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<p><strong><em>Bodo saltans</em></strong><strong> kill curve protocol using G418 (Gentamicin)</strong></p>
<p><strong> </strong></p>
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<ul>
<li>Gentamycin exhibits toxicity toward both eukaryotic and prokaryotic cells by disrupting ribosome function, thereby blocking the elongation step in protein synthesis.</li>
<li>G418 is most commonly used as a selection agent for eukaryotic cells genetically engineered to express a neomycin resistance gene (NeoR), which is encoded by either transposon Tn601 (903) or Tn5.</li>
<li>The resistant cells grow in medium containing G418, and may be used to establish stably transfected cell lines as all the non-resistant cells die due to G418 toxicity, typically within 6 – 14 days.</li>
<li>To establish kill curve assay for <em> saltans</em> various concentrations of G418 were tested. The concentration range from 1 to 50 μg/mL for selection of eukaryotic, and then higher concentrations can be used for maintaining stable transfected cell line.</li>
</ul>
<p><strong> </strong></p>
<p><strong>Kill Curve Assay </strong></p>
<ol>
<li>Harvest the <em>B. saltans</em> cells from a culture that is at peak density (1 – 3.0 x 10<sup>5</sup>) by centrifugation at 800 x g for 5 min., discard the supernatant (medium).</li>
</ol>
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<ol start="2">
<li>Replace the growth medium with fresh medium containing 0 – 50 μg/mL. For each concentration, test in triplicate.</li>
</ol>
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<ol start="3">
<li>Using 6 wells plates, replace the medium in the wells every 3 – 4 days using fresh medium with the appropriate G418 concentration. Perform a daily visual inspection for evidence of toxicity, also cell count using hemocytometer.</li>
</ol>
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<ol start="4">
<li>Note that the optimal dose of G418 for selection is the lowest one for which all cells have died after one week. A low dose is the concentration which has minimal effects on cells after 2 weeks of antibiotic selection. Conversely, a high dose is a concentration which is highly toxic to cells within 2-3 days of starting antibiotic selection.</li>
</ol>
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<p>5- The results indicate that a G418 concentration of  2 μg/mL kill the entire <em>B. saltans</em> population in 12 days which is considered enough time for selecting the resistant cells after transfection.</p>
<p><strong> </strong></p>
<p><strong>G418 Sensitivity for <em>B. saltans</em></strong></p>
<table width="316">
<tbody>
<tr>
<td width="118">
<p>G418 (μg/ml)</p>
</td>
<td width="198">
<p>Cells survival (days)</p>
</td>
</tr>
<tr>
<td width="118">
<p>50</p>
</td>
<td width="198">
<p>2</p>
</td>
</tr>
<tr>
<td width="118">
<p>20</p>
</td>
<td width="198">
<p>2</p>
</td>
</tr>
<tr>
<td width="118">
<p>15</p>
</td>
<td width="198">
<p>3</p>
</td>
</tr>
<tr>
<td width="118">
<p>10</p>
</td>
<td width="198">
<p>4</p>
</td>
</tr>
<tr>
<td width="118">
<p>5</p>
</td>
<td width="198">
<p>6</p>
</td>
</tr>
<tr>
<td width="118">
<p>3</p>
</td>
<td width="198">
<p>8</p>
</td>
</tr>
<tr>
<td width="118">
<p>2</p>
</td>
<td width="198">
<p>12</p>
</td>
</tr>
<tr>
<td width="118">
<p>1</p>
</td>
<td width="198">
<p>&gt;16</p>
</td>
</tr>
<tr>
<td width="118">
<p>0</p>
</td>
<td width="198">
<p>&gt;16</p>
</td>
</tr>
</tbody>
</table>
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<p> </p>
<table width="100%">
<tbody>
<tr>
<td>
<p>G418 (μg/ml)</p>
</td>
</tr>
</tbody>
</table>
<table width="100%">
<tbody>
<tr>
<td>
<p>Cell survival (days)</p>
</td>
</tr>
</tbody>
</table>
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<p><strong>Cell Selection </strong></p>
<ol>
<li>In all of our plasmids constructs we included the <em>NeoR </em>gene (Neomycine resistant gene) to select our transfected cells.</li>
<li>Twenty-four hours after electroporation, the growth medium of transfected cells were supplemented with G418 antibiotic Solution (2 μg/mL) based on the kill curve results (above).</li>
</ol>
<p> </p>
<ol start="3">
<li>3<em>. </em>Replace the G418-containing medium every 3 – 5 days and examine cells for visual toxicity. Most non-transfected (non-resistant) cells will die within 10- 12 days, leaving the transfected cells to expand.</li>
<li>Once cells grow to high confluence, they may be maintained, or frozen as a polyclonal line or plated by limited dilution to select for single clones.</li>
</ol>
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