"
Team:OUC-China/Project Result
From 2014.igem.org
(Difference between revisions)
| Line 192: | Line 192: | ||
<div id="mainContent" style="width:1024px;background-color:#d2e9fd;padding:10px;margin:0 auto"> | <div id="mainContent" style="width:1024px;background-color:#d2e9fd;padding:10px;margin:0 auto"> | ||
<h3 class="text-muted">mini plasmid & double plasmid system</h3> | <h3 class="text-muted">mini plasmid & double plasmid system</h3> | ||
| - | + | <h4>mini plasmid</h4> | |
<h4>We get the oriT region of plasmid RP4 by means of PCR, and ligate it with the reporter gene BBa_J04450, then a non-self-controllable conjugated part is constructed.</h4> | <h4>We get the oriT region of plasmid RP4 by means of PCR, and ligate it with the reporter gene BBa_J04450, then a non-self-controllable conjugated part is constructed.</h4> | ||
<img src="https://static.igem.org/mediawiki/2014/1/19/OUC-China_result_K1439001.jpg" /> | <img src="https://static.igem.org/mediawiki/2014/1/19/OUC-China_result_K1439001.jpg" /> | ||
| Line 198: | Line 198: | ||
<img src="https://static.igem.org/mediawiki/2014/a/a4/OUC-China_result_K1439002.jpg" /> | <img src="https://static.igem.org/mediawiki/2014/a/a4/OUC-China_result_K1439002.jpg" /> | ||
| - | + | <h4>RP4 OriT 位点的失活</h4> | |
<h4>这一部分我们利用同源重组去失活RP4的OriT位点，很遗憾我们第一次的实验失败了，在比赛之前我们没有时间做第二次实验。在比赛结束后，我们会继续这部分的实验。但利用同源重组去失活OriT位点在理论上是完全行的通的。</h4> | <h4>这一部分我们利用同源重组去失活RP4的OriT位点，很遗憾我们第一次的实验失败了，在比赛之前我们没有时间做第二次实验。在比赛结束后，我们会继续这部分的实验。但利用同源重组去失活OriT位点在理论上是完全行的通的。</h4> | ||
| + | <h4>We make two plasmids enter in E.coli HB101, one is plasmid RP4 (the oriT site was deactivated) and another is mini plasmid carrying oriTRP4-RFP or oriTR-RFP. Firstly, oriTRP4-RFP and oriTR-RFP transfer in competent cells HB101 (by inducing artificially, from Qiu Zhigang) respectively. Secondly, by means of conjugation, we make the plasmid RP4 get in HB101 which mentioned above and already has a mini plasmid. Thirdly, we screen cells with two plasmids by chloramphenicol and kanamycin. Eventually, we acquire HB101 strains carrying two plasmids (RP4, oriTRP4-RFP or oriTR-RFP).</h4> | ||
| + | |||
| + | <h3>Conjugation</h3> | ||
| + | <h4>Test1. Conjugation between E.coli HB101 and Top10</h4> | ||
| + | |||
| + | <h4>We did conjugation experiment between HB101 and Top10 to test the conjugation ability of OriTRP4 (BBa_K1439000). We got a special strain from other lab which is sensitive to Streptomycin, while Top10 has streptomycin resistance. In order to test BBa_K1439000 better, we ligated OriTRP4 with the report gene RFP. After conjugation, we picked the red colonies by LB medium with resistance.</h4> | ||
| + | <h4>We designed a orthogonal experiment, it is showed in the table. The first column shows the strains used in coating, and the first row shows the antibiotics added in medium. Besides, the table shows the result ideally. The second row proves that HB101, plasmid RP4 and the backbone pSB1C3 don’t contain streptomycin resistant gene. The third row proves that Top10 is sensitive to chloramphenicol. And the forth row proves that Top10 can receive the mini plasmid after conjugation.</h4> | ||
| + | Conjugation between E.coli HB101 and Top10 | ||
| + | {| class="wikitable" | ||
| + | |- | ||
| + | ! !! Conjugation (HB101 and Top10 mixed system) !! Top10(no plasmid) !! HB101(double plasmids system) | ||
| + | |- | ||
| + | | Streptomycin || White colony || White colony || No colony | ||
| + | |- | ||
| + | | chloramphenicol || Red colony || No colony || Red colony | ||
| + | |- | ||
| + | | chloramphenicol & streptomycin || Red colony || No colony || No colony | ||
| + | |} | ||
| + | Table.1 We constructed a new part BBa_K1439001 to test the conjugation result. BBa_K1439001 is used in conjugation experiments below. | ||
</div> | </div> | ||
<div id="childNavigator" style="width:180px"> | <div id="childNavigator" style="width:180px"> | ||
Revision as of 08:50, 17 October 2014
mini plasmid & double plasmid system
mini plasmid
We get the oriT region of plasmid RP4 by means of PCR, and ligate it with the reporter gene BBa_J04450, then a non-self-controllable conjugated part is constructed.
In the meantime, we ligate BBa_J04450 with BBa_J01003 to test the conjugated ability of BBa_J01003.
RP4 OriT 位点的失活
这一部分我们利用同源重组去失活RP4的OriT位点，很遗憾我们第一次的实验失败了，在比赛之前我们没有时间做第二次实验。在比赛结束后，我们会继续这部分的实验。但利用同源重组去失活OriT位点在理论上是完全行的通的。
We make two plasmids enter in E.coli HB101, one is plasmid RP4 (the oriT site was deactivated) and another is mini plasmid carrying oriTRP4-RFP or oriTR-RFP. Firstly, oriTRP4-RFP and oriTR-RFP transfer in competent cells HB101 (by inducing artificially, from Qiu Zhigang) respectively. Secondly, by means of conjugation, we make the plasmid RP4 get in HB101 which mentioned above and already has a mini plasmid. Thirdly, we screen cells with two plasmids by chloramphenicol and kanamycin. Eventually, we acquire HB101 strains carrying two plasmids (RP4, oriTRP4-RFP or oriTR-RFP).
Conjugation
Test1. Conjugation between E.coli HB101 and Top10
We did conjugation experiment between HB101 and Top10 to test the conjugation ability of OriTRP4 (BBa_K1439000). We got a special strain from other lab which is sensitive to Streptomycin, while Top10 has streptomycin resistance. In order to test BBa_K1439000 better, we ligated OriTRP4 with the report gene RFP. After conjugation, we picked the red colonies by LB medium with resistance.
We designed a orthogonal experiment, it is showed in the table. The first column shows the strains used in coating, and the first row shows the antibiotics added in medium. Besides, the table shows the result ideally. The second row proves that HB101, plasmid RP4 and the backbone pSB1C3 don’t contain streptomycin resistant gene. The third row proves that Top10 is sensitive to chloramphenicol. And the forth row proves that Top10 can receive the mini plasmid after conjugation.
Conjugation between E.coli HB101 and Top10 {| class="wikitable" |- ! !! Conjugation (HB101 and Top10 mixed system) !! Top10(no plasmid) !! HB101(double plasmids system) |- | Streptomycin || White colony || White colony || No colony |- | chloramphenicol || Red colony || No colony || Red colony |- | chloramphenicol & streptomycin || Red colony || No colony || No colony |} Table.1 We constructed a new part BBa_K1439001 to test the conjugation result. BBa_K1439001 is used in conjugation experiments below.
