Difference between revisions of "Team:NCKU Tainan/Notebook"

Line 210: Line 210:
 
                             <p class="pcontent">8/1</br> </p>
 
                             <p class="pcontent">8/1</br> </p>
 
                             <ul>
 
                             <ul>
−
                                 <li>Ligate the RbcL with PlacI.</li>
+
                                 <li>Ligate the RbcL with P<sub>LacI</sub>.</li>
−
                                 <li>Transformed the pSB1C3-P<sub>T7</sub>-RbcL and pSB1C3-PlacI-RbcL into DH5 alpha.</li>
+
                                 <li>Transformed the pSB1C3-P<sub>T7</sub>-RbcL and pSB1C3-P<sub>LacI</sub>-RbcL into DH5 alpha.</li>
 
                             </ul></br>
 
                             </ul></br>
  
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                             <p class="pcontent">8/1</br> </p>
 
                             <p class="pcontent">8/1</br> </p>
 
                             <ul>
 
                             <ul>
−
                                 <li>Ligated PRK with PlacI and PT7.</li>
+
                                 <li>Ligated PRK with P<sub>LacI</sub> and P<sub>T7</sub>.</li>
 
                                 <li>Transformed into DH5 alpha.
 
                                 <li>Transformed into DH5 alpha.
  
Line 290: Line 290:
 
                             <p class="pcontent">8/8</br> </p>
 
                             <p class="pcontent">8/8</br> </p>
 
                             <ul>
 
                             <ul>
−
                                 <li>Ligated PT7-CA with PLacI-PRK on pSB1C3</li>
+
                                 <li>Ligated P<sub>T7</sub>-CA with P<sub>LacI</sub>-PRK on pSB1C3</li>
 
                             </ul></br>
 
                             </ul></br>
  
 
                             <p class="pcontent">8/9</br> </p>
 
                             <p class="pcontent">8/9</br> </p>
 
                             <ul>
 
                             <ul>
−
                                 <li>Ligated PT7-RbcL with PT7-RbcXS on pSB1C3</li>
+
                                 <li>Ligated P<sub>T7</sub>>-RbcL with P<sub>T7</sub>-RbcXS on pSB1C3</li>
 
                             </ul></br>
 
                             </ul></br>
  
 
                             <p class="pcontent">8/28</br> </p>
 
                             <p class="pcontent">8/28</br> </p>
 
                             <ul>
 
                             <ul>
−
                                 <li>Inserted PT7-CA with PLacI-PRK into pSB3K3</li>
+
                                 <li>Inserted P<sub>T7</sub>-CA with P<sub>LacI</sub>-PRK into pSB3K3</li>
 
                             </ul></br>
 
                             </ul></br>
 
                         </div>
 
                         </div>
Line 332: Line 332:
 
                             <p class="pcontent">9/2</br> </p>
 
                             <p class="pcontent">9/2</br> </p>
 
                             <ul>
 
                             <ul>
−
                                 <li> Ran SDS-PAGE of pSB1C3-PT7-RbcL-PT7-RbcXS and pSB3K3-Plac-PRK-PT7-CA</li>
+
                                 <li> Ran SDS-PAGE of pSB1C3-P<sub>T7</sub>-RbcL-P<sub>T7</sub>-RbcXS and pSB3K3-Plac-PRK-P<sub>T7</sub>-CA</li>
 
                             </ul></br>
 
                             </ul></br>
  
 
                             <p class="pcontent">9/3</br> </p>
 
                             <p class="pcontent">9/3</br> </p>
 
                             <ul>
 
                             <ul>
−
                                 <li> Transformed pSB1C3-PT7-RbcL-PT7-RbcXS and pSB3K3-Plac-PRK-PT7-CA into W3110</li>
+
                                 <li> Transformed pSB1C3-P<sub>T7</sub>-RbcL-P<sub>T7</sub>-RbcXS and pSB3K3-Plac-PRK-P<sub>T7</sub>-CA into W3110</li>
 
                             </ul></br>
 
                             </ul></br>
  

Revision as of 16:13, 5 October 2018

Notebook





Construction of Pasr and PgadA


4/26~4/27

  • PCR Pasr and PgadA from E. coli MG1655 chromosome. Confirmed and extracted products by DNA gel.

4/28

  • Digested the pSB1C3-sfGFP plasmid. Ligase the plasmid with Pasr / PgadA and RiboJ by PCR. Confirmed by DNA gel electrophoresis. Transformed the gene in DH5 alpha and spread the bacteria on LB plates.

4/30~5/1

  • Confirmed the transformation by colony PCR ,enzyme digestion and DNA gel electrophoresis. Selected the correct colonies for sequencing and cultured them on LB plates.

