Difference between revisions of "Team:SKLMT-China/Demonstrate"
Zhaowenxue (Talk | contribs) |
Zhaowenxue (Talk | contribs) |
||
| Line 5: | Line 5: | ||
<div class="paragraph shadow"> | <div class="paragraph shadow"> | ||
<h2 class="title">Demonstrate</h2> | <h2 class="title">Demonstrate</h2> | ||
| − | <p>test</p> | + | <p>Based on our promoter library, we selected three promoters (promoter 4, promoter 5 and promoter11) of different intensities to regulate the expression of the key nicotine-degrading gene <i>nicA2</i>.We’ve constructed lasmid, pBBR1-km-amp-cm-promoter-nic, and electroporated it into our new chassis bacteria,<latin> Pseudomonas fluorescences pf-5</latin>. </p> |
| + | <span class="image fit"> | ||
| + | <img src="https://static.igem.org/mediawiki/2018/2/20/T--SKLMT-China--demonstrationfig1.png | ||
| + | " alt=" " /> | ||
| + | </span> | ||
| + | |||
| + | |||
| + | <p>We found that there are only a few colonies of <latin> P. fluorescences pf-5</latin> (containing <i>nicA2 </i>with promoters in front of it)on the LB plate,but lots of colonies on the <i>CK </i>plate(control group electroporated with pBBR1-km-amp-cm-nic without promoter). Combining the fact that theE.coliusually lost a part of (sometimes the whole plasmid)when we want to use high-copy vector to carry this gene cluster,we wondered if it<i> NicA2 </i>is a poisonous enzyme so that <latin>E.coli </latin>lost this plasmid during DNA replication under stress. </p> | ||
| + | <div style="top: 0px;"></div><span style="font-family:宋体;font-size:16px;"></span><table style="background: white; border: currentColor; border-image: none; border-collapse: collapse; mso-yfti-tbllook: 1184; mso-padding-alt: 0cm 5.4pt 0cm 5.4pt; mso-border-top-alt: solid black 1.0pt; mso-background-themecolor: background1; mso-border-top-themecolor: text1; mso-border-bottom-alt: solid black 1.0pt; mso-border-bottom-themecolor: text1;" border="1" cellspacing="0" cellpadding="0"><span style="font-family:宋体;"> </span><tbody><tr style="mso-yfti-irow: -1; mso-yfti-firstrow: yes;"><span style="font-family:宋体;"> </span><td width="304" valign="top" style="border-width: 1pt 0px; border-style: solid none; border-color: black rgb(0, 0, 0); padding: 0cm 5.4pt; width: 182.6pt; background-color: transparent; mso-border-top-themecolor: text1; mso-border-bottom-themecolor: text1;"><span style="font-family:宋体;font-size:16px;"> </span><p align="center" style="margin: 0cm 0cm 0pt; text-align: center; mso-yfti-cnfc: 5;"><span lang="EN-US" style="color: black; font-family: 'Times New Roman','serif'; mso-bidi-font-size: 10.5pt; mso-fareast-font-family: 仿宋; mso-themecolor: text1; mso-themeshade: 191; mso-bidi-font-weight: bold;"><span style="font-size:16px;">Plasmid in </span></span><em><span lang="EN-US" style="color: black; font-family: 'Times New Roman','serif'; font-size: 10pt; mso-fareast-font-family: 仿宋; mso-themecolor: text1; mso-themeshade: 191; mso-bidi-font-weight: bold;">P. fluorescences </span></em><span lang="EN-US" style="color: black; font-family: 'Times New Roman','serif'; font-size: 10pt; mso-fareast-font-family: 仿宋; mso-themecolor: text1; mso-themeshade: 191; mso-bidi-font-weight: bold; mso-bidi-font-style: italic;">pf-5</span></p><span style="font-family:宋体;font-size:16px;"> </span></td><span style="font-family:宋体;"> </span><td width="213" valign="top" style="border-width: 1pt 0px; border-style: solid none; border-color: black rgb(0, 0, 0); padding: 0cm 5.4pt; width: 127.6pt; background-color: transparent; mso-border-top-themecolor: text1; mso-border-bottom-themecolor: text1;"><span style="font-family:宋体;font-size:16px;"> </span><p align="center" style="margin: 0cm 0cm 0pt; text-align: center; mso-yfti-cnfc: 1;"><span lang="EN-US" style="color: black; font-family: 'Times New Roman','serif'; mso-bidi-font-size: 10.5pt; mso-fareast-font-family: 仿宋; mso-themecolor: text1; mso-themeshade: 191; mso-bidi-font-weight: bold;"><span style="font-size:16px;">Strength of