Difference between revisions of "Team:CKWA-China/Contribution"

 
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                     <li>Contribution</li>
 
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                         <h2 class="point" id="point-1">Literature Review</h2>
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                         <h2 class="point" id="point-1">Contribution</h2>
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                        <p>As we all know, potato (Solanum tuberosum) is a globally important high-yield crop that produces nutrient-rich tubers. This non-grain crop is the third most important food crop, after wheat and rice (Patil et al.2017). One of the major threats to potato production is soft rot disease caused by Erwinia bacteria, which generally occur during cultivation, harvesting or transportation and storage of farm produce, resulting in considerable yield reduction, poor quality of produce, and economic loss. In Kenya, it causes up to 50% total crop loss (Onkendi and Moleleki 2014; Muturi et al.2018).</p>
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                      <h2 class="point" id="point-1"> <a href="http://parts.igem.org/Part:BBa_C0012#CKWA-China_2021_Contribution">Part BBa_C0012</a></h2>
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                        <p>Different approaches to soft rot disease control have been developed and applied. The effectiveness of the phages in preventing infection of potato tubers by P. carotovorum was tested in laboratory experiments The management of potato diseases is based on a massive use of chemical pesticides, causing environmental pollution and ecological destruction. Although some researches about environmental-friendly pest management approaches have been reported, there are still many problems in effect and safety aspects.</p>
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                             <h3 class="point" id="point-2">Interview（Potato Center Asia-Pacific Headquarter）</h3>
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                            <p>We met with senior staffs of the potato center Asia-Pacific Headquarter to learn about the current detection and treatments of potato diseases. We then visited their workplace, which accounted for a large proportion of the current potato-agricultural development. We presented our initial concept to the experts (including the chairman of the potato center, Mr. Lu) and audited a group meeting on agricultural pharmacology. Through the discussions with them, we became more aware of the consequences of Soft Rot disease and the importance of creating an effective bio-control solution.</p>
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We were able to complete experiments and made measurement on our parts. For Bronze medal, we contributed to the part <a href="http://parts.igem.org/Part:BBa_C0012#CKWA-China_2021_Contribution">Part BBa_C0012</a>
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We use LacI for expression of Lon protease. The E. coli we used are BL21, Rosetta and similar bateria. According to the reference articles, this Lon protease decomposes C-Myc protein commonly found in cancer cells. Because of this potential, the protease is planned to be used to make medicine to suppress cancer recurrence.
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We designed to use LacI and LacO together to produce the Lon protease in a commonly used plasmid. The plasmid structure works as shown in the figure below.
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<p><center>Figure.1.The designed use of the part LacI.</center></p>
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For the measurement, E. coli Rosetta containing the designed plasmid was incubated at 37C overnight with 1mM IPTG added. Then the cells are lysed by 200 μL 4% SDS for 10 minutes in room temperature, then 10 min at 95C. Then loading buffer is added. The SDS-PAGE was carried with a 12% precast polyacrylamide gel. Coomassie bright blue stain is used to show the bands.
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With this induction conditions, first test failed to observe a clear band of Lon protein. The bacteria culture was also relatively clear. It is possible that Lon protease has been decomposing the proteins needed for E. coli growth. Because the bacteria growth was suppressed by the Lon protease, its own production was low.
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<p>Figure.2. Bacteria Culture overnight and the SDS-PAGE results with/without iPTG.</p></center>
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When there is LacI and there is LacO before the Lon gene sequence, IPTG can be used to control the expression. The control can be tuned by the concentration and timing of induction. We then changed our condition: the bacteria were cultured on LB plate overnight at 37C, and then pick a single colony and incubate in LB medium until the OD600 reached 0.6-0.8. At this time, IPTG was added at 0.5 mM, and the medium was again incubated at 37C for another 4 hours. This time the SDS-PAGE results showed remarkable expression of the Lon protease. The results confirmed the capability of this part, and also showed for better protein expression and better bateria growth, the timing for induction should also be considered and experimented.
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<h3 class="point" id="point-2">Summary</h3>
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This part LacI can work with LacO in E. coli Rosseta to control the protein production. The control power has important meanings for some special proteins like Lon protease, which can suppress bacteria growth. Our results suggested at 0.5mM IPTG concentration and an induction timing at OD600=0.6~0.8 can help improve the production target protein.</p>
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                                src="https://static.igem.org/mediawiki/parts/0/06/T--CKWA-China-Lon2.jpg" width="500" alt="">
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<p>Figure.3. Expression of Lon protease confirmed in SDS-PAGE.</p></center>
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Latest revision as of 09:32, 21 October 2021

Contribution

Contribution

Part BBa_C0012

We were able to complete experiments and made measurement on our parts. For Bronze medal, we contributed to the part Part BBa_C0012


We use LacI for expression of Lon protease. The E. coli we used are BL21, Rosetta and similar bateria. According to the reference articles, this Lon protease decomposes C-Myc protein commonly found in cancer cells. Because of this potential, the protease is planned to be used to make medicine to suppress cancer recurrence.

We designed to use LacI and LacO together to produce the Lon protease in a commonly used plasmid. The plasmid structure works as shown in the figure below.

Figure.1.The designed use of the part LacI.

For the measurement, E. coli Rosetta containing the designed plasmid was incubated at 37C overnight with 1mM IPTG added. Then the cells are lysed by 200 μL 4% SDS for 10 minutes in room temperature, then 10 min at 95C. Then loading buffer is added. The SDS-PAGE was carried with a 12% precast polyacrylamide gel. Coomassie bright blue stain is used to show the bands.

With this induction conditions, first test failed to observe a clear band of Lon protein. The bacteria culture was also relatively clear. It is possible that Lon protease has been decomposing the proteins needed for E. coli growth. Because the bacteria growth was suppressed by the Lon protease, its own production was low.

Figure.2. Bacteria Culture overnight and the SDS-PAGE results with/without iPTG.


When there is LacI and there is LacO before the Lon gene sequence, IPTG can be used to control the expression. The control can be tuned by the concentration and timing of induction. We then changed our condition: the bacteria were cultured on LB plate overnight at 37C, and then pick a single colony and incubate in LB medium until the OD600 reached 0.6-0.8. At this time, IPTG was added at 0.5 mM, and the medium was again incubated at 37C for another 4 hours. This time the SDS-PAGE results showed remarkable expression of the Lon protease. The results confirmed the capability of this part, and also showed for better protein expression and better bateria growth, the timing for induction should also be considered and experimented.

Summary

This part LacI can work with LacO in E. coli Rosseta to control the protein production. The control power has important meanings for some special proteins like Lon protease, which can suppress bacteria growth. Our results suggested at 0.5mM IPTG concentration and an induction timing at OD600=0.6~0.8 can help improve the production target protein.

Figure.3. Expression of Lon protease confirmed in SDS-PAGE.