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<font size="3">Bicistronic device (BCD) is a modified ribosome binding site (RBS) with another cistron. The device consists of another cistron (cistron 1) with another RBS (SD2) | <font size="3">Bicistronic device (BCD) is a modified ribosome binding site (RBS) with another cistron. The device consists of another cistron (cistron 1) with another RBS (SD2) | ||
between RBS (SD1) and gene of interest (cistron 2). Also, the stop codon of cistron 1 overlaps the start codon of cistron 2. Ribosome binding efficiency and translation rate will be affected after the secondary structure near the RBS has changed due to the change of gene of interest. This device can maintain the ribosome binding efficiency and translation rate even though the gene of interest has changed. Therefore, it is used to control the amount of fluorescence in this study. BCD is expected to generate a more reliable and precise gene expression.</font> | between RBS (SD1) and gene of interest (cistron 2). Also, the stop codon of cistron 1 overlaps the start codon of cistron 2. Ribosome binding efficiency and translation rate will be affected after the secondary structure near the RBS has changed due to the change of gene of interest. This device can maintain the ribosome binding efficiency and translation rate even though the gene of interest has changed. Therefore, it is used to control the amount of fluorescence in this study. BCD is expected to generate a more reliable and precise gene expression.</font> | ||
+ | |||
+ | <h3><font size="6">Method</font></h3> | ||
+ | <table width="80%"> | ||
+ | <thead> | ||
+ | <tr> | ||
+ | <th colspan="2"><u>We follow exactly the interlab protocol provided by iGEM(link) with the below plate reader setting: </u></th> | ||
+ | </tr> | ||
+ | </thead> | ||
+ | |||
+ | <tr> | ||
+ | <th colspan="2">Microplate used: Corning® clear flat bottom black 96 well plates</th> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <th colspan="2">Instrument used: BMG LABTECH’s CLARIOstar®</th> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <th colspan="2">Instrument Settings for measuring OD600 of LUDOX and Cells:</th> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <th colspan="2">Endpoint settings</th> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <th>No. of flashes per well</th> | ||
+ | <th>10</th> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <th>Scan mode</th> | ||
+ | <th>orbital averaging</th> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <th>Scan diameter [mm]</th> | ||
+ | <th>3</th> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <th colspan="2">Optic settings</th> | ||
+ | <tr> | ||
+ | |||
+ | <tr> | ||
+ | <th>Excitation</th> | ||
+ | <th>600</th> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <th colspan="2">General settings</th> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <th colspan="2">Top optic used</th> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <th>Settling time [s]</th> | ||
+ | <th>0.1</th> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <th>Reading direction</th> | ||
+ | <th>bidirectional, horizontal left to right, top to bottom</th> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <th>Target temperature [°C]</th> | ||
+ | <th>25</th> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <th colspan="2">Instrument Settings for measuring fluorecence of Fluorescein and GFP:</th> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <th colspan="2">Endpoint settings</th> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <th>No. of flashes per well</th> | ||
+ | <th>8</th> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <th>Scan mode</th> | ||
+ | <th>orbital averaging</th> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <th>Scan diameter [mm]</th> | ||
+ | <th>3</th> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <th colspan="2">Optic settings</th> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <th>Excitation:</th> | ||
+ | <th>470-15</th> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <th>Emission</th> | ||
+ | <th>515-20</th> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <th>Gain</th> | ||
+ | <th>500</th> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <th>Focal height [mm]</th> | ||
+ | <th>9</th> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <th colspan="2">General settings</th> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <th colspan="2">Top optic used</th> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <th>Reading direction</th> | ||
+ | <th>bidirectional, horizontal left to right, top to bottom</th> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <th>Target temperature [°C]</th> | ||
+ | <th>25 | ||
+ | </th> | ||
+ | </tr> | ||
+ | |||
+ | </table> | ||
Revision as of 09:53, 2 October 2017