promoter</span></span></p><span style="font-family:宋体;font-size:16px;"> </span></td><span style="font-family:宋体;"> </span><td width="193" valign="top" style="border-width: 1pt 0px; border-style: solid none; border-color: black rgb(0, 0, 0); padding: 0cm 5.4pt; width: 115.9pt; background-color: transparent; mso-border-top-themecolor: text1; mso-border-bottom-themecolor: text1;"><span style="font-family:宋体;font-size:16px;"> </span><p align="center" style="margin: 0cm 0cm 0pt; text-align: center; mso-yfti-cnfc: 1;"><span lang="EN-US" style="color: black; font-family: 'Times New Roman','serif'; mso-bidi-font-size: 10.5pt; mso-fareast-font-family: 仿宋; mso-themecolor: text1; mso-themeshade: 191; mso-bidi-font-weight: bold;"><span style="font-size:16px;">CFU on LB plate</span></span></p><span style="font-family:宋体;font-size:16px;"> </span></td><span style="font-family:宋体;"> </span></tr><span style="font-family:宋体;"> </span><tr style="mso-yfti-irow: 0;"><span style="font-family:宋体;"> </span><td width="304" valign="top" style="padding: 0cm 5.4pt; border: 0px rgb(0, 0, 0); border-image: none; width: 182.6pt; background-color: transparent;"><span style="font-family:宋体;font-size:16px;"> </span><p align="center" style="margin: 0cm 0cm 0pt; text-align: center; mso-yfti-cnfc: 68;"><span lang="EN-US" style="color: black; font-family: 'Times New Roman','serif'; mso-bidi-font-size: 10.5pt; mso-fareast-font-family: 仿宋; mso-themecolor: text1; mso-themeshade: 191; mso-bidi-font-weight: bold;"><span style="font-size:16px;">pBBR1-km-amp-cm-nic</span></span></p><span style="font-family:宋体;font-size:16px;"> </span></td><span style="font-family:宋体;"> </span><td width="213" valign="top" style="padding: 0cm 5.4pt; border: 0px rgb(0, 0, 0); border-image: none; width: 127.6pt; background-color: transparent;"><span style="font-family:宋体;font-size:16px;"> </span><p align="center" style="margin: 0cm 0cm 0pt; text-align: center; mso-yfti-cnfc: 64;"><span lang="EN-US" style="color: black; font-family: 'Times New Roman','serif'; mso-bidi-font-size: 10.5pt; mso-fareast-font-family: 仿宋; mso-themecolor: text1; mso-themeshade: 191;"><span style="font-size:16px;">none</span></span></p><span style="font-family:宋体;font-size:16px;"> </span></td><span style="font-family:宋体;"> </span><td width="193" valign="top" style="padding: 0cm 5.4pt; border: 0px rgb(0, 0, 0); border-image: none; width: 115.9pt; background-color: transparent;"><span style="font-family:宋体;font-size:16px;"> </span><p align="center" style="margin: 0cm 0cm 0pt; text-align: center; mso-yfti-cnfc: 64;"><span lang="EN-US" style="color: black; font-family: 'Times New Roman','serif'; mso-bidi-font-size: 10.5pt; mso-fareast-font-family: 仿宋; mso-themecolor: text1; mso-themeshade: 191;"><span style="font-size:16px;">>200</span></span></p><span style="font-family:宋体;font-size:16px;"> </span></td><span style="font-family:宋体;"> </span></tr><span style="font-family:宋体;"> </span><tr style="mso-yfti-irow: 1;"><span style="font-family:宋体;"> </span><td width="304" valign="top" style="padding: 0cm 5.4pt; border: 0px rgb(0, 0, 0); border-image: none; width: 182.6pt; background-color: transparent;"><span style="font-family:宋体;font-size:16px;"> </span><p align="center" style="margin: 0cm 0cm 0pt; text-align: center; mso-yfti-cnfc: 4;"><span lang="EN-US" style="color: black; font-family: 'Times New Roman','serif'; mso-bidi-font-size: 10.5pt; mso-fareast-font-family: 仿宋; mso-themecolor: text1; mso-themeshade: 191; mso-bidi-font-weight: bold;"><span style="font-size:16px;">pBBR1-km-amp-cm-promoter4-nic</span></span></p><span style="font-family:宋体;font-size:16px;"> </span></td><span style="font-family:宋体;"> </span><td width="213" valign="top" style="padding: 0cm 5.4pt; border: 0px rgb(0, 0, 0); border-image: none; width: 127.6pt; background-color: transparent;"><span style="font-family:宋体;font-size:16px;"> </span><p align="center" style="margin: 0cm 0cm 0pt; text-align: center;"><span lang="EN-US" style="color: black; font-family: 