Difference between revisions of "Team:GZHS-United/Notebook"

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<img src="https://static.igem.org/mediawiki/2017/7/75/T--GZHS-United--notebook3.png" class="img100" ><br>
 
<img src="https://static.igem.org/mediawiki/2017/7/75/T--GZHS-United--notebook3.png" class="img100" ><br>
 
2. A series of Experiment were performed successfully.
 
2. A series of Experiment were performed successfully.
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</td>
 
</td>
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<td>qqq</td>
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<td>/</td>
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<td>qqq</td>
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<td>/</td>
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</tr>
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<tr>
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<td>7-14</td>
 +
<td>
 +
1.  Use colony PCR to select positive colonies among white colonies that we got from the blue-white selection. We set three groups of colonies (A,B and C)<br>
 +
A: mtx1<br>
 +
B: Cry4Ba (primers with enzyme digestion sets)<br>
 +
C: Cry4Ba (primers without digestion sets)<br>
 +
Each group selected 14 colonies. Operation was conducted in super-clean bench. So as to preserve the bacterial of colonies, we took 42 centrifuge tubes and added 750uL of cultural medium in each tube. We mix the bacterial in the cultural medium before adding into the reaction system for PCR.<br>
 +
2. Make up new LB cultural medium (Cultural medium we used yesterday may not be reliable).<br>
 +
3. Spreading positive control (BBa_I20270) and negative control (BBa_R0040) on the plate so as to get transformant, each duplicated for two groups.<br>
 +
</td>
 +
<td>
 +
Result of colony PCR.<br>
 +
<img src="https://static.igem.org/mediawiki/2017/9/90/T--GZHS-United--notebook4.jpg" class="img100" ><br>
 +
According to this result, we selected one colony from group B, one colony from group C and two colonies from group A for sequencing.<br>
 +
The outcome of cry4Ba is really good, but we may need more test to figure out the best temperature for annealing.
 +
</td>
 +
<td>
 +
1. Be careful not to contaminate the samples during the operation on super-clean bench.<br>
 +
2, Be aware that once leave the super-clean bench, the operator has to extinguish the spirit lamp
 +
</td>
 +
<td>The whole team.</td>
 +
</tr>
 +
<tr>
 +
<td>7-15</td>
 +
<td>
 +
1. Transform BBa_I20270 and BBa_R0040 transformants into LB solution and culture in flasks.<br>
 +
2.  Distribute the missions of human practice and wiki.
 +
</td>
 +
<td>
 +
No experimental outcome, but made many plans for future work.
 +
</td>
 +
<td></td>
 +
<td>The whole team.</td>
 +
</tr>
 +
<tr>
 +
<td>7-16</td>
 +
<td>
 +
1. We received the result of sequencing for cry4Ba and Mtx1. According to the result, we have succeeded in assembling cry4Ba into the cloning vector but failed in Mtx1, so we have to redo the extraction of genome DNA in Bs. <br>
 +
2. Extract plasmid from cry4Ba transformant.
 +
</td>
 +
<td>
 +
The gel of genome and plasmid extraction:<br>
 +
<img src="https://static.igem.org/mediawiki/2017/0/04/T--GZHS-United--notebook5.jpg" class="img100" ><br>
 +
Lane 1：Marker DL5000<br>
 +
Lane 2：Bs genome (failed)<br>
 +
Lane 3：Bs genome (failed)<br>
 +
Lane 4：Extraction of cloning vector with <br>
 +
cry4Ba (Low in cconcentration)<br>
 +
Lane 5：cry4Ba amplified by PCR (use the <br>
 +
extracted plasmid as template)<br>
 +
summary: Failed in extracting Bs genome again. The cloning vector actually assembled cry4Ba, but the result of gel is not clear due to the low concentration of plasmid.
 +
</td>
 +
<td>
 +
Note for plasmid extraction: <br>
 +
1, Ensure that the rotor of centrifuge is balanced before centrifuge.<br>
 +
2, Be aware of the time for reaction in each steps.<br>
 +
3，One of the important steps which can determine the outcome of the extraction is standing and demix. Be careful not to mix the precipitation of protein with DNA in the supernatant.<br>
 +
4, control the speed of centrifuge precisely.
 +
</td>
 +
<td>The whole team.</td>
 +
</tr>
 +
<tr>
 +
<td>07-30</td>
 +
<td>We got the eGFP sequences (BBa_K404316) from the iGEM registry and redesign nest-PCR primer.</td>
 +
<td>Figure 1</td>
 +
<td>No control, try again</td>
 +
<td>
 +
Control-1: BL21，<br>
 +
Control-2:BL21+ cry4Ba
 +
</td>
 +
</tr>
 +
<tr>
 +
<td>07-31</td>
 +
<td>
 +
1. Explore the best ratio of fodder<br>
 +
2. Feed larva<br>
 +
3. Test toxicity of bacterias
 +
</td>
 +
<td>
 +
1. 0.0125g agar+1mL H2O+0.02g cat food<br>
 +
2.  table1<br>
 +
3.  table2-result<br>
 +
<img src="https://static.igem.org/mediawiki/2017/3/31/T--GZHS-United--notebook6.png" class="img100" ><br>
 +
</td>
 +
<td></td>
 +
<td></td>
 
