Team:Aix-Marseille/Notebook 08

From 2014.igem.org

(Difference between revisions)
 
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                       </div>
                       </div>
                     </div>
                     </div>
 +
                  </li>
 +
                  <li>
 +
                    <p>Clean-up of PCR product number 82 and 83 from 8-8-14 (50µL)</p>
 +
                  </li>
 +
                  <li>
 +
                    <p>SOE reaction (= overlap PCR):</p>
 +
                    <p>
 +
                      Mix:
 +
                      <ul>
 +
                        <li>PCR product 82= 3µL</li>
 +
                        <li>PCR product 83 = 3µL</li>
 +
                        <li>dNTP 10mM = 2µL</li>
 +
                        <li>Q5 buffer = 8µL</li>
 +
                        <li>Q5 polymerase = 0,5µL</li>
 +
                        <li>H2O qsp 40µL</li>
 +
                      </ul>
 +
                    </p><br>
 +
                    <p>
 +
                      Program:
 +
                      <ol>
 +
                        <li>98°C 2’</li>
 +
                        <li>98°C 20”</li>
 +
                        <li>45°C 25”</li>
 +
                        <li>72°C 1’</li>
 +
                        <li>72°C 10’</li>
 +
                      </ol>
 +
                      Repeat the steps 3-4-5, 10 times.
 +
                    </p>
                   </li>
                   </li>
                 </ul>
                 </ul>
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                   <li>
                   <li>
                     <p>We realize day 4 of Lambda Red protocol on  W3110 sdaBC- strain CheA- KanR. strain.</p>
                     <p>We realize day 4 of Lambda Red protocol on  W3110 sdaBC- strain CheA- KanR. strain.</p>
 +
                  </li>
 +
                  <li>
 +
                    <p>PCR2: Second PCR round to amplification of serA from the SOE product (26-08-14) with primers 5 and 6, using the iGEM PCR protocol with Q5 polymerase. The temperature of annealing is 60°C</p>
 +
                    <div class="media notes-media">
 +
                      <img class="media-object img-rounded pull-right" src="https://static.igem.org/mediawiki/2014/8/80/AMU_Team-serA-0827.png" style="height:350px">
 +
                      <div class="media-body" style="padding-left: 10px;">
 +
                        <p>Result: the amplification of serA is good. Clean-up of serA (PCR2) and elution with 45µL of water.</p>
 +
                      </div>
 +
                    </div>
 +
                  </li>
 +
                  <li>
 +
                    <p>Digestion of pSB1A3 and pSB1C3 during 1h30 at 37°C. Then, inactivation of enzymes during 20’ at 80°C</p>
 +
                    <p>
 +
                      Mix:
 +
                      <ul>
 +
                        <li>10µL of plasmid</li>
 +
                        <li>1µL EcoRI</li>
 +
                        <li>1µL PstI</li>
 +
                        <li>5µL Buffer 2.1</li>
 +
                        <li>33 µL H2O</li>
 +
                      </ul>
 +
                    </p>
 +
                  </li>
 +
                  <li>
 +
                    <p>Digestion of the insert (serA) during 1h at 37°C. Then inactivation of enzymes during 20’ at 80°C</p>
 +
                    <p>
 +
                      Mix:
 +
                      <ul>
 +
                        <li>20µL of serA</li>
 +
                        <li>1µL EcoRI</li>
 +
                        <li>1µL PstI</li>
 +
                        <li>5µL Buffer 2.1</li>
 +
                        <li>13 µL H2O</li>
 +
                      </ul>
 +
                    </p>
 +
                  </li>
 +
                  <li>
 +
                    <p>NEB ligation:</p>
 +
                    <p>
 +
                      <ul>
 +
                        <li>2µL of digested plasmid </li>
 +
                        <li>2µL of digested serA insert</li>
 +
                        <li>2µL ligase buffer</li>
 +
                        <li>1µL ligase (NEB)</li>
 +
                        <li>13 µL H2O</li>
 +
                      </ul>
 +
                    </p><br>
 +
                    <p>Incubation overnight at 16°C</p>
                   </li>
                   </li>
                 </ul>
                 </ul>
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                   <li>
                   <li>
                     <p>We realize day 5 of Lambda Red protocol on W3110 sdaBC- CheA- KanR strain. </p>
                     <p>We realize day 5 of Lambda Red protocol on W3110 sdaBC- CheA- KanR strain. </p>
 +
                  </li>
 +
                  <li>
 +
                    <p>Transformation of 90µL of DH5α super competent cells with 10µL of ligation (pSB1C3-SerA or pSB1A2-SerA). The selective medium is LB + Ampicillin (Ap) 100µg/mL or LB + Chloramphenicol (Cm) 30µg/mL</p>
 +
                    <div class="media notes-media">
 +
                      <img class="media-object img-rounded pull-right" src="https://static.igem.org/mediawiki/2014/6/6e/AMU_Team-serA-0828.png" style="height:350px">
 +
                      <div class="media-body" style="padding-left: 10px;">
 +
                        <p>pSB1C3-SerA-1 seemed good. It was sent to sequencing. It’s OK</p>
 +
                      </div>
 +
                    </div>
                   </li>
                   </li>
                 </ul>
                 </ul>

Latest revision as of 21:55, 13 October 2014

Notebook: August



Week 5 : 07/28/2014 ➡ 08/03/2014

  • Several DNA fragments were amplified thanks to iGEM PCR protocol (Q5 polymerase).

    # DNA Template Primers Numbers Primers Names Weight of amplified DNA
    2 Genomic DNA of W3110 6 - 7 serA_mut_ A849G_up
    serA_tronq_SufRFC23_down    		 171 bp
    3 Genomic DNA of W3110 5 - 8 serA_PrefRFC10RBS_up
    sera_mut_A849_down    		 861 bp
    6 Genomic DNA of W3110 10 - 13 cheA_SufRFC23_down
    cheA_mut_T1290C_up    		 726 bp
    8 PiGEM_02.05 16 - 18 relA_mut_C1366_down
    relA_sans_stop_SufRFC23_down    		 882 bp
    9 PiGEM_02.05 16 - 19 relA_mut_C1366_down
    relA_2_stop_SufRFC23_down    		 885 bp
    10 PiGEM_02.06 20 - 24 Mesh_1_mut_A255T_up
    Mesh_1_RFC10-RBS_up    		 267 bp
    11 PiGEM_02.06 21 - 22 Mesh_1_mut_A255T_down
    Mesh_1_mut_A343G_up    		 120 bp
    12 PiGEM_02.06 23 - 25 Mesh_1_mut_A343G_down
    Mesh1_sans_stop_SufRFC23_down    		 192 bp
    13 PiGEM_02.06 23 - 26 Mesh_1_mut_A343G_down
    Mesh_1_2_stop_SufRFC23_down    		 192 bp

    The efficiency of the amplification was tested thanks to agarose gel electrophoresis. Results are presented below.

    All DNA fragments seemed to be correctly amplified.

  • PCR products were purified thanks to Macherey Nagel PCR Clean up Kit.

  • The DNA concentration of PCR products was tested thanks to the Nanodrop. Results are presented below.

    # Concentration of DNA (ng/µL)
    2 52.28
    3 44.86
    6 49.93
    8 73.38
    9 76.77
    10 57.00
    11 39.87
    12 50.88
    13 62.42
  • PCR products number 6, 8 and 9 were stocked at -20°C whereas PCR products number 2, 3, 10, 11, 12 and 13 were used to realize SLIC protocol. This protocol permits to assemble all parts of SerA and Mesh1 genes with their directed mutation. The destination plasmid DNA used is the backbone pSB1A3 linearized plasmid. DH5α supercompetent bacteria were transformed with SLIC products and spread over Amp dish.
  • Several plasmids were purified thanks to Macherey Nagel MiniPrep Kit :
    • Brick 200 (3)
    • Brick 200 (4)
    • Brick 200 (5)
    • Brick 200 (6)
  • The efficiency of the purification was tested thanks to agarose gel electrophoresis. Results are presented following.
    Brick 200 (6) well did not present any DNA fragment. Other wells contain one or two fragments of DNA, so Brick 200 (3), 200 (4) and 200 (5) were successfully purified.

  • Several control PCR were realized to verify if SLIC worked. Taq polymerase was used. Three clones of each SLIC were tested. They were also pricked up on Amp dish.

    # DNA Template Primers Numbers Primers Names Weight of amplified DNA
    S1 Clone 1 of SLIC SerA 5 - 6 serA_PrefRFC10RBS_up
    serA_tronq_SufRFC23_down    		 1032 bp
    S2 Clone 2 of SLIC SerA 5 - 6 serA_PrefRFC10RBS_up
    serA_tronq_SufRFC23_down    		 1032 bp
    S3 Clone 3 of SLIC SerA 5 - 6 serA_PrefRFC10RBS_up
    serA_tronq_SufRFC23_down    		 1032 bp
    S4 Clone 4 of SLIC Mesh1 without STOP 24 - 25 Mesh_1_RFC10-RBS_up
    relA_sans_stop_SufRFC23_down    		 579 bp
    S5 Clone 5 of SLIC Mesh1 without STOP 24 - 25 Mesh_1_RFC10-RBS_up
    relA_sans_stop_SufRFC23_down    		 579 bp
    S6 Clone 6 of SLIC Mesh1 without STOP 24 - 25 Mesh_1_RFC10-RBS_up
    relA_sans_stop_SufRFC23_down    		 579 bp
    S7 Clone 7 of SLIC Mesh1 with STOP 24 - 26 Mesh_1_RFC10-RBS_up
    Mesh_1_2_stop_SufRFC23_down    		 582 bp
    S8 Clone 8 of SLIC Mesh1 with STOP 24 - 26 Mesh_1_RFC10-RBS_up
    Mesh_1_2_stop_SufRFC23_down    		 582 bp
    S9 Clone 9 of SLIC Mesh1 with STOP 24 - 26 Mesh_1_RFC10-RBS_up
    Mesh_1_2_stop_SufRFC23_down    		 582 bp

  • Several DNA fragments were amplified thanks to iGEM PCR protocol (Q5 polymerase).

