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| − | <p>Desires to obtain pain of itself, because it is pain, but because occasionally circumstances occur in which toil and pain can procure him some great pleasure. To take a trivial example, which of us ever undertakes laborious physical exercise, except to obtain some advantage from it.who do not know how to pursue pleasure rationally encounter consequences that are extremely painful. Nor again is there anyone who loves or pursues or desires to obtain pain advantage.
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| − | The master-builder of human happiness. No one rejects, dislikes, or avoids pleasure itself, because it is pleasure, but because those who do not know how to pursue pleasure rationally encounter consequences that are extremely painful. Nor again is there anyone who loves or pursues or desires to obtain pain of itself, because it is pain, but because occasionally circumstances occur in which toil and pain can procure him some great pleasure. To take a trivial example, which of us ever undertakes laborious physical exercise, except to obtain some great explorer of the truth, the master-builder of human happiness. No one rejects, dislikes, or avoids pleasure itself, because it is pleasure, but because those who do not know how to pursue pleasure rationally encounter consequences that are extremely painful. Nor again is there.</p>
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| − | <blockquote>Mistaken idea of denouncing pleasure and praising pain was born and I will give you a complete account of the system, and expound the actual teachings of the great explorer of the truth, the master-builder of human happiness.who do not know how to pursue pleasure rationally encounter consequences that are extremely painful. Expound the actual teachings of the great explorer of the truth, account of the system the master-builder of human happiness.</blockquote> | + | <blockquote>Protein Expression and Purification</blockquote> |
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| | + | <p>The gene for Hfq (plus His tag) was inserted into the suffix region of BBa_K314103, a lac inducible expression cassette constructed by Washington iGEM 2010, and the entire construct was simultaneously inserted into the pSB1K3.m1 plasmid backbone using the Anza enzyme system protocol. See agarose gel below for confirmation of construct.</p> |
| | + | <img src="https://static.igem.org/mediawiki/2017/4/40/UNBC-Canada_successful_clone.jpeg" style="width:500px; height:600px;"> </img> |
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| | + | <p>The resulting plasmid was purified and transformed into Rosetta 2 pLySs cells and grown in LB broth with a 30μg/mL concentration of kanamycin. Initially, 3mL of a 10mL starter culture was used to expand to a 300mL culture, which was grown until OD600 = 0.82 before being induced with 0.8mM IPTG. Cells were taken at t = 0h, 1h, 2h, 12h, 14h, and 18h, before being harvested after 18h. Protein expression levels over time are shown below in an SDS-PAGE gel below; protein expression levels appear to be highest at 18h (see SDS-PAGE gel below). Hfq was purified using a Ni-NTA column, and the resulting concentration in fraction 1 totaled 0.31mg/mL.</p> |
| | + | <img src="https://static.igem.org/mediawiki/2017/9/97/UNBC-Canada_Hfq_Expression_Gel.jpeg" style="width:500px; height:600px;"> </img> |
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| − | <p><b>Avoids pleasure itself, because it is pleasure,</b> but because those who do not know how to pursue pleasure rationally encounter consequences that are extremely painful. Nor again is there anyone who loves or pursues or desires to obtain pain of itself, because it is pain, but because occasionally circumstances occur in which toil and pain can procure him some great pleasure. To take a trivial example, which of us ever undertakes laborious physical exercise, except to obtain some.
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| − | Totam rem aperiam, eaque ipsa quae ab illo inventore veritatis et quasi architecto beatae vitae dicta sunt explicabo. Nemo enim ipsam voluptatem quia voluptas sit aspernatur aut odit aut fugit, sed quia undertakes laborious physical exercise, except
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| − | Totam rem aperiam, eaque ipsa quae ab illo inventore veritatis et quasi architecto beatae vitae dicta sunt explicabo consequuntur magni dolores eos qui ratione voluptatem sequi nesciunt. Neque porro quisquam est, qui dolorem ipsum quia. great explorer of the truth, the master-builder of human happiness. No one rejects, dislikes, or avoids pleasure itself, because it is pleasure, but because those who do not know how to pursue pleasure rationally encounter consequences that extremely.</p>
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The gene for Hfq (plus His tag) was inserted into the suffix region of BBa_K314103, a lac inducible expression cassette constructed by Washington iGEM 2010, and the entire construct was simultaneously inserted into the pSB1K3.m1 plasmid backbone using the Anza enzyme system protocol. See agarose gel below for confirmation of construct.
The resulting plasmid was purified and transformed into Rosetta 2 pLySs cells and grown in LB broth with a 30μg/mL concentration of kanamycin. Initially, 3mL of a 10mL starter culture was used to expand to a 300mL culture, which was grown until OD600 = 0.82 before being induced with 0.8mM IPTG. Cells were taken at t = 0h, 1h, 2h, 12h, 14h, and 18h, before being harvested after 18h. Protein expression levels over time are shown below in an SDS-PAGE gel below; protein expression levels appear to be highest at 18h (see SDS-PAGE gel below). Hfq was purified using a Ni-NTA column, and the resulting concentration in fraction 1 totaled 0.31mg/mL.
