Difference between revisions of "Part:BBa K2003011:Experience"

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===Results===
 
===Results===
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<html>
 
<html>
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</br><SPAN style='font-size: 110%; font-weight: bold;'>Small-scale Expression of UnaG</SPAN>
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<p>
  
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<p class=MsoNormal style='text-align:justify;text-justify:inter-ideograph'><span
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Since summers are always too short in Sweden, there was not enough time for the Uppsala 2016 iGEM team to test this UnaG BioBrick as thoroughly as the <span
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style='font-size:10.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman"'>In
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style='font-size:10.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";
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addition to mutagenesis experiments, small-scale expression tests were
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mso-bidi-theme-font:minor-bidi'><a
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performed to verify that <span class=SpellE>UnaG</span> expresses under our
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laboratory conditions (originally the protein was purified and crystalized using
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Escherichia coli, so it should be producible in prokaryotes). <span
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class=SpellE>BioBricks</span> </span><u><span style='font-size:10.0pt;
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font-family:Arial;mso-bidi-font-family:"Times New Roman";mso-bidi-theme-font:
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minor-bidi'><a href="https://parts.igem.org/Part:BBa_K880005"><span
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style='mso-bidi-font-family:"Times New Roman";color:windowtext'>BBa_K880005</span></a></span></u><span
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style='font-size:10.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman"'>
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and </span><u><span style='font-size:10.0pt;font-family:Arial;mso-bidi-font-family:
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"Times New Roman";mso-bidi-theme-font:minor-bidi'><a
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href="https://parts.igem.org/Part:BBa_K2003010"><span style='mso-bidi-font-family:
 