    # DNA Template Primers Numbers Primers Names Weight of amplified DNA
    1 pKD4 plasmid 1 - 2 sdaCsdaB_mut_pKD4_up
    sdaCsdaB_ mut_pKD4_down    		 1032 bp
    4 pKD4 plasmid 3 - 4 cheA_mut_pKD4_up
    cheA_mut_pKD4_down    		 1032 bp
    5 Genomic DNA of W3110 9 - 14 cheA_PrefRFC23_up
    cheA_mut_T1290C_down    		 1032 bp
    7 Genomic DNA of W3110 15 - 17 relA_mut_C1366G_up
    relA_RFC10-RBS_up    		 579 bp
    PCR product 1 and 4 were correctly amplified. Thus, they were purified thanks to Macherey Nagel PCR Clean up Kit. PCR product 5 and 7 seemed to be compound with too many different DNA fragments. That's why, a new amplification by PCR was made overnight (iGEM PCR protocol Q5 polymerase).
  • Realization of "Day 2" of lambda red pKOBEG protocol. W3110 with active recombinase was electroporated by PCR product 1 (sdaCsdaB) and 4 (cheA). Transformed bacteria were spread over a Kan dish.

  • The efficiency of the purification of PCR product number S1 to S9 was tested thanks to agarose gel electrophoresis. At the same time, the efficiency of the amplification of the beginning sequences of CheA and RelA (PCR product 5 and 7) was tested thanks to agarose gel electrophoresis. Results are presented below.

    PCR products 5 and 7 were good for SLIC. Clone 1 and 2 (serA), clone 4 and 6 (Mesh1 without STOP) and clone 7 and 8 (Mesh1 with STOP) were put in culture in LB with Amp.
  • The DNA concentration of PCR products 5 and 7 was tested thanks to the Nanodrop. Results are presented below.
    # Concentration of DNA (ng/µL)
    5 29.78
    7 85.51
  • PCR products number 5, 6, 7, 8 and 9 were used to realize SLIC protocol. This protocol permits to assemble all parts of CheA (5 and 6) and RelA (7 and 8 without STOP, 7 and 9 with STOP) genes with their directed mutation. The destination plasmid DNA used is the backbone pSB1A3 linearized plasmid. DH5α supercompetent bacteria were transformed with SLIC products and spread over Amp dish.
  • SLIC assembly of RelA was a success but SLIC assembly of CheA failed. That's why, a new SLIC assembly of CheA with PCR products 5 and 6 was made. The destination plasmid DNA used is the backbone pSB1A3 linearized plasmid. DH5α supercompetent bacteria were transformed with SLIC products and spread over Amp dish.
  • Realization of "Day 2" of lambda red pKOBEG protocol a second time. PCR product 1 and 4 were purified a second time. The efficiency of the purification was tested thanks to agarose gel electrophoresis. Results are presented below. W3110 with active recombinase was electroporated by PCR product 1 (sdaCsdaB) and 4 (cheA). Transformed bacteria were spread over a Kan dish.

  • Several control PCR were realized to verify whether RelA SLIC worked. Taq polymerase was used. Three clones of each SLIC were tested. They were also pricked up on Amp dish.

    # DNA Template Primers Numbers Primers Names Weight of amplified DNA
    S22 Clone 22 of SLIC RelA without Stop 17 - 18 relA_RFC10-RBS_up
    relA_sans_stop_SufRFC23_down    		 2259 bp
    S23 Clone 22 of SLIC RelA without Stop 17 - 18 relA_RFC10-RBS_up
    relA_sans_stop_SufRFC23_down    		 2259 bp
    S24 Clone 22 of SLIC RelA without Stop 17 - 18 relA_RFC10-RBS_up
    relA_sans_stop_SufRFC23_down    		 2259 bp
    S25 Clone 25 of SLIC RelA with STOP 17 - 19 relA_RFC10-RBS_up
    relA_2_stop_SufRFC23_down    		 2262 bp
    S26 Clone 26 of SLIC RelA with STOP 17 - 19 relA_RFC10-RBS_up
    relA_2_stop_SufRFC23_down    		 2262 bp
    S27 Clone 27 of SLIC RelA with STOP 17 - 19 relA_RFC10-RBS_up
    relA_2_stop_SufRFC23_down    		 2262 bp

    The efficiency of the amplification was tested thanks to agarose gel electrophoresis. Results are presented below.

    PCR products 1 and 4 were correctly amplified. Thus, they were purified thanks to Macherey Nagel PCR Clean up Kit. Transplanting of S22 to S27 failed, those clones were gave up.
  • Several plasmids were purified thanks to Macherey Nagel MiniPrep Kit :
    • Clone 1 and 2 (SerA) ⇒ PiGEM_02.10 and PiGEM_02.11
    • Clone 4 and 6 (Mesh1 without STOP) ⇒ PiGEM_02.12 and PiGEM_02.13
    • Clone 7 and 8 (Mesh1 with STOP) ⇒ PiGEM_02.14 and PiGEM_02.15
  • SLIC assembly of CheA failed. That's why, a new SLIC assembly of CheA with PCR products 5 and 6 was made. The destination plasmid DNA used is the backbone pSB1A3 linearized plasmid. DH5α supercompetent bacteria were transformed with SLIC products and spread over Amp dish.

  • Several control PCR were realized to verify whether RelA SLIC worked. Taq polymerase was used. Three clones of each SLIC were tested. They were also pricked up on Amp dish.

    # DNA Template Primers Numbers Primers Names Weight of amplified DNA
    S28 Clone 28 of SLIC RelA without Stop 17 - 18 relA_RFC10-RBS_up
    relA_sans_stop_SufRFC23_down    		 2259 bp
    S29 Clone 28 of SLIC RelA without Stop 17 - 18 relA_RFC10-RBS_up
    relA_sans_stop_SufRFC23_down    		 2259 bp
    S30 Clone 28 of SLIC RelA without Stop 17 - 18 relA_RFC10-RBS_up
    relA_sans_stop_SufRFC23_down    		 2259 bp
    S31 Clone 31 of SLIC RelA with STOP 17 - 19 relA_RFC10-RBS_up
    relA_2_stop_SufRFC23_down    		 2262 bp
    S32 Clone 31 of SLIC RelA with STOP 17 - 19 relA_RFC10-RBS_up
    relA_2_stop_SufRFC23_down    		 2262 bp
    S33 Clone 31 of SLIC RelA with STOP 17 - 19 relA_RFC10-RBS_up
    relA_2_stop_SufRFC23_down    		 2262 bp

    The efficiency of the amplification was tested thanks to agarose gel electrophoresis. Results are presented below.

    PCR product S28, S29, S31, S32 and S33 were good for SLIC. Clones 28 and 29 (RelA without STOP) and clones 31, 32 and 33 (RelA with STOP) were put in culture in LB with Amp.
  • Realization of "Day 2" of lambda red pKOBEG protocol for the third time. PCR products 1 and 4 were purified a third time. pKOBEG electrocompetent were prepared a third time. W3110 with active recombinase was electroporated with PCR products 1 (sdaCsdaB) and 4 (cheA). Transformed bacteria were spread over a Kan dish.
  • The efficiency of the purification of PiGEM_02.10, PiGEM_02.11, PiGEM_02.12, PiGEM_02.13, PiGEM_02.14 and PiGEM_02.15 was tested thanks to agarose gel electrophoresis. Results are presented below.
    These plasmids seemed to be in low quantity. The DNA concentration of plasmids was tested thanks to the Nanodrop. Results are presented following
    # Concentration of DNA (ng/µL)
    PiGEM_02.10 30.6
    PiGEM_02.11 37.8
    PiGEM_02.12 12.2
    PiGEM_02.13 15.11
    PiGEM_02.14 14.11
    PiGEM_02.15 19.22
  • The W3110 with active recombinase was electroporated with PCR products 1 (sdaCsdaB) and 4 (cheA). Transformed bacteria were spread over a Kan dish.
  • Bacteria transformed with PCR products 1 (sdaCsdaB) and 4 (cheA) put in culture on Kan dish on the 08/01/14 showed colonies.
  • 5 different colonies of SdaCsdaB and CheA mutants were spread over Kan dish and Cm30 dish in order to verify whether the pKOBEG plasmid was eliminated (by the previously heat shock) or not as we wanted to, and whether the transformed bacteria contained the kanamycin resistance. The bacteria we want must show colonies on Kan dish and no colonies on Cm 30 dish at the same time.
Protocols used : ...

Week 6 : 08/04/2014 ➡ 08/10/2014

  • Several plasmids were purified thanks to Macherey Nagel MiniPrep Kit :
    • SLIC RelA without STOP PCR product 7/8 Clone 28 and 29 ⇒ PiGEM_02.16 and PiGEM_02.17
    • SLIC RelA with STOP PCR product 7/8 Clone 31, 32 and 33 ⇒ PiGEM_02.18, PiGEM_02.19 and PiGEM_02.20
    All plasmids seemed to be correctly purified. No DNA contamination was noticeable.

  • Several DNA fragments were amplified thanks to iGEM PCR protocol (Q5 polymerase).

    # DNA Template Primers Numbers Primers Names Weight of amplified DNA
    14 Genomic DNA of W3110 33 - 34 CusR_iProm-up
    CusR_iProm-down    		 117 bp
    15 PiGEM_01.04 37 - 38 BBa_E1010-up
    BBa_E1010_NoStop-down    		 708 bp
    16 PiGEM_02.03 39 - 40 BBa_E0040-up
    BBa_E0040_NoStop-down    		 714 bp
    17 PiGEM_01.09 45 - 46 (SH3)4-up
    (SH3)4-down    		 932 bp
    18 PiGEM_02.08 47 - 48 cusR_L_LZA-up
    cusR_L_LZA_down    		 857 bp
    19 PiGEM_02.07 49 - 50 SH3pep_L_LZA-up
    SH3pep_L_LZA-down    		 192 bp

    The efficiency of the amplification was tested thanks to agarose gel electrophoresis. Results are presented below.

    Fragments 19, 15, 16 and 18 seemed to be correctly amplified. They were going to be cloned in iGEM plasmids. Unfortunately, fragments 14 and 17 were not amplified. Another PCR had to be made.

  • Several control PCR were realized to verify whether SLIC, bricks 200 and bricks 202 worked. Taq polymerase was used.

