<p> The expression and purification of CasRX were based on this <ahref="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5910255/">article</a>
<ol>
<listyle="margin-left: 5%;">Streak LB-Agarose plate containing 100 µL of Kanamycin and 100 µL of Chloramphenicol (plates already in the fridge) with cell suspension containing the plasmid. Incubate the plates overnight at 37°C.</li>
<listyle="margin-left: 5%;">The next day inoculate 15 mL of LB media containing 15 µL of Kanamycin with one single colony and incubate overnight at 37°C on a rotary shaker at 190 RPM.</li>
<listyle="margin-left: 5%;">The next day inoculate 750 mL of TB media containing 765 µL of kanamycin and 765 µL of chloramphenicol with 15 mL of starter culture. Then shake at 37°C, 190 RPM.</li>
<listyle="margin-left: 5%;">Monitor OD every hour until cells reach an optical density of 0.4 – 0.6, then transfer flasks to 4°C for 30 min to allow flasks to cool prior to induction.</li>
<listyle="margin-left: 5%;">Critical step: For optimal expression, it is important to strictly follow the indicated OD of 0.4–0.6 at the time-point of induction.</li>
<listyle="margin-left: 5%;">Cool on ice for 15 mins </li>
<listyle="margin-left: 5%;">Take 20 µL of the flask in a tube (non-induced sample)</li>
<listyle="margin-left: 5%;">Induce expression by adding 765 µL of 0.2M IPTG (0.2 mM final concentration) and shake cultures for 20 hours on a rotary shaker at 190 RPM, in a pre-chilled 18°C incubator.</li>
</ol>
<p>1. Streak LB-Agarose plate containing 100 µL of Kanamycin and 100 µL of Chloramphenicol (plates already in the fridge) with cell suspension containing the plasmid. Incubate the plates overnight at 37°C.</p>
<p> 2. The next day inoculate 15 mL of LB media containing 15 µL of Kanamycin with one single colony and incubate overnight at 37°C on a rotary shaker at 190 RPM.</p>
<p> 3.The next day inoculate 750 mL of TB media containing 765 µL of kanamycin and 765 µL of chloramphenicol with 15 mL of starter culture. Then shake at 37°C, 190 RPM.</p>
<p> 4. Monitor OD every hour until cells reach an optical density of 0.4 – 0.6, then transfer flasks to 4°C for 30 min to allow flasks to cool prior to induction.</p>
<p> 5. Critical step: For optimal expression, it is important to strictly follow the indicated OD of 0.4–0.6 at the time-point of induction.</p>
<p> 6.Cool on ice for 15 mins </p>
<p> 7. Take 20 µL of the flask in a tube (non-induced sample)</lp>
<p> 8. Induce expression by adding 765 µL of 0.2M IPTG (0.2 mM final concentration) and shake cultures for 20 hours on a rotary shaker at 190 RPM, in a pre-chilled 18°C incubator.</p>
<p> Then we proceeded to purify, however, due to availability of the machines, we purified protein at room temperature, instead of 4°C, which is one of the reasons we did not manage to purify it. Once the purification process was finished, we abandoned this idea, and made an expression test in small volumes.</p>
<ol>
<listyle="margin-left: 5%;">We prepared 4 cultures of 10 ml in TB, with 10 µl of Kanamycin and Chloramphenicol. Then cultures were left to grow at 37°C, expected OD is between 0.4-0.6. </li>
<listyle="margin-left: 5%;">Of these 4 cultures, 1 was used as “non induced”, 3 others were induced at different IPTG concentrations, precisely 0.25mM, 0.5 mM and 1 mM. </li>
<listyle="margin-left: 5%;">After 20h at 18°C on 190 RPM, we took 1 ml of each culture, sonicate it up until the solution clarified. 45 µl were taken from each tube, then mixed with 15 µl of the blue.These were our TOTAL fractions, the ones called “supernatant + pellet”.</li>
<listyle="margin-left: 5%;">Secondly, we used the rest of the volume, 955 µL, we centrifuged it for 7 min, took 45 µL of the supernatant and added 15 µL of the blue. These were “supernatant only” fractions. </li>
<p>1. We prepared 4 cultures of 10 ml in TB, with 10 µl of Kanamycin and Chloramphenicol. Then cultures were left to grow at 37°C, expected OD is between 0.4-0.6. </p>
<p>2. Of these 4 cultures, 1 was used as “non induced”, 3 others were induced at different IPTG concentrations, precisely 0.25mM, 0.5 mM and 1 mM. </p>
<p>3. After 20h at 18°C on 190 RPM, we took 1 ml of each culture, sonicate it up until the solution clarified. 45 µl were taken from each tube, then mixed with 15 µl of the blue.These were our TOTAL fractions, the ones called “supernatant + pellet”.</p>
<p>4. Secondly, we used the rest of the volume, 955 µL, we centrifuged it for 7 min, took 45 µL of the supernatant and added 15 µL of the blue. These were “supernatant only” fractions. <p>
</ol>
<p> In total we got 8 fractions, including 2 non induced, and 6 others induced at different IPTG concentrations. However, once we’ve done SDS PAGE, we didn’t see any significant difference between non induced and induced fractions. We started by quantifying it in ImageJ, to be sure that there is no difference. However, each fraction, for example, was not sonicated in the same manner, sometimes it took more time, sometimes less. There was no lysis buffer in this experiment, so maybe the lysis was not finished. We put 10 µl of each fraction of the gel, but some columns are more intense than others, as if different conditions were applied to them. So the quantification was not very useful. </p>