</tr>
 
</tr>
 
<tr>
 
<tr>

Revision as of 18:25, 28 October 2017

Notebook

Date objection result Analysis Solution
7-10 1. Extract the genomic DNA of Bt and Bs. 1. Only the genomic DNA of Bs were obtained. The Bt haven’t been actived. Culture Bt in LB to active it.
7-11 1. Extract the genomic DNA of Bt.
2. Acquire mtx gene form genomic DNA of Bs by PCR. 		1. DNA electrophoresis showed that the strip appeared at 150bp.
2. A peal DNA strip in agarose gel electrophoresis were observed. 		1. The fragment we extracted were RNA. 1. Use a more gentle method to broke the Gram-negative bacterium cell wall.
2. Optimize the PCR condition and try to use the diluted production as template.
7-12 1. Extract the genomic DNA of Bt.
2. Amplification of mtx1 gene. 		1. The genomic DNA of Bt were successfully obtained.

2. The mtx1 gene was successfully obtained. 		Boiling water-bath is a good method to broke the bacterium cell wall. /
7-13 1. Acquire Cry4Ba gene form genomic DNA of Bt by PCR.
2. Purification the PCR product of mtx1 .
3. Construction of cloning vector for direct ligation with mtx1’s purified product .
4. Transform the plasmid into E.coli. 		1. The Cry4Ba gene was successfully obtained.

2. A series of Experiment were performed successfully. 		/ /
7-14 1. Use colony PCR to select positive colonies among white colonies that we got from the blue-white selection. We set three groups of colonies (A,B and C)
A: mtx1
B: Cry4Ba (primers with enzyme digestion sets)
C: Cry4Ba (primers without digestion sets)
Each group selected 14 colonies. Operation was conducted in super-clean bench. So as to preserve the bacterial of colonies, we took 42 centrifuge tubes and added 750uL of cultural medium in each tube. We mix the bacterial in the cultural medium before adding into the reaction system for PCR.
2. Make up new LB cultural medium (Cultural medium we used yesterday may not be reliable).
3. Spreading positive control (BBa_I20270) and negative control (BBa_R0040) on the plate so as to get transformant, each duplicated for two groups.
 Result of colony PCR.

According to this result, we selected one colony from group B, one colony from group C and two colonies from group A for sequencing.
The outcome of cry4Ba is really good, but we may need more test to figure out the best temperature for annealing. 		1. Be careful not to contaminate the samples during the operation on super-clean bench.
2, Be aware that once leave the super-clean bench, the operator has to extinguish the spirit lamp 		The whole team.
7-15 1. Transform BBa_I20270 and BBa_R0040 transformants into LB solution and culture in flasks.
2. Distribute the missions of human practice and wiki. 		No experimental outcome, but made many plans for future work. The whole team.
7-16 1. We received the result of sequencing for cry4Ba and Mtx1. According to the result, we have succeeded in assembling cry4Ba into the cloning vector but failed in Mtx1, so we have to redo the extraction of genome DNA in Bs.
2. Extract plasmid from cry4Ba transformant. 		The gel of genome and plasmid extraction:

Lane 1：Marker DL5000
Lane 2：Bs genome (failed)
Lane 3：Bs genome (failed)
Lane 4：Extraction of cloning vector with
cry4Ba (Low in cconcentration)
Lane 5：cry4Ba amplified by PCR (use the
extracted plasmid as template)
summary: Failed in extracting Bs genome again. The cloning vector actually assembled cry4Ba, but the result of gel is not clear due to the low concentration of plasmid. 		Note for plasmid extraction:
1, Ensure that the rotor of centrifuge is balanced before centrifuge.
2, Be aware of the time for reaction in each steps.
3，One of the important steps which can determine the outcome of the extraction is standing and demix. Be careful not to mix the precipitation of protein with DNA in the supernatant.
4, control the speed of centrifuge precisely. 		The whole team.
07-30 We got the eGFP sequences (BBa_K404316) from the iGEM registry and redesign nest-PCR primer. Figure 1 No control, try again Control-1: BL21，
Control-2:BL21+ cry4Ba
07-31 1. Explore the best ratio of fodder
2. Feed larva
3. Test toxicity of bacterias 		1. 0.0125g agar+1mL H2O+0.02g cat food
2. table1
3. table2-result
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