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

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===Results===
 
===Results===
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<p>
  
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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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</br><SPAN style='font-size: 110%; font-weight: bold;'>Designing the UnaG BioBricks</SPAN>
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style='font-size:10.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";
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<p>The IDT plasmid contains the lac-promoter so it is possible to do test expression directly with IPTG. However due to the way the final product is created, the UnaG sequence had a 50% chance to be incorporated in reverse, which was the case. Therefore different BioBrick promoters were extracted from the 2016 distribution to test for expression. Those include a medium constitutive promoter (<span style='font-size:10.0pt;font-family:Arial;mso-bidi-font-family:
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mso-bidi-theme-font:minor-bidi'><a
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"Times New Roman";mso-bidi-theme-font:minor-bidi'><a href="https://parts.igem.org/Part:BBa_K608006"><span style='mso-bidi-font-family: "Times New Roman";color:windowtext'><u>BBa_K608006</u></span></a></span>), a strong constitutive promoter (<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'><u>BBa_K880005</u></span></a></span>), IPTG inducible promoter (<span style='font-size:10.0pt;font-family:Arial;
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mso-bidi-font-family:"Times New Roman";mso-bidi-theme-font:minor-bidi'><a
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href="https://parts.igem.org/Part:BBa_J04500"><span style='mso-bidi-font-family:
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"Times New Roman";color:windowtext'><u>BBa_J04500</u></span></a></span>), and a T7 promoter (<span style='font-size:10.0pt;font-family:Arial;
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mso-bidi-font-family:"Times New Roman";mso-bidi-theme-font:minor-bidi'><a
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href="https://parts.igem.org/Part:BBa_K525998"><span style='mso-bidi-font-family:
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"Times New Roman";color:windowtext'><u>BBa_K525998</u></span></a></span>). All of those contain an RBS already assembled in order to speed up work. The UnaG sequence itself was excised from the IDT plasmid using EcoRI and PstI and ligated into <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:pSB1C3"><span style='mso-bidi-font-family:
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"Times New Roman";color:windowtext'><u>pSB1C3</u></span></a></span> backbone cut with the same set of enzymes. </p>
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<p>After successfully obtaining UnaG assembled with a variety of promoters, mutagenesis was performed in order to create several variants of the BioBrick for future use. The “stock” option contains the UnaG coding sequence. Upstream of it lies a 6xHis affinity tag, separated by an additional Serine amino acid to increase flexibility between the tag and the protein. In the registry it is annotated as <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'><u>BBa_K2003010</u></span></a></span>. Note that this part is designed with </span><span style='font-size:10.0pt;
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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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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/Help:Assembly_standard_25"><span
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style='mso-bidi-font-family:"Times New Roman";color:windowtext'><u>RFC25 (Freiburg
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Standard)</u></span></a></span><span style='font-size:10.0pt;font-family:Arial;
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mso-bidi-font-family:"Times New Roman"'> prefix and suffix in mind, hence it contains additional restriction sites, that do not affect normal 3A assembly but enable in-frame protein fusion without creating stop codons. Downstream of the part lies a short Glycine-rich flexible linker to minimize the effects of possible protein fusion. However the base part still contains the double TAA codons before this flexible linker.</p>
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<p>The stop signal has been removed with PCR in <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_K2003011"><span
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style='mso-bidi-font-family:"Times New Roman";color:windowtext'><u>BBa_K2003011</u></span></a></span>. This is part retains all the features, including the RFC25 prefix and suffix, but now the flexible linker is properly expressed and in case of fusing to another CDS would not cause premature termination.</p>
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<p>Finally, to avoid potential interference from the 6xHistidine affinity tag, <span
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style='font-size:10.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";
 
style='font-size:10.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";
 
mso-bidi-theme-font:minor-bidi'><a
 
mso-bidi-theme-font:minor-bidi'><a
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href="https://parts.igem.org/Part:BBa_K2003012"><span style='mso-bidi-font-family:
 
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"Times New Roman";color:windowtext'><u>BBa_K2003012</u></span></a></span> was created based on <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_K2003011"><span
 
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style='mso-bidi-font-family:"Times New Roman";color:windowtext'><u>BBa_K2003011</u></span></a></span>. In this part, the six amino acids were removed (again through PCR), as well as the Serine linker in between. This part has been designed for studying the properties of the protein in vivo, since the high positive charge of the affinity tag could interfere with its function or localization. At each step of the experiments, sequencing was performed using the iGEM standard VF2 and VR verification primers to ensure CDS integrity and especially to avoid introduced frameshift mutations.</p>
 
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</br><SPAN style='font-size: 110%; font-weight: bold;'>Small-scale Expression of UnaG</SPAN>
 
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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"'>In
 
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addition to mutagenesis experiments, small-scale expression tests were
 
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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
 
 
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> BioBrick as template. BBa_K2003011 is therefore expected  to function similarly to BB_K2003010 during expression. </p>
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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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water, so the stock 1mM solution was prepared in DMSO instead. It seems the
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molecule does not permeate the cell easily, so appropriate amounts were added
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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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</br><SPAN style='font-size: 110%; font-weight: bold;'>Large-scale expression of UnaG</SPAN>
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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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<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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Latest revision as of 14:25, 19 October 2016


This experience page is provided so that any user may enter their experience using this part.
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

UNIQ46ae74749c0f5798-partinfo-00000001-QINU UNIQ46ae74749c0f5798-partinfo-00000002-QINU