    # DNA Template Primers Numbers Primers Names Weight of amplified DNA
    20 Clone 1 of SLIC SerA 43 - 44 Prefix_up
    Suffix_down    		 1032 bp
    21 Clone 2 of SLIC SerA 43 - 44 Prefix_up
    Suffix_down    		 1032 bp
    22 Clone 28 of SLIC SerA 43 - 44 Prefix_up
    Suffix_down    		 2260 bp
    23 Clone 29 of SLIC SerA 43 - 44 Prefix_up
    Suffix_down    		 2260 bp
    24 Clone 31 of SLIC SerA 43 - 44 Prefix_up
    Suffix_down    		 2260 bp
    25 Clone 32 of SLIC SerA 43 - 44 Prefix_up
    Suffix_down    		 2260 bp
    26 Clone 33 of SLIC SerA 43 - 44 Prefix_up
    Suffix_down    		 2260 bp
    27 Clone 34 of SLIC CheA 43 - 44 Prefix_up
    Suffix_down    		 2432 bp
    28 Clone 35 of SLIC CheA 43 - 44 Prefix_up
    Suffix_down    		 2432 bp
    29 Clone 36 of SLIC CheA 43 - 44 Prefix_up
    Suffix_down    		 2432 bp
    30 Clone 37 of SLIC CheA 43 - 44 Prefix_up
    Suffix_down    		 2432 bp
    31 Clone 38 of SLIC CheA 43 - 44 Prefix_up
    Suffix_down    		 2432 bp
    32 Clone 39 of SLIC CheA 43 - 44 Prefix_up
    Suffix_down    		 2432 bp
    33 Brick 202 (1) 43 - 44 Prefix_up
    Suffix_down    		 730 bp
    34 Brick 202 (2) 43 - 44 Prefix_up
    Suffix_down    		 769 bp
    35 Brick 200 (3) 43 - 44 Prefix_up
    Suffix_down    		 769 bp
    36 Brick 200 (4) 43 - 44 Prefix_up
    Suffix_down    		 769 bp
    37 Brick 200 (5) 43 - 44 Prefix_up
    Suffix_down    		 769 bp

    The efficiency of the amplification was tested thanks to agarose gel electrophoresis. Results are presented below.

    No fragment seemed to be correctly amplified. SLIC were going to be done again with another protocol. Bricks 200 and 202 were also going to be remade.

  • PiGEM_01.03 (pSB1C3, BBa_B0033) was digested with XbaI and SpeI to be used for stability label cloning.

    The efficiency of the digestion was tested thanks to agarose gel electrophoresis. Results are presented below.

    PSB1C3 of PiGEM_01.03 seemed to be correctly digested.

  • PiGEM_01.03 (pSB1C3, BBa_B0033) was digested with EcoRI and SpeI to be used for PCR products 15 and 16 cloning. PCR products 15 and 16 (respectively RFP without STOP codon and GFP without STOP codon) were digested by EcoRI and SpeI. Then, PCR products 15 and 16 were leagued in pSB1C3 digested. DH5 alpha supercompetent were transformed with these ligation products and spread on Cm dish.

  • PiGEM_02.27 (pSB1A2, BBa_J04450) was digested with EcoRI and PstI to be used for PCR products 18 and 19 cloning. PCR products 18 and 19 (respectively cusR-L-LZa and SH3pep-L-LZa) were digested by EcoRI and PstI. Then, PCR products 18 and 19 were leagued in pSB1A2 digested. DH5 alpha supercompetent were transformed with these ligation products and spread on Amp dish.

  • Because PCR 17 did not work, (SH3)4 was amplified again thanks to iGEM PCR protocol (Q5 polymerase).

    # DNA Template Primers Numbers Primers Names Weight of amplified DNA
    38 PiGEM_02.07 49 - 50 SH3pep_L_LZA-up
    SH3pep_L_LZA-down    		 192 bp

    The efficiency of the amplification was tested thanks to agarose gel electrophoresis. Results are presented below.

    (SH3)4 amplification did not work again. A specific treatment of this cloning was needed.

  • PiGEM_01.03 (pSB1C3, BBa_B0033) had be digested with XbaI and SpeI the day before to be used for stability label cloning. A gel extract was made this day to collect only the backbone. The backbone obtained was digested by PstI because XbaI and SpeI are compatible restriction sites. Stability labels were assembled by annealing.

    Primers numbers Primers names Brick created
    27 - 28 BBa_K1051206-up
    BBa_K1051206-down    		 BBa_K1051206
    29 -30 BBa_K1051207-up
    BBa_K1051207-down    		 BBa_K1051207
    31 -32 BBa_K1051208-up
    BBa_K1051208-down    		 BBa_K1051208

    Each annealing oligonucleotides were leagued in pSB1C3 digested XSP. DH5 alpha supercompetent were transformed with these ligation products and spread on Cm dish.

  • Several plasmids were purified thanks to Macherey Nagel MiniPrep Kit :

    SLIC CheA PCR product 5/6 Clone 34, 35, 36, 37, 38 and 39 ⇒ PiGEM_02.21, PiGEM_02.22, PiGEM_02.23, PiGEM_02.24, PiGEM_02.25, PiGEM_02.26

  • Several control PCR were realized to verify one more time whether SLIC worked. Besides, several control PCR were realized to verify whether bricks 200 and bricks 202 worked. Taq polymerase was used.

    # DNA Template Primers Numbers Primers Names Weight of amplified DNA
    39 PiGEM_02.10 5 - 6 serA_PrefRFC10RBS_up
    serA_tronq_SufRFC23_down    		 1032 bp
    40 PiGEM_02.11 5 - 6 serA_PrefRFC10RBS_up
    serA_tronq_SufRFC23_down    		 1032 bp
    41 PiGEM_02.16 17 - 18 relA_RFC10-RBS_up
    relA_sans_stop_SufRFC23_down    		 2259 bp
    42 PiGEM_02.17 17 - 18 relA_RFC10-RBS_up
    relA_sans_stop_SufRFC23_down    		 2259 bp
    43 PiGEM_02.18 17 - 19 relA_RFC10-RBS_up
    relA_2_stop_SufRFC23_down    		 2259 bp
    44 PiGEM_02.19 17 - 19 relA_RFC10-RBS_up
    relA_2_stop_SufRFC23_down    		 2259 bp
    45 PiGEM_02.20 9 - 10 cheA_PrefRFC23_up
    cheA_SufRFC23_down    		 2432 bp
    46 PiGEM_02.21 9 - 10 cheA_PrefRFC23_up
    cheA_SufRFC23_down    		 2432 bp
    47 PiGEM_02.22 9 - 10 cheA_PrefRFC23_up
    cheA_SufRFC23_down    		 2432 bp
    48 PiGEM_02.23 9 - 10 cheA_PrefRFC23_up
    cheA_SufRFC23_down    		 2432 bp
    49 PiGEM_02.24 9 - 10 cheA_PrefRFC23_up
    cheA_SufRFC23_down    		 2432 bp
    50 PiGEM_02.25 9 - 10 cheA_PrefRFC23_up
    cheA_SufRFC23_down    		 2432 bp
    51 PiGEM_02.26 9 - 10 cheA_PrefRFC23_up
    cheA_SufRFC23_down    		 2432 bp
    52 PiGEM_01.04 43 - 44 Prefix_up
    Suffix_down    		 706 bp
    53 PiGEM_02.10 43 - 44 Prefix_up
    Suffix_down    		 1032 bp
    54 Brick 202 (1) 40 - 51 BBa_E0040_NoStop-down
    BBa_B0034-up    		 730 bp
    55 Brick 202 (2) 40 - 51 BBa_E0040_NoStop-down
    BBa_B0034-up    		 730 bp
    56 Brick 202 (3) 40 - 51 BBa_E0040_NoStop-down
    BBa_B0034-up    		 730 bp
    57 Brick 202 (4) 40 - 51 BBa_E0040_NoStop-down
    BBa_B0034-up    		 730 bp
    58 Brick 200 (3) 38 - 51 BBa_E0040_NoStop-down
    BBa_B0034-up    		 711 bp
    59 Brick 200 (4) 38 - 51 BBa_E0040_NoStop-down
    BBa_B0034-up    		 730 bp
    60 Brick 200 (6) 38 - 51 BBa_E0040_NoStop-down
    BBa_B0034-up    		 711 bp

  • Because PCR 14 did not work, cusR promoter was amplified again thanks to iGEM PCR protocol (Q5 polymerase).

    # DNA Template Primers Numbers Primers Names Weight of amplified DNA
    62 Genomic DNA of W3110 33 - 34 CusR_iProm-up
    CusR_iProm-down    		 117 bp

  • The efficiency of the amplification was tested thanks to agarose gel electrophoresis. Results are presented below.

    SLIC had to be made again. Bricks 200 were correctly amplified but bricks 202 were not. CusR promoter was not amplified.

  • Because PCR 62 did not work, cusR promoter was amplified again thanks to iGEM PCR protocol (Q5 polymerase).

    # DNA Template Primers Numbers Primers Names Weight of amplified DNA
    63 Genomic DNA of W3110 33 - 34 CusR_jProm-up
    CusR_jProm-down    		 117 bp

  • Several control PCR were realized to verify whether bricks 200 worked. Taq polymerase was used.

    # DNA Template Primers Numbers Primers Names Weight of amplified DNA
    64 Brick 202 (5) 40 - 51 BBa_E0040_NoStop-down
    BBa_B0034-up    		 730 bp
    65 Brick 202 (6) 40 - 51 BBa_E0040_NoStop-down
    BBa_B0034-up    		 730 bp
    646 Brick 202 (7) 40 - 51 BBa_E0040_NoStop-down
    BBa_B0034-up    		 730 bp
    67 Brick 202 (8) 40 - 51 BBa_E0040_NoStop-down
    BBa_B0034-up    		 730 bp
    68 Brick 202 (9) 40 - 51 BBa_E0040_NoStop-down
    BBa_B0034-up    		 730 bp
    69 Brick 202 (10) 40 - 51 BBa_E0040_NoStop-down
    BBa_B0034-up    		 730 bp
    70 Brick 202 (11) 40 - 51 BBa_E0040_NoStop-down
    BBa_B0034-up    		 730 bp

  • A gel extract of PCR product 38 (SH3)4 was made.

  • Several control PCR were realized to verify whether RFP without STOP codon and GFP without STOP codon worked. Taq polymerase was used.

    # DNA Template Primers Numbers Primers Names Weight of amplified DNA
    72 RFP without STOP codon
    Clone 40    		 37 - 38 BBa_E1010-up
    BBa_E1010_NoStop-down    		 708 bp
    73 RFP without STOP codon
    Clone 41    		 37 - 38 BBa_E1010-up
    BBa_E1010_NoStop-down    		 708 bp
    74 RFP without STOP codon
    Clone 42    		 37 - 38 BBa_E1010-up
    BBa_E1010_NoStop-down    		 708 bp
    75 RFP without STOP codon
    Clone 43    		 37 - 38 BBa_E1010-up
    BBa_E1010_NoStop-down    		 708 bp
    76 RFP without STOP codon
    Clone 44    		 37 - 38 BBa_E1010-up
    BBa_E1010_NoStop-down    		 708 bp
    77 GFP without STOP codon
    Clone 45    		 39 - 40 BBa_E0040-up
    BBa_E0040_NoStop-down    		 714 bp
    78 GFP without STOP codon
    Clone 46    		 39 - 40 BBa_E0040-up
    BBa_E0040_NoStop-down    		 714 bp
    79 GFP without STOP codon
    Clone 47    		 39 - 40 BBa_E0040-up
    BBa_E0040_NoStop-down    		 714 bp
    80 GFP without STOP codon
    Clone 48    		 39 - 40 BBa_E0040-up
    BBa_E0040_NoStop-down    		 714 bp
    81 GFP without STOP codon
    Clone 49    		 39 - 40 BBa_E0040-up
    BBa_E0040_NoStop-down    		 714 bp

  • The efficiency of the purification was tested thanks to agarose gel electrophoresis. Results are presented following.