href="https://parts.igem.org/Part:BBa_K2003010"><span style='mso-bidi-font-family:
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"Times New Roman";color:windowtext'>BBa_K2003010</span></a></span></u><span
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"Times New Roman";color:windowtext'><u>BBa_K2003010</u></span></a></span> was tested. However, BBa_K2003011 was made by PCR mutagenesis with the <span
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style='font-size:10.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman"'>
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style='font-size:10.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";
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were 3A assembled and grown overnight in LB. Bilirubin is not very soluble in
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mso-bidi-theme-font:minor-bidi'><a
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water, so the stock 1mM solution was prepared in DMSO instead. It seems the
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href="https://parts.igem.org/Part:BBa_K2003010"><span style='mso-bidi-font-family:
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molecule does not permeate the cell easily, so appropriate amounts were added
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"Times New Roman";color:windowtext'><u>BBa_K2003010</u></span></a></span> BioBrick as template. BBa_K2003011 is therefore expected  to function similarly to BB_K2003010 during expression. </p>
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to crude cell extracts instead, to a final concentration of 100 µM. Those were
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obtained by replacing the growth medium with room temperature PBS pH 7.4
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containing 1mg/ml lysozyme and breaking open the cells through incubation for
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30 minutes and occasional mixing. To avoid unspecific fluorescence, cell debris
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were pelleted before the experiment and small aliquots of the supernatant were
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added to 200 µL thin-walled PCR tubes. Results are displayed in the Figures 1 and 2:<o:p></o:p></span></p>
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</html>[[File:--Uppsala--protein-test.png |300px|thumb|left|'''Figure 1.''' Preliminary test of UnaG.]]<html>
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</html>[[File:--Uppsala--fluorescence-UnaG.jpg|530px|thumb|right|'''Figure 2.''' Comparative fluorescence of crude cell extracts of DH5a cells expressing UnaG or RFP.]]<html>
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</br><SPAN style='font-size: 110%; font-weight: bold;'>Large-scale expression of UnaG</SPAN>
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</html>[[File:--Uppsala--IMAC.png|350px|thumb|left|'''IMAC Chromatography Setup''']]<html>
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<p class=MsoNormal style='text-align:justify;text-justify:inter-ideograph'><span
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style='font-size:10.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman"'>After
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observing the small-scale expression results, plans for large-scale purification
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were designed. <span class=SpellE>E.coli</span> BL21DE3 cells were used in
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conjunction with T7 promoter-driven <span class=SpellE>UnaG</span> in TB medium
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to achieve peak levels of expression. T7 expression is induced using IPTG at
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18° for 16 hours. Breaking open the cells was performed using <span
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class=SpellE>Qsonica</span> Q700 titanium-tip <span class=SpellE>sonicator</span>
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since lysozyme has nearly the same size as <span class=SpellE>UnaG</span> and
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would make purification more difficult, as well as just slower and less efficient
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compared to sonication. It also breaks DNA, reducing overall lysate viscosity.
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After centrifugation at 4°C and 10 000 g for 1 hour, the supernatant was
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filtered through 0.2 µM pore filter and loaded to a Ni<sup>2+</sup> affinity
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column. </span><span style='font-size:10.0pt;font-family:Arial;mso-fareast-font-family:
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"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
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</html>[[File:--Uppsala--sdspage.png |350px|thumb|right|'''Figure 3. SDS Page Results''' Coomasie stained gel representing the eluted fractions of protein; L - PageRuler™ Prestained Protein Ladder, 10 to 180 kDa; 1 to 7 = incteasing concentrations of imidazole: from 50 to 350 mM in 50 mM steps respectively;]]<html>
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<p class=MsoNormal style='text-align:justify;text-justify:inter-ideograph'><span
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style='font-size:10.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman"'>First
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the column had to be packed using Nickel <span class=SpellE>Sepharose</span>
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Fast Flow resin (GE Healthcare). The calculated column volume for this setup was
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5 ml. Afterwards the column had to be charged with Ni<sup>2+</sup> this is done
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with a ion binding buffer (50 <span class=SpellE>mM</span> Na<sup>+</sup>CH<sub>3</sub>COO<sup>-</sup>,
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300 <span class=SpellE>mM</span> <span class=SpellE>NaCl</span>, pH4). The
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column was equilibrated by pumping through 5 column volumes (CV) with a <span
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class=SpellE>flowrate</span> of 0.7ml/min. After the column is equilibrated
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with ion binding buffer, the buffer it switched out with 0.3 M NiSO<sub>4</sub>
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and a new equilibration step is made with 5CV’s of ion solution. The final
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equilibration is done with 5CV ion binding buffer to wash away all of the
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excess Ni<sup>+2</sup> ions.<o:p></o:p></span></p>
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<p class=MsoNormal style='text-align:justify;text-justify:inter-ideograph'><span
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style='font-size:10.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman"'>Three
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others buffers were prepared, having a base composition of 50 <span
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class=SpellE>mM</span> Na<sup>+</sup>CH<sub>3</sub>COO<sup>-</sup> and 300 <span
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class=SpellE>mM</span> <span class=SpellE>NaCl</span>. The binding buffer for <span
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class=SpellE>UnaG</span> contains 10 <span class=SpellE>mM</span> imidazole,
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the washing buffer (to remove the excess protein from the column) contains 20 <span
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class=SpellE>mM</span> imidazole and the elution buffer contains 350 <span
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class=SpellE>mM</span> imidazole. The column is equilibrated with 5 CV of
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binding buffer, afterward 25 ml of the binding buffer is mixed with the <span
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class=SpellE>UnaG</span> lysate and loaded onto the column. Afterwards 5 CV of
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wash buffer is pumped through to remove of all the excess protein still in the
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column. Finally the elution buffer is used and fractions are collected. All of
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the fractions were then tested for the target protein using SDS-PAGE (Figure
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3).<o:p></o:p></span></p>
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<p class=MsoNormal style='text-align:justify;text-justify:inter-ideograph'><span
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style='font-size:10.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman"'>Samples
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were boiled with 2x <span class=SpellE>Laemmli</span> Sample Buffer with DTT
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and 10 µL loaded on each start. The protein ladder was 5 µL, not boiled. After
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staining the gel with <span class=SpellE>Coomassie</span> Brilliant Blue and <span
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class=SpellE>destaining</span>, the band corresponding to <span class=SpellE>UnaG’s</span>
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expected size (15.6 <span class=SpellE>kDa</span>) was not observed anywhere.
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In addition, none of the fractions fluoresced when bilirubin was added.<o:p></o:p></span></p>
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<p class=MsoNormal style='text-align:justify;text-justify:inter-ideograph'><span
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style='font-size:10.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman"'>Since
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sequencing confirmed that there are no mutations, and we previously managed to
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obtain impure functioning <span class=SpellE>UnaG</span> during the small-scale
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experiments of <span class=SpellE>UnaG</span>, the conclusion was that the IPTG
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solution was faulty and did not manage to induce the T7 promoter driven <span
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class=SpellE>UnaG</span> expression.<o:p></o:p></span></p>
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</html>
 
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Latest revision as of 14:25, 19 October 2016


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Please enter how you used this part and how it worked out.

Results

Since summers are always too short in Sweden, there was not enough time for the Uppsala 2016 iGEM team to test this UnaG BioBrick as thoroughly as the BBa_K2003010 was tested. However, BBa_K2003011 was made by PCR mutagenesis with the BBa_K2003010 BioBrick as template. BBa_K2003011 is therefore expected to function similarly to BB_K2003010 during expression.

User Reviews

UNIQ1a780f03973fafbb-partinfo-00000001-QINU UNIQ1a780f03973fafbb-partinfo-00000002-QINU