, washing and lysis

  • cells were grown in a 2 litre chicanery flask containing 1 litre of Terrific Broth medium 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 = 6.5, 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 after-wards resuspended in 1.3 ml of buffer B
  • feed: 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.

Harvesting, washing and lysis

  • obtained lysate is pelleted via ultracentrifugation at 30,000 g for 30 min at 4°C
  • The expression of T7 RNA polymerase then was induced by adding 0.5 ml of 1 M IPTG to the medium
  • 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