    PCR product 63 was correctly amplified. PiGEM_02.27 (pSB1A2, BBa_J04450) was digested with EcoRI and Pst. PCR product 63 (cusR promoter) was digested by EcoRI and PstI. Then, PCR product 63 was leagued in pSB1A2 digested. DH5 alpha supercompetent were transformed with this ligation product and spread on Amp dish.

  • To confirm the cloning of stability label and SLIC, each plasmid containing constructions were digested with specific restriction enzymes.

    Plasmid Numbers Plasmid Names Digestion Weight of expected bands if constructions are correct
    EcoRI PstI SpeI XbaI
    PiGEM_01.10 BBa_K1051207
    Clone St1    		 X X
    • 2082 pb
    • 80 pb
    PiGEM_01.11 BBa_K1051207
    Clone St2    		 X X
    • 2082 pb
    • 80 pb
    PiGEM_01.12 BBa_K1051208
    Clone St3    		 X X
    • 2082 pb
    • 80 pb
    PiGEM_02.10 SLIC SerA produit PCR 2/3
    Clone 1    		 X
    • 2352 pb
    • 847 pb
    PiGEM_02.11 SLIC SerA produit PCR 2/3
    Clone 2    		 X
    • 2352 pb
    • 847 pb
    PiGEM_02.12 SLIC Mesh1 sans STOP produit PCR 10/11/12
    Clone 4    		 X X
    • 2422 pb
    • 180 pb
    • 84
    PiGEM_02.13 SLIC Mesh1 sans STOP produit PCR 10/11/12
    Clone 6    		 X X
    • 2422 pb
    • 180 pb
    • 84
    PiGEM_02.14 SLIC Mesh1 sans STOP produit PCR 10/11/13
    Clone 7    		 X X
    • 2422 pb
    • 180 pb
    • 84
    PiGEM_02.15 SLIC Mesh1 sans STOP produit PCR 10/11/13
    Clone 8    		 X X
    • 2422 pb
    • 180 pb
    • 84
    PiGEM_02.16 SLIC RelA sans STOP produit PCR 7/8
    Clone 28    		 X
    • 3530 pb
    • 872 pb
    PiGEM_02.17 SLIC RelA sans STOP produit PCR 7/8
    Clone 29    		 X
    • 3530 pb
    • 872 pb
    PiGEM_02.18 SLIC RelA sans STOP produit PCR 7/8
    Clone 31    		 X
    • 3530 pb
    • 872 pb
    PiGEM_02.19 SLIC RelA sans STOP produit PCR 7/8
    Clone 32    		 X
    • 3530 pb
    • 872 pb
    PiGEM_02.24 SLIC CheA produit PCR 5/6
    Clone 37    		 X
    • 3457 pb
    • 675 pb
    PiGEM_02.25 SLIC CheA produit PCR 5/6
    Clone 38    		 X
    • 3457 pb
    • 675 pb
    PiGEM_02.26 SLIC CheA produit PCR 5/6
    Clone 39    		 X
    • 3457 pb
    • 675 pb

    The efficiency of the digestion was tested thanks to agarose gel electrophoresis. Results are presented below.

    For stability label, linearized plasmid was the only fragment visible. For SLIC, no fragment matched with the weight of expected bands. All SLIC construction had to be made again.

  • PiGEM_02.27 (pSB1A2, BBa_J04450) was digested with XbaI, SpeI and PstI to be used for stability label cloning. Stability labels were assembled again by annealing.

    Primers numbers Primers names Brick created
    27 - 28 BBa_K1051206-up
    BBa_K1051206-down    		 BBa_K1051206
    29 -30 BBa_K1051207-up
    BBa_K1051207-down    		 BBa_K1051207
    31 -32 BBa_K1051208-up
    BBa_K1051208-down    		 BBa_K1051208

    Each annealing oligonucleotides were leagued in pSB1A2 digested XSP. DH5 alpha supercompetent were transformed with these ligation products and spread on Amp dish.

  • PiGEM_02.27 (pSB1A2, BBa_J04450) was digested with XbaI and SpeI. CusR box and Flag were assembled by annealing.

    Primers numbers Primers names Brick created
    35 - 36 CusR_Box-up
    CusR_Box-down    		 CusR Box
    41 - 42 Flag-up
    Flag-down    		 Flag

    Each annealing oligonucleotides were leagued in pSB1A2 digested XS. DH5 alpha supercompetent were transformed with these ligation products and spread on Amp dish.

  • SLIC assembly of CheA, SerA, RelA with and without STOP codon and Mesh 1 with and without STOP codon were restarted. DNA fragments were amplified thanks to iGEM PCR protocol (Q5 polymerase).

    # DNA Template Primers Numbers Primers Names Weight of amplified DNA
    82 Genomic DNA of W3110 6 - 7 serA_mut_ A849G_up
    serA_tronq_SufRFC23_down    		 171 bp
    83 Genomic DNA of W3110 5 - 8 serA_PrefRFC10RBS_up
    serA_mut_A849_down    		 861 bp
    84 PiGEM_02.05 15 - 17 relA_mut_C1366G_up
    relA_RFC10-RBS_up    		 1377 bp
    85 PiGEM_02.05 16 - 18 relA_mut_C1366_down
    relA_sans_stop_SufRFC23_down    		 882 bp
    86 PiGEM_02.05 16 - 19 relA_mut_C1366_down
    relA_2_stop_SufRFC23_down    		 885 bp
    87 PiGEM_02.06 20 - 24 Mesh_1_mut_A255T_up
    Mesh_1_RFC10-RBS_up    		 267 bp
    88 PiGEM_02.06 21 - 22 Mesh_1_mut_A255T_down
    Mesh_1_mut_A343G_up    		 120 bp
    89 PiGEM_02.06 23 - 25 Mesh_1_mut_A343G_down
    Mesh1_sans_stop_SufRFC23_down    		 192 bp
    90 PiGEM_02.06 23 - 26 Mesh_1_mut_A343G_down
    Mesh_1_2_stop_SufRFC23_down    		 192 bp

    The efficiency of the amplification was tested thanks to agarose gel electrophoresis. Results are presented below.

    All DNA fragments were correctly amplified. Thus, SLIC protocol was realized to assemble each fragment of CheA, SerA, RelA and Mesh1. Plasmid pSB1A2 digested XbaI and SpeI was used.

  • Several control PCR had be realized the day before to verify whether RFP without STOP codon and GFP without STOP codon worked. The efficiency of the amplification was tested thanks to agarose gel electrophoresis. Results are presented following.

    Only clones 41, 42 43 and 44 of RPF without STOP codon and clones 47 and 48 of GFP without STOP codon seemed to be correct.

  • Several plasmids were purified thanks to Macherey Nagel MiniPrep Kit :

    • PCR product 15 RFP without STOP Clone 41 ⇒ PiGEM_01.14
    • PCR product 15 RFP without STOP Clone 43 ⇒ PiGEM_01.15
    • PCR product 16 GFP without STOP Clone 47 ⇒ PiGEM_01.16
    • PCR product 16 GFP without STOP Clone 48 ⇒ PiGEM_01.17
Protocols used : ...

Week 7 : 08/11/2014 ➡ 08/17/2014

  • Several control PCR were realized to verify whether cusR promoter, RFP and GFP without STOP codon, cusR-L-LZa, SH3pep, cusR box and SLIC worked. Taq polymerase was used.

