Team:OUC-China/Project Result

From 2014.igem.org

(Difference between revisions)
Line 193: Line 193:
         <h3 class="text-muted">mini plasmid & double plasmid system</h3>
         <h3 class="text-muted">mini plasmid & double plasmid system</h3>
         <h4 id="Mini_Plasmid">mini plasmid</h4>
         <h4 id="Mini_Plasmid">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>
+
         <p>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.</p>
         <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" />
-
         <h4>In the meantime, we ligate BBa_J04450 with BBa_J01003 to test the conjugated ability of BBa_J01003.</h4>
+
         <p>In the meantime, we ligate BBa_J04450 with BBa_J01003 to test the conjugated ability of BBa_J01003.</p>
         <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>deactivate the OriTRP4</h4>
+
       
-
        <h4>
+
         <h4>Deactivate the OriTRP4</h4>
-
            We attempt to deactivate the OriT of plasmid RP4 by homologous recombination,
+
            <p>We attempt to deactivate the OriT of plasmid RP4 by homologous recombination,
-
             unfortunately, the first experiment failed. And we have no time to do experiment before the deadline.But, it is theoretical to deactivate the OriT. We will continue our experiment after the competition.
+
             unfortunately, the first experiment failed. And we have no time to do experiment before the deadline.But, it is theoretical to deactivate the OriT. We will continue our experiment after the competition.</p>
-
        </h4>
+
     
-
 
+
         <p>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).</p>
-
         <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>
+

Revision as of 09:22, 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.

Deactivate the OriTRP4

We attempt to deactivate the OriT of plasmid RP4 by homologous recombination, unfortunately, the first experiment failed. And we have no time to do experiment before the deadline.But, it is theoretical to deactivate the OriT. We will continue our experiment after the competition.

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
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.

Result

Retrieved from "http://2014.igem.org/Team:OUC-China/Project_Result"