5/9

  • Store the correct colonies in glycerol in -80°C.

6/1

  • Transformed each plasmid into BL21(DE3)





The pH sensor test


5/9

  • Prepared the M9 medium in different pH value.

5/11

  • Pre-cultured the DH5 alpha-pSB1C3 -Pasr-sfGFP strain.

5/12

  • Cultured the DH5 alpha-pSB1C3 -Pasr-sfGFP strain in M9 medium in different pH value. Measured the O.D. 600 value and fluorescence every hour.

5/18

  • Pre-cultured DH5 alpha-pSB1C3 –PgadA-RiboJ-sfGFP strain.
  • Prepared the M9 medium in different pH value.

5/19

  • Cultured the DH5 alpha-pSB1C3 –PgadA-RiboJ-sfGFP strain in M9 medium in different pH value. Measured the O.D. 600 value and fluorescence every hour.

6/2~6/3

  • Repeated the experiment on 5/11~5/12

7/29

  • Pre-culture the BL21(DE3)-pSB1C3 –Pasr-sfGFP strain

7/30

  • Cultured the BL21(DE3)-pSB1C3 –Pasr -sfGFP strain on 96 well plate in different pH value. Measure the fluorescence every 5 minutes in 30 minutes.

9/4

  • Pre-culture the BL21(DE3)-pSB1C3 –PgadA-RiboJ-sfGFP strain

9/5

  • Cultured the BL21(DE3)-pSB1C3 –PgadA-RiboJ-sfGFP strain strain on 96 well plate in different pH value. Measure the fluorescence every 5 minutes in 30 minutes.





Construction of CA


7/2

  • PCR IDT products (RbcL, RbcXS, PRK, CA). Confirmed by DNA gel electrophoresis. Extracted the gene from DNA gel.

7/5

  • PCR the CA. Confirmed by DNA gel electrophoresis and extracted the gene from DNA gel.

7/6

  • Digested and ligase the pSB1C3 plasmid and CA gene.

7/10~7/11

  • Repeated the PCR of CA, digestion, ligation.

7/12

  • Transformed the pSB1C3-PT7-CA into DH5 alpha.

7/19

  • Send the plasmid to sequencing





Construction of RbcL


7/2

  • PCR IDT products (RbcL, RbcXS, PRK, CA). Confirmed by DNA gel electrophoresis.
  • Extracted the gene from DNA gel.

7/11

  • PCR the RbcL with new primer.

7/20

  • PCR the RbcL with old and new primer.

7/21~24

  • Repeated the PCR of RbcL

8/1

  • Ligate the RbcL with PLacI.
  • Transformed the pSB1C3-PT7-RbcL and pSB1C3-PLacI-RbcL into DH5 alpha.

8/3

  • Confirmed the pSB1C3-PT7-RbcL construct by colony PCR.
  • Sent the gene to sequencing





Construction of RbcXS


7/2

  • Prepared the M9 medium in different pH value.

7/3~7/21

  • PCR IDT products (RbcL, RbcXS, PRK, CA). Confirmed by DNA gel electrophoresis.
  • Extracted the gene from DNA gel.

7/23

  • PCR normal/mutant RbcXS with old and new primer.

7/24

  • Confirmed by DNA gel.





Construction of PRK


7/2

  • PCR IDT products (RbcL, RbcXS, PRK, CA). Confirmed by DNA gel electrophoresis.
  • Extracted the gene from DNA gel.

7/3~8/2

  • PCR PRK.

8/1

  • Ligated PRK with PLacI and PT7.
  • Transformed into DH5 alpha.

8/2

  • Confirmed by colony PCR. Selected the colony with correct length and sent to sequencing.





Whole construction


8/8

  • Ligated PT7-CA with PLacI-PRK on pSB1C3

8/9

  • Ligated PT7>-RbcL with PT7-RbcXS on pSB1C3

8/28

  • Inserted PT7-CA with PLacI-PRK into pSB3K3





SDS PAGE, Functional test and Total solution


7/25~7/30, 8/4~8/5, 8/11~8/12

  • Ran SDS-PAGE of CA, normal-RbcXS

8/7, 8/9, 8/10

  • Ran SDS-PAGE of RbcL and PRK

8/16

  • Ran the SDS-PAGE of RbcL, RbcXS and PRK

8/18~8/19

  • Activity test of CA

9/2

  • Ran SDS-PAGE of pSB1C3-PT7-RbcL-PT7-RbcXS and pSB3K3-Plac-PRK-PT7-CA

9/3

  • Transformed pSB1C3-PT7-RbcL-PT7-RbcXS and pSB3K3-Plac-PRK-PT7-CA into W3110

9/5~9/17

  • Total solution.

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