    # DNA Template Primers Numbers Primers Names Weight of amplified DNA
    106 cusR promoter
    Clone 1    		 43 - 44 Prefix_up
    Suffix_down    		 117 bp
    107 cusR promoter
    Clone 2    		 43 - 44 Prefix_up
    Suffix_down    		 117 bp
    108 cusR promoter
    Clone 3    		 43 - 44 Prefix_up
    Suffix_down    		 117 bp
    109 RFP without STOP codon
    Clone 41    		 43 - 44 Prefix_up
    Suffix_down    		 708 bp
    110 RFP without STOP codon
    Clone 42    		 43 - 44 Prefix_up
    Suffix_down    		 708 bp
    111 RFP without STOP codon
    Clone 43    		 43 - 44 Prefix_up
    Suffix_down    		 708 bp
    112 RFP without STOP codon
    Clone 44    		 43 - 44 Prefix_up
    Suffix_down    		 708 bp
    113 RFP without STOP codon
    Clone 47    		 43 - 44 Prefix_up
    Suffix_down    		 708 bp
    114 RFP without STOP codon
    Clone 48    		 43 - 44 Prefix_up
    Suffix_down    		 708 bp
    126 SH3pep
    Clone 4    		 43 - 44 Prefix_up
    Suffix_down    		 192 bp
    127 SH3pep
    Clone 6    		 43 - 44 Prefix_up
    Suffix_down    		 192 bp
    128 SH3pep
    Clone 8    		 43 - 44 Prefix_up
    Suffix_down    		 192 bp
    129 SH3pep
    Clone 10    		 43 - 44 Prefix_up
    Suffix_down    		 192 bp
    130 K1051207
    Clone St1    		 43 - 44 Prefix_up
    Suffix_down    		 86 bp
    131 K1051207
    Clone St2    		 43 - 44 Prefix_up
    Suffix_down    		 86 bp
    132 K1051207
    Clone St3    		 43 - 44 Prefix_up
    Suffix_down    		 80 bp
    133 cusR box
    Clone 1    		 43 - 44 Prefix_up
    Suffix_down    		 117 bp
    134 cusR box
    Clone 4    		 43 - 44 Prefix_up
    Suffix_down    		 117 bp
    135 cusR box
    Clone 5    		 43 - 44 Prefix_up
    Suffix_down    		 117 bp
    136 cusR box
    Clone 7    		 43 - 44 Prefix_up
    Suffix_down    		 117 bp
    137 SLIC bis CheA
    CA5    		 43 - 44 Prefix_up
    Suffix_down    		 2432 bp
    138 SLIC bis CheA
    CA6    		 43 - 44 Prefix_up
    Suffix_down    		 2432 bp
    139 SLIC bis CheA
    CA8    		 43 - 44 Prefix_up
    Suffix_down    		 2432 bp
    140 SLIC bis CheA
    CA9    		 43 - 44 Prefix_up
    Suffix_down    		 2432 bp
    141 SLIC bis CheA
    CA10    		 43 - 44 Prefix_up
    Suffix_down    		 2432 bp
    142 SLIC bis SerA
    SA1    		 43 - 44 Prefix_up
    Suffix_down    		 1032 bp
    143 SLIC bis SerA
    SA3    		 43 - 44 Prefix_up
    Suffix_down    		 1032 bp
    144 SLIC bis SerA
    SA4    		 43 - 44 Prefix_up
    Suffix_down    		 1032 bp
    145 SLIC bis SerA
    SA5    		 43 - 44 Prefix_up
    Suffix_down    		 1032 bp
    146 SLIC bis SerA
    SA6    		 43 - 44 Prefix_up
    Suffix_down    		 1032 bp
    147 SLIC bis RelA with STOP codon
    RA2    		 43 - 44 Prefix_up
    Suffix_down    		 2260 bp
    148 SLIC bis RelA with STOP codon
    RA5    		 43 - 44 Prefix_up
    Suffix_down    		 2260 bp
    149 SLIC bis RelA with STOP codon
    RA6    		 43 - 44 Prefix_up
    Suffix_down    		 2260 bp
    150 SLIC bis RelA with STOP codon
    RA7    		 43 - 44 Prefix_up
    Suffix_down    		 2260 bp
    151 SLIC bis RelA with STOP codon
    RA8    		 43 - 44 Prefix_up
    Suffix_down    		 2260 bp
    152 SLIC bis RelA without STOP codon
    RAS2    		 43 - 44 Prefix_up
    Suffix_down    		 2260 bp
    153 SLIC bis RelA without STOP codon
    RAS3    		 43 - 44 Prefix_up
    Suffix_down    		 2260 bp
    154 SLIC bis RelA without STOP codon
    RAS4    		 43 - 44 Prefix_up
    Suffix_down    		 2260 bp
    155 SLIC bis RelA without STOP codon
    RAS5    		 43 - 44 Prefix_up
    Suffix_down    		 2260 bp
    156 SLIC bis RelA without STOP codon
    RAS6    		 43 - 44 Prefix_up
    Suffix_down    		 2260 bp
    157 SLIC bis Mesh1 without STOP
    MSS1    		 43 - 44 Prefix_up
    Suffix_down    		 579 bp
    158 SLIC bis Mesh1 without STOP
    MSS2    		 43 - 44 Prefix_up
    Suffix_down    		 579 bp
    159 SLIC bis Mesh1 without STOP
    MSS3    		 43 - 44 Prefix_up
    Suffix_down    		 579 bp
    160 SLIC bis Mesh1 without STOP
    MSS4    		 43 - 44 Prefix_up
    Suffix_down    		 579 bp
    161 SLIC bis Mesh1 without STOP
    MSS5    		 43 - 44 Prefix_up
    Suffix_down    		 579 bp
    162 SLIC bis Mesh1 with STOP
    MS1    		 43 - 44 Prefix_up
    Suffix_down    		 579 bp
    163 SLIC bis Mesh1 with STOP
    MS2    		 43 - 44 Prefix_up
    Suffix_down    		 579 bp
    164 SLIC bis Mesh1 with STOP
    MS3    		 43 - 44 Prefix_up
    Suffix_down    		 579 bp
    165 SLIC bis Mesh1 with STOP
    MS4    		 43 - 44 Prefix_up
    Suffix_down    		 579 bp
    166 SLIC bis Mesh1 with STOP
    MS5    		 43 - 44 Prefix_up
    Suffix_down    		 579 bp
    167 PiGEM_01.04 43 - 44 Prefix_up
    Suffix_down    		 708 bp
    167 PiGEM_01.04 43 - 44 Prefix_up
    Suffix_down    		 708 bp

    The efficiency of the amplification was tested thanks to agarose gel electrophoresis. Results are presented below.

    Neither cusR promoter no stability label clone seemed to present the right construction, these cloning had to be remade. Nevertheless, every SH3pep and cusR box clones seemed to be correctly amplified. Clones 1 and 4 of cusR box (CB1 and CB4) and clones 4 and 10 of SH3pep (SP4 and SP10) were chosen to go on brick construction.

    Clones 42, 43, 44, 47 and 48 of RFP and GFP without STOP codon seemed to be correctly amplified. Only clones 1,2 and 3 of cusR-L-LZa seemed to present the right construction. Clones 42, 43, 47 and 48 of RFP and GFP without STOP codon (RFP42, RFP43, GFP47 and GFP48) and clones 2 and 3 of cusR-L-LZa (CZ2 and CZ3) were chosen to go on brick construction.

    No (SH3)4 clone seemed to present the right construction, this cloning had to be remade. Only clones 3 and 4 of Mesh1 without STOP codon (MSS3 and MSS4) seemed to present the right construction, these clones were chosen to go on brick construction. No clone of Mesh1 with STOP codon was correctly amplified, other clones were going to be tested by PCR.

    Only clone 5 of SLIC CheA (CA5), clones 2 and 5 of RelA with STOP codon (RA2 and RA5) and clones 4 and 5 of RelA without STOP codon (RAS4 and RAS5) seemed to present the right construction. Thus, only these clones were chosen to go on brick construction.

    Clone 1 was the only clone of SLIC SerA correctly amplified. SA1 was chosen to go on brick construction.

  • TG1 competent cells were prepared to substitute DH5 alpha that grows up too slowly.

  • PiGEM_02.27 (pSB1A2, BBa_J04450) was digested with XbaI, SpeI and PstI to be used for stability label cloning. Stability labels were assembled a third time by annealing.

    Primers numbers Primers names Brick created
    27 - 28 BBa_K1051206-up
    BBa_K1051206-down    		 BBa_K1051206
    29 -30 BBa_K1051207-up
    BBa_K1051207-down    		 BBa_K1051207
    31 -32 BBa_K1051208-up
    BBa_K1051208-down    		 BBa_K1051208

    Each annealing oligonucleotides were leagued in pSB1A2 digested XSP. DH5 alpha supercompetent were transformed with these ligation products and spread on Amp dish.

  • Because PCR 106 to 108 did not work, cusR promoter was amplified again thanks to iGEM PCR protocol (Q5 polymerase).

    # DNA Template Primers Numbers Primers Names Weight of amplified DNA
    169 Genomic DNA of W3110 33 - 34 CusR_iProm-up
    CusR_iProm-down    		 117 bp

    The efficiency of the amplification was tested thanks to agarose gel electrophoresis. Results are presented below.

    Once again, the amplification of cusR promoter did not work.

  • Several control PCR were realized to verify whether SLIC Mesh1 with STOP codon, (SH3)4 worked. Taq polymerase was used.

    # DNA Template Primers Numbers Primers Names Weight of amplified DNA
    170 SLIC bis Mesh1 with STOP
    MS6    		 43 - 44 CusR_iProm-up
    CusR_iProm-down    		 579 bp
    171 SLIC bis Mesh1 with STOP
    MS7    		 43 - 44 Prefix_up
    Suffix_down    		 579 bp
    172 SLIC bis Mesh1 with STOP
    MS8    		 43 - 44 Prefix_up
    Suffix_down    		 579 bp
    173 SLIC bis Mesh1 with STOP
    MS9    		 43 - 44 Prefix_up
    Suffix_down    		 579 bp
    174 SLIC bis Mesh1 with STOP
    MS10    		 43 - 44 Prefix_up
    Suffix_down    		 579 bp
    175 (SH3)4
    Clones 11, 12, 13, 14, 15    		 43 - 44 Prefix_up
    Suffix_down    		 932 bp
    176 (SH3)4
    Clones 16, 17, 18, 19, 20    		 43 - 44 Prefix_up
    Suffix_down    		 932 bp
    177 (SH3)4
    Clones 21, 22, 23, 24, 25    		 43 - 44 Prefix_up
    Suffix_down    		 932 bp
    178 (SH3)4
    Clones 26, 27, 28, 29, 30    		 43 - 44 Prefix_up
    Suffix_down    		 932 bp
    179 (SH3)4
    Clones 31, 32, 33, 34, 35    		 43 - 44 Prefix_up
    Suffix_down    		 932 bp

    Clone 8 was the only clone of SLIC Mesh1 with STOP codon correctly amplified. MS8 was chosen to go on brick construction. No clone of (SH3)4 seemed to be correctly amplified.

  • We have control if the insert of FRT-Kan-FRT is in the gene sdaBC and CheA by PCR.

    # DNA Template Primers Name Weight of amplified DNA
    180 Genomic DNA of W3110 CheA_up
    CheA_down    		 1700
    181 CheA- CheA_up
    CheA_down    		 1500
    182 Genomic DNA of W3110 sdaBC_up
    sdaBC_down    		 3100
    183 sdaBC- KanR sdaBC_up
    sdaBC_down    		 1500
    184 sdaBC- KanR + pCP20 sdaBC_up
    sdaBC_down    		 1500

    CheA- control is unsuccessful, we see a weight of amplification for #180 and #181 of 1700bp the gene FRT-Kan-FRT isn't insert in cheA.

    >SdaBC- control have a problem we don't have amplification for #182 to #184.

    Results not shown.

  • Several control PCR were realized to verify whether stability label and (SH3)4 cloning worked. Taq polymerase was used.