'Times New Roman','serif'; mso-bidi-font-size: 10.5pt; mso-fareast-font-family: 仿宋; mso-themecolor: text1; mso-themeshade: 191;"><span style="font-size:16px;">normal</span></span></p><span style="font-family:宋体;font-size:16px;"> </span></td><span style="font-family:宋体;"> </span><td width="193" valign="top" style="padding: 0cm 5.4pt; border: 0px rgb(0, 0, 0); border-image: none; width: 115.9pt; background-color: transparent;"><span style="font-family:宋体;font-size:16px;"> </span><p align="center" style="margin: 0cm 0cm 0pt; text-align: center;"><span lang="EN-US" style="color: black; font-family: 'Times New Roman','serif'; mso-bidi-font-size: 10.5pt; mso-fareast-font-family: 仿宋; mso-themecolor: text1; mso-themeshade: 191;"><span style="font-size:16px;">5</span></span></p><span style="font-family:宋体;font-size:16px;"> </span></td><span style="font-family:宋体;"> </span></tr><span style="font-family:宋体;"> </span><tr style="mso-yfti-irow: 2;"><span style="font-family:宋体;"> </span><td width="304" valign="top" style="padding: 0cm 5.4pt; border: 0px rgb(0, 0, 0); border-image: none; width: 182.6pt; background-color: transparent;"><span style="font-family:宋体;font-size:16px;"> </span><p align="center" style="margin: 0cm 0cm 0pt; text-align: center; mso-yfti-cnfc: 68;"><span lang="EN-US" style="color: black; font-family: 'Times New Roman','serif'; mso-bidi-font-size: 10.5pt; mso-fareast-font-family: 仿宋; mso-themecolor: text1; mso-themeshade: 191; mso-bidi-font-weight: bold;"><span style="font-size:16px;">pBBR1-km-amp-cm-promoter5-nic</span></span></p><span style="font-family:宋体;font-size:16px;"> </span></td><span style="font-family:宋体;"> </span><td width="213" valign="top" style="padding: 0cm 5.4pt; border: 0px rgb(0, 0, 0); border-image: none; width: 127.6pt; background-color: transparent;"><span style="font-family:宋体;font-size:16px;"> </span><p align="center" style="margin: 0cm 0cm 0pt; text-align: center; mso-yfti-cnfc: 64;"><span lang="EN-US" style="color: black; font-family: 'Times New Roman','serif'; mso-bidi-font-size: 10.5pt; mso-fareast-font-family: 仿宋; mso-themecolor: text1; mso-themeshade: 191;"><span style="font-size:16px;">weak</span></span></p><span style="font-family:宋体;font-size:16px;"> </span></td><span style="font-family:宋体;"> </span><td width="193" valign="top" style="padding: 0cm 5.4pt; border: 0px rgb(0, 0, 0); border-image: none; width: 115.9pt; background-color: transparent;"><span style="font-family:宋体;font-size:16px;"> </span><p align="center" style="margin: 0cm 0cm 0pt; text-align: center; mso-yfti-cnfc: 64;"><span lang="EN-US" style="color: black; font-family: 'Times New Roman','serif'; mso-bidi-font-size: 10.5pt; mso-fareast-font-family: 仿宋; mso-themecolor: text1; mso-themeshade: 191;"><span style="font-size:16px;">5</span></span></p><span style="font-family:宋体;font-size:16px;"> </span></td><span style="font-family:宋体;"> </span></tr><span style="font-family:宋体;"> </span><tr style="mso-yfti-irow: 3; mso-yfti-lastrow: yes;"><span style="font-family:宋体;"> </span><td width="304" valign="top" style="border-width: 0px 0px 1pt; border-style: none none solid; border-color: rgb(0, 0, 0) rgb(0, 0, 0) black; padding: 0cm 5.4pt; border-image: none; width: 182.6pt; background-color: transparent; mso-border-bottom-themecolor: text1;"><span style="font-family:宋体;font-size:16px;"> </span><p align="center" style="margin: 0cm 0cm 0pt; text-align: center; mso-yfti-cnfc: 4;"><span lang="EN-US" style="color: black; font-family: 'Times New Roman','serif'; mso-bidi-font-size: 10.5pt; mso-fareast-font-family: 仿宋; mso-themecolor: text1; mso-themeshade: 191; mso-bidi-font-weight: bold;"><span style="font-size:16px;">pBBR1-km-amp-cm-promoter11-nic</span></span></p><span style="font-family:宋体;font-size:16px;"> </span></td><span style="font-family:宋体;"> </span><td width="213" valign="top" style="border-width: 0px 0px 1pt; border-style: none none solid; border-color: rgb(0, 0, 0) rgb(0, 0, 0) black; padding: 0cm 