    # DNA Template Primers Numbers Primers Names Weight of amplified DNA
    195 K1051206
    Clone St4    		 43 - 44 Prefix_up
    Suffix_down    		 80 bp
    196 K1051206
    Clone St5    		 43 - 44 Prefix_up
    Suffix_down    		 80 bp
    197 K1051206
    Clone St6    		 43 - 44 Prefix_up
    Suffix_down    		 80 bp
    198 K1051206
    Clone St7    		 43 - 44 Prefix_up
    Suffix_down    		 80 bp
    199 K1051206
    Clone St8    		 43 - 44 Prefix_up
    Suffix_down    		 80 bp
    200 K1051206
    Clone St9    		 43 - 44 Prefix_up
    Suffix_down    		 80 bp
    201 K1051206
    Clone St10    		 43 - 44 Prefix_up
    Suffix_down    		 80 bp
    202 K1051206
    Clone St11    		 43 - 44 Prefix_up
    Suffix_down    		 80 bp
    203 K1051206
    Clone St12    		 43 - 44 Prefix_up
    Suffix_down    		 80 bp
    204 K1051206
    Clone St13    		 43 - 44 Prefix_up
    Suffix_down    		 80 bp
    205 K1051207
    Clone St14    		 43 - 44 Prefix_up
    Suffix_down    		 86 bp
    206 K1051207
    Clone St15    		 43 - 44 Prefix_up
    Suffix_down    		 86 bp
    207 K1051207
    Clone St16    		 43 - 44 Prefix_up
    Suffix_down    		 86 bp
    208 K1051207
    Clone St17    		 43 - 44 Prefix_up
    Suffix_down    		 86 bp
    209 K1051207
    Clone St18    		 43 - 44 Prefix_up
    Suffix_down    		 86 bp
    210 K1051207
    Clone St19    		 43 - 44 Prefix_up
    Suffix_down    		 86 bp
    211 K1051207
    Clone St20    		 43 - 44 Prefix_up
    Suffix_down    		 86 bp
    212 K1051207
    Clone St21    		 43 - 44 Prefix_up
    Suffix_down    		 86 bp
    213 K1051207
    Clone St22    		 43 - 44 Prefix_up
    Suffix_down    		 86 bp
    214 K1051207
    Clone St23    		 43 - 44 Prefix_up
    Suffix_down    		 86 bp
    215 K1051208
    Clone St24    		 43 - 44 Prefix_up
    Suffix_down    		 80 bp
    216 K1051208
    Clone St25    		 43 - 44 Prefix_up
    Suffix_down    		 80 bp
    217 K1051208
    Clone St26    		 43 - 44 Prefix_up
    Suffix_down    		 80 bp
    218 K1051208
    Clone St27    		 43 - 44 Prefix_up
    Suffix_down    		 80 bp
    219 K1051208
    Clone St28    		 43 - 44 Prefix_up
    Suffix_down    		 80 bp
    220 K1051208
    Clone St29    		 43 - 44 Prefix_up
    Suffix_down    		 80 bp
    221 K1051208
    Clone St30    		 43 - 44 Prefix_up
    Suffix_down    		 80 bp
    222 K1051208
    Clone St31    		 43 - 44 Prefix_up
    Suffix_down    		 80 bp
    223 K1051208
    Clone St32    		 43 - 44 Prefix_up
    Suffix_down    		 80 bp
    224 K1051208
    Clone St33    		 43 - 44 Prefix_up
    Suffix_down    		 80 bp
    225 (SH3)4Clones sc1 43 - 44 Prefix_up
    Suffix_down    		 932 bp
    226 (SH3)4Clones sc2 43 - 44 Prefix_up
    Suffix_down    		 932 bp
    227 (SH3)4Clones sc3 43 - 44 Prefix_up
    Suffix_down    		 932 bp
    228 (SH3)4Clones sc4 43 - 44 Prefix_up
    Suffix_down    		 932 bp
    229 (SH3)4Clones sc5 43 - 44 Prefix_up
    Suffix_down    		 932 bp
    230 (SH3)4Clones sc6 43 - 44 Prefix_up
    Suffix_down    		 932 bp
    231 (SH3)4Clones sc7 43 - 44 Prefix_up
    Suffix_down    		 932 bp
    232 (SH3)4Clones sc8 43 - 44 Prefix_up
    Suffix_down    		 932 bp
    233 (SH3)4Clones sc9 43 - 44 Prefix_up
    Suffix_down    		 932 bp
    234 (SH3)4Clones sc10 43 - 44 Prefix_up
    Suffix_down    		 932 bp

    Clone St8 was the only clone of K1051206 correctly amplified. St8 was chosen to go on brick construction.

    Only clones St16, 17, 19, 20 and 21 of K1051207 seemed to present the right construction. These clones were chosen to go on brick construction.

    Clones 24, 26, 27, 28, 29 and 33 of K1051208 seemed to be correctly amplified. These clones were chosen to go on brick construction.

    No clone of (SH3)4 seemed to present the right construction.

  • We made a PCR with new oligo to control mutants.

    # DNA Template Primers Name Weight of amplified DNA
    185 Genomic DNA of W3110 CheA_check_up
    CheA_check_down    		 1700
    186 CheA- CheA_check_up
    CheA_check_down    		 1500
    187 Genomic DNA of W3110 sdaBC_check_up
    sdaBC_check_down    		 3100
    188 sdaBC- KanR sdaBC_check_up
    sdaBC_check_down    		 1500
    189 sdaBC- KanR + pCP20 sdaBC_check_up
    sdaBC_check_down    		 1500
    190 Genomic DNA of W3110 FRT_up
    sdaBC_check_down    		 0
    191 sdaBC- KanR FRT_up
    sdaBC_check_down    		 1600
    192 sdaBC- KanR + pCP20 FRT_up
    sdaBC_check_down    		 1600

    Second control of CheA- show us the gene FRT-Kan-FRT isn't in the gene cheA

    SdaBC control have an other problem with amplification

    Results not shown.

  • Several plasmids were purified thanks to Macherey Nagel MiniPrep Kit :

    • PCR product 15 RFP without STOP codon Clones 42 and 43 ⇒ PiGEM_01.18 and PiGEM_01.19
    • PCR product 16 GFP without STOP codon Clones 47 and 48 ⇒ PiGEM_01.20 and PiGEM_01.21
    • CusRbox clones 1 and 4 (CB1 and CB4) ⇒ PiGEM_02.33 and PiGEM_02.34
    • SH3 pep L-LZa clones 4 and 10 (SP4 and SP10) ⇒ PiGEM_02.35 and PiGEM_02.36
    • CusR-L-LZa clones 2 and 3 (CZ2 and CZ3) ⇒ PiGEM_02.37 and PiGEM_02.38
    • SLIC CheA Bis Clone CA5 ⇒ PiGEM_02.39
    • SLIC SerA Bis Clone SA1 ⇒ PiGEM_02.40
    • SLIC RelA Bis avec STOP Clones RA2 and RA5 ⇒ PiGEM_02.41 and PiGEM_02.42
    • SLIC RelA Bis sans STOP Clone RAS4 and RAS5 ⇒ PiGEM_02.43 and PiGEM_02.44
    • SLIC Mesh1 Bis sans STOP Clone MSS3 and MSS4 ⇒ PiGEM_02.45 and PiGEM_02.46
    • SLIC CheA Clone 38 ⇒ PiGEM_02.47
    • SLIC Mesh1 avec STOP MS8 ⇒ PiGEM_02.48

  • CusR promoter was amplified again thanks to iGEM PCR protocol (Q5 polymerase) by switching matrix.

    # DNA Template Primers Numbers Primers Names Weight of amplified DNA
    238 Genomic DNA of W3110 33 - 34 CusR_iProm-up
    CusR_iProm-down    		 117 bp
    239 PiGEM_02.08 33 - 34 CusR_iProm-up
    CusR_iProm-down    		 117 bp

    The efficiency of the amplification was tested thanks to agarose gel electrophoresis. Results are presented below.

    The amplification from the genomique template did not work again but the amplification from the Berkeley plasmid worked very well. CusR promoter cloning could be done again.

  • Several control PCR were realized to verify whether (SH3)4 worked. Taq polymerase was used.

    # DNA Template Primers Numbers Primers Names Weight of amplified DNA
    240 (SH3)4
    Clones C4.1    		 43 - 44 CusR_iProm-up
    CusR_iProm-down    		 932 bp
    240 (SH3)4
    Clones C4.2    		 43 - 44 CusR_iProm-up
    CusR_iProm-down    		 932 bp
    240 (SH3)4
    Clones C4.3    		 43 - 44 CusR_iProm-up
    CusR_iProm-down    		 932 bp
    240 (SH3)4
    Clones C4.4    		 43 - 44 CusR_iProm-up
    CusR_iProm-down    		 932 bp
    240 (SH3)4
    Clones C4.5    		 43 - 44 CusR_iProm-up
    CusR_iProm-down    		 932 bp
    240 (SH3)4
    Clones C5.1    		 43 - 44 CusR_iProm-up
    CusR_iProm-down    		 932 bp
    240 (SH3)4
    Clones C5.2    		 43 - 44 CusR_iProm-up
    CusR_iProm-down    		 932 bp
    240 (SH3)4
    Clones C5.3    		 43 - 44 CusR_iProm-up
    CusR_iProm-down    		 932 bp
    240 (SH3)4
    Clones C5.4    		 43 - 44 CusR_iProm-up
    CusR_iProm-down    		 932 bp
    240 (SH3)4
    Clones C5.5    		 43 - 44 CusR_iProm-up
    CusR_iProm-down    		 932 bp
    240 (SH3)4
    Clones C6.1    		 43 - 44 CusR_iProm-up
    CusR_iProm-down    		 932 bp
    240 (SH3)4
    Clones C6.2    		 43 - 44 CusR_iProm-up
    CusR_iProm-down    		 932 bp
    240 (SH3)4
    Clones C6.3    		 43 - 44 CusR_iProm-up
    CusR_iProm-down    		 932 bp
    240 (SH3)4
    Clones C6.4    		 43 - 44 CusR_iProm-up
    CusR_iProm-down    		 932 bp
    240 (SH3)4
    Clones C6.5    		 43 - 44 CusR_iProm-up
    CusR_iProm-down    		 932 bp

    The efficiency of the amplification was tested thanks to agarose gel electrophoresis. Results are presented below.

    No clone of (SH3)4 seemed to present the right construction.

  • Several control PCR were realized to verify whether stability label K1051206 and flag cloning worked. Taq polymerase was used.

    # DNA Template Primers Numbers Primers Names Weight of amplified DNA
    255 K1051206
    Clone St34    		 43 - 44 Prefix_up
    Suffix_down    		 80 bp
    256 K1051206
    Clone St35    		 43 - 44 Prefix_up
    Suffix_down    		 80 bp
    257 K1051206
    Clone St36    		 43 - 44 Prefix_up
    Suffix_down    		 80 bp
    258 K1051206
    Clone St37    		 43 - 44 Prefix_up
    Suffix_down    		 80 bp
    259 K1051206
    Clone St39    		 43 - 44 Prefix_up
    Suffix_down    		 80 bp
    260 Flag
    Clone 1    		 43 - 44 Prefix_up
    Suffix_down    		 80 bp
    261 Flag
    Clone 2    		 43 - 44 Prefix_up
    Suffix_down    		 80 bp
    262 Flag
    Clone 3    		 43 - 44 Prefix_up
    Suffix_down    		 80 bp
    263 Flag
    Clone 4    		 43 - 44 Prefix_up
    Suffix_down    		 80 bp
    264 Flag
    Clone 5    		 43 - 44 Prefix_up
    Suffix_down    		 80 bp
    265 Flag
    Clone 6    		 43 - 44 Prefix_up
    Suffix_down    		 86 bp
    266 Flag
    Clone 7    		 43 - 44 Prefix_up
    Suffix_down    		 86 bp
    267 Flag
    Clone 8    		 43 - 44 Prefix_up
    Suffix_down    		 86 bp
    268 Flag
    Clone 9    		 43 - 44 Prefix_up
    Suffix_down    		 86 bp
    269 Flag
    Clone 10    		 43 - 44 Prefix_up
    Suffix_down    		 86 bp

    The efficiency of the amplification was tested thanks to agarose gel electrophoresis. Results are presented below.

    No clone of K1051206 seemed to be correctly amplified. Nevertheless, every Flag clone seemed to present the right construction. Fl1 and Fl3 clones were chosen to go on brick construction.

  • We made a gradient PCR 55° to 65° with oligo sdaBC_checkdo/FRTup and W3110 sdaBC- strain and W3110 Wild type.

    No amplification shown by the electrophoresis for all these PCR.

    Results not shown

  • Several plasmids were purified thanks to Macherey Nagel MiniPrep Kit :

    • Stability label 27/28 K1051206 clone St8 ⇒ PiGEM_02.49
    • Stability label 29/30 K1051207 clones St16, 17, 19, 20 and 21 ⇒ PiGEM_02.50, PiGEM_02.51, PiGEM_02.52, PiGEM_02.53 and PiGEM_02.54
    • Stability label 31/32 K1051208 clones St24, 26, 27, 28, 29 and 33 ⇒ PiGEM_02.55, PiGEM_02.56, PiGEM_02.57, PiGEM_02.58, PiGEM_02.59 and PiGEM_02.60

  • We made Q5 PCR with oligo 1-2 and 3-4 on pKD4 template for prepare new transformation sdaBC and cheA respectively #293 and #294. After we supress the template pKD4 with

  • We purified PCR products #293 thanks to Macherey Nagel PCR Clean up Kit.

    29322.65 ng.mL-1

  • We made 2 new PCR on transformed strains. With specific hybridation temperature.

    # DNA Template Primers Name Weight of amplified DNA Hybridation Temperature
    300A CheA- CheA_check_up
    CheA_check_down    		 1500 55°C
    301A sdaBC- KanR FRT_up
    sdaBC_check_down    		 1600 50°C

Week 8 : 08/18/2014 ➡ 08/24/2014

  • Plasmids containing the stability labels 27/28 K1051206 clone St8, the stability labels 29/30 K1051207 clones St16, 17, 19, 20 and 21 and the stability labels 31/32 K1051208 clones St24, 26, 27, 28, 29 and 33 were digested by XbaI to determine whether they presented the expected construction.

    The efficiency of the digestion was tested thanks to agarose gel electrophoresis. Results are presented below.

    The weight of digested fragment were too heavy. It was impossible that only one stability label was inserted in one iGEM plasmid. Certainly, several stability labels were associated in a same plasmid. The cloning of stability label must have be remade with new ratios of stability label and plasmid.

  • PiGEM_02.27 (pSB1A2, BBa_J04450) was digested with EcoRI and PstI to be used for PCR product 239 cloning. PCR product 239 (cusR promoter) was digested by EcoRI and PstI. Then, PCR product 239 was leagued in pSB1A2 digested. Tg1 competent cells were transformed with these ligation products and spread on Amp dish.

  • Bricks 200 and 202 were built again by changing destination plasmid. 3A protocol was implemented to assemble the upstream part with downstream part and with the destination plasmid. Tg1 competent cells were transformed with these ligation products and spread on Kan or Cm dish. The cloning plan is presented below.

    Part Number Description Upstream Part Downstream Part Destination Plasmid
    200 Reference RBS with RFP with stop codon PiGEM_02.29 Amp PiGEM_01.04 Cm PiGEM_02.28 Kan
    202 Reference RBS with GFP with stop codon PiGEM_02.29 Amp PiGEM_02.03 Amp PiGEM_01.13 Cm

  • Several control PCR were realized to verify whether cusR promoter cloning worked. Taq polymerase was used.

    # DNA Template Primers Numbers Primers Names Weight of amplified DNA
    AS1 cusR promoter
    Clone 1    		 43 - 44 Prefix_up
    Suffix_down    		 120 bp
    AS2 cusR promoter
    Clone 2    		 43 - 44 Prefix_up
    Suffix_down    		 120 bp
    AS3 cusR promoter
    Clone 3    		 43 - 44 Prefix_up
    Suffix_down    		 120 bp
    AS4 cusR promoter
    Clone 4    		 43 - 44 Prefix_up
    Suffix_down    		 120 bp
    AS5 cusR promoter
    Clone 5    		 43 - 44 Prefix_up
    Suffix_down    		 120 bp
    AS6 cusR promoter
    Clone 6    		 43 - 44 Prefix_up
    Suffix_down    		 120 bp
    AS7 cusR promoter
    Clone 7    		 43 - 44 Prefix_up
    Suffix_down    		 120 bp
    AS8 cusR promoter
    Clone 8    		 43 - 44 Prefix_up
    Suffix_down    		 120 bp
    AS9 cusR promoter
    Clone 9    		 43 - 44 Prefix_up
    Suffix_down    		 120 bp
    AS10 cusR promoter
    Clone 10    		 43 - 44 Prefix_up
    Suffix_down    		 120 bp

    The efficiency of the amplification was tested thanks to agarose gel electrophoresis. Results are presented below.

    The majority of clones seemed to present the right construction. Clones 2 and 8 (PCU2 and PCU8) were chosen to go on brick construction.

  • Bricks 201, 203 to 207, 211, 301 and 303 were built. 3A protocol was implemented to assemble the upstream part with downstream part and with the destination plasmid. Tg1 competent cells were transformed with these ligation products and spread on Kan or Cm dish. The cloning plan is presented below.

    Part Number Description Upstream Part Downstream Part Destination Plasmid
    201 Reference RBS with RFP without stop codon PiGEM_02.29 Amp PiGEM_01.18 Cm
    PiGEM_01.19 Cm    		 PiGEM_02.28 Kan
    203 Reference RBS with GFP without stop codon PiGEM_02.29 Amp PiGEM_01.20 Cm
    PiGEM_01.21 Cm    		 PiGEM_02.28 Kan
    204 Reference RBS with RelA with stop codon PiGEM_02.29 Amp PiGEM_01.40 Amp
    PiGEM_01.41 Amp    		 PiGEM_02.13 Cm
    205 Reference RBS with RelA without stop codon PiGEM_02.29 Amp PiGEM_01.43 Amp
    PiGEM_01.44 Amp    		 PiGEM_02.13 Cm
    206 Reference RBS with Mesh1 with stop codon PiGEM_02.29 Amp PiGEM_01.48 Amp PiGEM_02.13 Cm
    207 Reference RBS with Mesh1 without stop codon PiGEM_02.29 Amp PiGEM_01.45 Amp
    PiGEM_01.46 Amp    		 PiGEM_02.13 Cm
    211 Stability 3 with double terminator PiGEM_02.29 Amp PiGEM_01.33 Amp
    PiGEM_01.34 Amp    		 PiGEM_02.13 Cm
    301 SH3pep-LZA with double terminator PiGEM_02.35 Amp
    PiGEM_02.36 Amp    		 PiGEM_01.07 Cm PiGEM_02.28 Kan
    303 RBS with CusR-LZa protein PiGEM_02.29 Amp PiGEM_02.37 Amp
    PiGEM_02.38 Amp    		 PiGEM_01.13 Cm

  • PiGEM_02.27 (pSB1A2, BBa_J04450) was digested with XbaI, SpeI and PstI to be used for stability label cloning. Stability labels were assembled again by annealing.

    Primers numbers Primers names Brick created
    27 - 28 BBa_K1051206-up
    BBa_K1051206-down    		 BBa_K1051206
    29 -30 BBa_K1051207-up
    BBa_K1051207-down    		 BBa_K1051207
    31 -32 BBa_K1051208-up
    BBa_K1051208-down    		 BBa_K1051208

    Each annealing oligonucleotides were leagued in pSB1A2 digested XSP with ratio 1:1 (0.0047 ng of stability label for 117 ng of plasmid) and 1:2 (0.0094 ng of stability label for 117 ng of plasmid). Tg1 competent cells were transformed with these ligation products and spread on Amp dish.

  • We made a PCR with new oligo to control mutants.

    # DNA Template Primers Name Weight of amplified DNA
    185 Genomic DNA of W3110 CheA_check_up
    CheA_check_down    		 1700
    186 CheA- CheA_check_up
    CheA_check_down    		 1500
    187 Genomic DNA of W3110 sdaBC_check_up
    sdaBC_check_down    		 3100
    188 sdaBC- KanR sdaBC_check_up
    sdaBC_check_down    		 1500
    189 sdaBC- KanR + pCP20 sdaBC_check_up
    sdaBC_check_down    		 1500
    190 Genomic DNA of W3110 FRT_up
    sdaBC_check_down    		 0
    191 sdaBC- KanR FRT_up
    sdaBC_check_down    		 1600
    192 sdaBC- KanR + pCP20 FRT_up
    sdaBC_check_down    		 1600

    After the electrophoresis, we can confirm The strain sdaBC- KanR is successful. We select 3 strains with electrophoresis control

    C.23 and C.33 are contaminated. So we select C.43, C.53 and C.63.

  • Several control PCR were realized to verify whether Bricks 200 and 202 constructions worked. Taq polymerase was used.