5.4pt; border-image: none; width: 127.6pt; background-color: transparent; mso-border-bottom-themecolor: text1;"><span style="font-family:宋体;font-size:16px;"> </span><p align="center" style="margin: 0cm 0cm 0pt; text-align: center;"><span lang="EN-US" style="color: black; font-family: 'Times New Roman','serif'; mso-bidi-font-size: 10.5pt; mso-fareast-font-family: 仿宋; mso-themecolor: text1; mso-themeshade: 191;"><span style="font-size:16px;">strong</span></span></p><span style="font-family:宋体;font-size:16px;"> </span></td><span style="font-family:宋体;"> </span><td width="193" valign="top" style="border-width: 0px 0px 1pt; border-style: none none solid; border-color: rgb(0, 0, 0) rgb(0, 0, 0) black; padding: 0cm 5.4pt; border-image: none; width: 115.9pt; background-color: transparent; mso-border-bottom-themecolor: text1;"><span style="font-family:宋体;font-size:16px;"> </span><p align="center" style="margin: 0cm 0cm 0pt; text-align: center;"><span lang="EN-US" style="color: black; font-family: 'Times New Roman','serif'; mso-bidi-font-size: 10.5pt; mso-fareast-font-family: 仿宋; mso-themecolor: text1; mso-themeshade: 191;"><span style="font-size:16px;">1</span></span></p><span style="font-family:宋体;font-size:16px;"> </span></td><span style="font-family:宋体;"> </span></tr><span style="font-family:宋体;"></span></tbody></table><span style="font-family:宋体;font-size:16px;"></span> | ||
| + | |||
| + | |||
| + | |||
| + | <p>In order to test if our plasmids really work in <latin> P. fluorescences pf-5</latin>, we demonstrate our project in three levels, transcription, protein expression, and substrate degradation efficiency. </p> | ||
| + | <p>We used <i>SDS-PAGE</i>, to demonstrate the expression of <i>NicA2</i> (52.5 kDa). In the picture,we can see the clear expression of <i>NicA2 </i>in <latin> P. fluorescences pf-5</latin> containing plasmid with promoter. It can be qualitatively known that our promoter can initiate transcription normally, and the nicotine-degrading gene cluster has also been successfully heterologously expressed</p> | ||
| + | <p>As for transcription, we are going to use real-time <i>PCR</i> to compare the ability ofinitiating transcription of three promoters. </p> | ||
| + | In addition, we are comparing the degradation efficiency by<i> HPLC</i>. <i>NicA2</i> in can convert nicotine to pseudo-oxidation in a whole-cell reaction, and the rest of the gene cluster will convert pseudo-oxidation to 2,5-<i>DHP</i>. </p> | ||
| + | <p>However, due to time limitation, we were unable to complete the last two experiments, but we will do a supplementary explanation in our presentation. Please stay tuned. </p> | ||
Revision as of 10:17, 17 October 2018
Demonstrate
Based on our promoter library, we selected three promoters (promoter 4, promoter 5 and promoter11) of different intensities to regulate the expression of the key nicotine-degrading gene nicA2.We’ve constructed lasmid, pBBR1-km-amp-cm-promoter-nic, and electroporated it into our new chassis bacteria,
We found that there are only a few colonies of
| Plasmid in P. fluorescences pf-5 | Strength of promoter | CFU on LB plate |
| pBBR1-km-amp-cm-nic | none | >200 |
| pBBR1-km-amp-cm-promoter4-nic | normal | 5 |
| pBBR1-km-amp-cm-promoter5-nic | weak | 5 |
| pBBR1-km-amp-cm-promoter11-nic | strong | 1 |
In order to test if our plasmids really work in
We used SDS-PAGE, to demonstrate the expression of NicA2 (52.5 kDa). In the picture,we can see the clear expression of NicA2 in
As for transcription, we are going to use real-time PCR to compare the ability ofinitiating transcription of three promoters.
In addition, we are comparing the degradation efficiency by HPLC. NicA2 in can convert nicotine to pseudo-oxidation in a whole-cell reaction, and the rest of the gene cluster will convert pseudo-oxidation to 2,5-DHP.However, due to time limitation, we were unable to complete the last two experiments, but we will do a supplementary explanation in our presentation. Please stay tuned.