    # DNA Template Primers Numbers Primers Names Weight of amplified DNA
    AS11 Brick 200
    Clone RFP1    		 43 - 44 Prefix_up
    Suffix_down    		 758 bp
    AS12 Brick 200
    Clone RFP2    		 43 - 44 Prefix_up
    Suffix_down    		 758 bp
    AS13 Brick 200
    Clone RFP3    		 43 - 44 Prefix_up
    Suffix_down    		 758 bp
    AS14 Brick 200
    Clone RFP4    		 43 - 44 Prefix_up
    Suffix_down    		 758 bp
    AS15 Brick 200
    Clone RFP5    		 43 - 44 Prefix_up
    Suffix_down    		 758 bp
    AS16 Brick 202
    Clone GFP1    		 43 - 44 Prefix_up
    Suffix_down    		 772 bp
    AS17 Brick 202
    Clone GFP2    		 43 - 44 Prefix_up
    Suffix_down    		 772 bp
    AS18 Brick 202
    Clone GFP3    		 43 - 44 Prefix_up
    Suffix_down    		 772 bp
    AS19 Brick 202
    Clone GFP4    		 43 - 44 Prefix_up
    Suffix_down    		 772 bp
    AS20 Brick 202
    Clone GFP5    		 43 - 44 Prefix_up
    Suffix_down    		 772 bp
    AS21 Brick 200
    Clone RFP1    		 38 - 51 BBa_E1010_NoStop-down
    BBa_B0034-up    		 718 bp
    AS22 Brick 200
    Clone RFP2    		 38 - 51 BBa_E1010_NoStop-down
    BBa_B0034-up    		 718 bp
    AS23 Brick 200
    Clone RFP3    		 38 - 51 BBa_E1010_NoStop-down
    BBa_B0034-up    		 718 bp
    AS24 Brick 200
    Clone RFP4    		 38 - 51 BBa_E1010_NoStop-down
    BBa_B0034-up    		 718 bp
    AS25 Brick 200
    Clone RFP5    		 38 - 51 BBa_E1010_NoStop-down
    BBa_B0034-up    		 718 bp
    AS26 Brick 202
    Clone GFP1    		 40 - 51 BBa_E0040_NoStop-down
    BBa_B0034-up    		 732 bp
    AS27 Brick 202
    Clone GFP2    		 40 - 51 BBa_E0040_NoStop-down
    BBa_B0034-up    		 732 bp
    AS28 Brick 202
    Clone GFP3    		 40 - 51 BBa_E0040_NoStop-down
    BBa_B0034-up    		 732 bp
    AS29 Brick 202
    Clone GFP4    		 40 - 51 BBa_E0040_NoStop-down
    BBa_B0034-up    		 732 bp
    AS30 Brick 202
    Clone GFP5    		 40 - 51 BBa_E0040_NoStop-down
    BBa_B0034-up    		 732 bp

    The efficiency of the amplification was tested thanks to agarose gel electrophoresis. Results are presented below.

    All clones seemed to present the right construction. Clones 200(3), 200(5), 202(1) and 202(3) were chosen to go on brick construction.

  • Several plasmids were purified thanks to Macherey Nagel MiniPrep Kit :

    • CusR promoter clones 2 and 8 (PCU2 and PCU8) ⇒ PiGEM_02.61 and PiGEM_02.62

  • We prepare seven starter of W3110 sdaBC- KanR strain with Kan50.

  • Sequencing results of plasmids PiGEM_02.33, PiGEM_02.34, PiGEM_02.35, PiGEM_02.36, PiGEM_02.37, PiGEM_02.38, PiGEM_02.39, PiGEM_02.40, PiGEM_02.41, PiGEM_02.42, PiGEM_02.43, PiGEM_02.44, PiGEM_02.45, PiGEM_02.46, PiGEM_02.47, PiGEM_02.48 were received. Thus, clones CB1, SP10, CZ3, CA38, RA2, MS8, MSS4, GFP47, RFP43, RAS5 and PCU2 had the right construction. Because no clone of SLIC SerA was correct, several control PCR were realized to verify whether other clones of SLIC SerA had the right construction. Taq polymerase was used.

    # DNA Template Primers Numbers Primers Names Weight of amplified DNA
    AS31 SLIC bis SerA
    SA9    		 43 - 44 Prefix_up
    Suffix_down    		 1049 bp
    AS32 SLIC bis SerA
    SA11    		 43 - 44 Prefix_up
    Suffix_down    		 1049 bp
    AS33 SLIC bis SerA
    SA12    		 43 - 44 Prefix_up
    Suffix_down    		 1049 bp
    AS34 SLIC bis SerA
    SA13    		 43 - 44 Prefix_up
    Suffix_down    		 1049 bp
    AS35 SLIC bis SerA
    SA14    		 43 - 44 Prefix_up
    Suffix_down    		 1049 bp
    AS36 SLIC bis SerA
    SA15    		 43 - 44 Prefix_up
    Suffix_down    		 1049 bp

    The efficiency of the amplification was tested thanks to agarose gel electrophoresis. Results are presented below.

    There were bands at the right molecular weight but clone SA1 got the same band (AS36). Nevertheless, this clone did not have the right construction (bad sequencing result). Thus, SerA was going to be assembled again by another protocol (Gibson protocol).

  • Plasmids containing the flag (Fl1 and Fl3) were digested by XbaI to determine whether they presented the expected construction.

    The efficiency of the digestion was tested thanks to agarose gel electrophoresis. Results are presented below.

    Clone Fl1 and Fl3 seemed to present the expected construction.

  • We realize day 7 of Lambda Red protocol on W3110 sdaBC- pCP20 strain.

  • Several plasmids were purified thanks to Macherey Nagel MiniPrep Kit :

    • Bricks 200 (3) and 200 (5) ⇒ PiGEM_02.65 and PiGEM_02.66
    • Bricks 202 (1) and 202 (3) ⇒ PiGEM_02.67 and PiGEM_02.68

  • Unfortunately, colonies careering bricks 200 and 202 were respectively red and green while constructions of these bricks were not supposed to contain any promoter. Thus, PiGEM_02.65, PiGEM_02.66, PiGEM_02.67 and PiGEM_02.68 were sent in order to be sequenced. In the mean time, RFP and GFP were assembled to another RBS to test whether the RBS B0034 was correct. The cloning plan is presented below.

    Part Number Description Upstream Part Downstream Part Destination Plasmid
    A RBS B0030 with RFP with stop codon PiGEM_01.05 Cm PiGEM_01.04 Cm PiGEM_01.13 Cm
    B RBS B0030 with GFP with stop codon PiGEM_01.05 Cm PiGEM_02.03 Cm PiGEM_01.13 Cm

  • Several control PCR were realized to verify whether stability labels cloning with different ratios worked. Taq polymerase was used.

    # DNA Template Primers Numbers Primers Names Weight of amplified DNA
    CB1 K1051206 Ratio 1:1
    Clone St40    		 43 - 44 Prefix_up
    Suffix_down    		 80 bp
    CB2 K1051206 Ratio 1:1
    Clone St41    		 43 - 44 Prefix_up
    Suffix_down    		 80 bp
    CB3 K1051206 Ratio 1:1
    Clone St42    		 43 - 44 Prefix_up
    Suffix_down    		 80 bp
    CB4 K1051206 Ratio 1:1
    Clone St43    		 43 - 44 Prefix_up
    Suffix_down    		 80 bp
    CB5 K1051206 Ratio 1:1
    Clone St44    		 43 - 44 Prefix_up
    Suffix_down    		 80 bp
    CB6 K1051206 Ratio 1:2
    Clone St45    		 43 - 44 Prefix_up
    Suffix_down    		 80 bp
    CB7 K1051206 Ratio 1:2
    Clone St46    		 43 - 44 Prefix_up
    Suffix_down    		 80 bp
    C86 K1051206 Ratio 1:2
    Clone St47    		 43 - 44 Prefix_up
    Suffix_down    		 80 bp
    CB9 K1051206 Ratio 1:2
    Clone St48    		 43 - 44 Prefix_up
    Suffix_down    		 80 bp
    CB10 K1051206 Ratio 1:2
    Clone St49    		 43 - 44 Prefix_up
    Suffix_down    		 80 bp
    CB11 K1051207 Ratio 1:1
    Clone St50    		 43 - 44 Prefix_up
    Suffix_down    		 86 bp
    CB12 K1051207 Ratio 1:1
    Clone St51    		 43 - 44 Prefix_up
    Suffix_down    		 86 bp
    CB13 K1051207 Ratio 1:1
    Clone St52    		 43 - 44 Prefix_up
    Suffix_down    		 86 bp
    CB14 K1051207 Ratio 1:1
    Clone St53    		 43 - 44 Prefix_up
    Suffix_down    		 86 bp
    CB15 K1051207 Ratio 1:1
    Clone St54    		 43 - 44 Prefix_up
    Suffix_down    		 86 bp
    CB16 K1051207 Ratio 1:2
    Clone St55    		 43 - 44 Prefix_up
    Suffix_down    		 86 bp
    CB17 K1051207 Ratio 1:2
    Clone St56    		 43 - 44 Prefix_up
    Suffix_down    		 86 bp
    CB18 K1051207 Ratio 1:2
    Clone St57    		 43 - 44 Prefix_up
    Suffix_down    		 86 bp
    CB19 K1051207 Ratio 1:2
    Clone St58    		 43 - 44 Prefix_up
    Suffix_down    		 86 bp
    CB20 K1051207 Ratio 1:2
    Clone St59    		 43 - 44 Prefix_up
    Suffix_down    		 86 bp
    CB21 K1051208 Ratio 1:1
    Clone St60    		 43 - 44 Prefix_up
    Suffix_down    		 80 bp
    CB22 K1051208 Ratio 1:1
    Clone St61    		 43 - 44 Prefix_up
    Suffix_down    		 80 bp
    CB23 K1051208 Ratio 1:1
    Clone St62    		 43 - 44 Prefix_up
    Suffix_down    		 80 bp
    CB24 K1051208 Ratio 1:1
    Clone St63    		 43 - 44 Prefix_up
    Suffix_down    		 80 bp
    CB25 K1051208 Ratio 1:1
    Clone St64    		 43 - 44 Prefix_up
    Suffix_down    		 80 bp
    CB26 K1051208 Ratio 1:2
    Clone St65    		 43 - 44 Prefix_up
    Suffix_down    		 80 bp
    CB27 K1051208 Ratio 1:2
    Clone St66    		 43 - 44 Prefix_up
    Suffix_down    		 80 bp
    CB28 K1051208 Ratio 1:2
    Clone St67    		 43 - 44 Prefix_up
    Suffix_down    		 80 bp
    CB29 K1051208 Ratio 1:2
    Clone St68    		 43 - 44 Prefix_up
    Suffix_down    		 80 bp
    CB30 K1051208 Ratio 1:2
    Clone St69    		 43 - 44 Prefix_up
    Suffix_down    		 80 bp

    The efficiency of the amplification was tested thanks to agarose gel electrophoresis. Results are presented below.

    Clone Fl1 and Fl3 seemed to present the expected construction.

    Unfortunately, there was not any expected result. Stability labels constructions did not success once again. New oligonucleotides were going to be ordered to try a new protocol.

  • We realize day 8 of Lambda Red protocol on W3110 sdaBC- strain.

Week 9 : 08/25/2014 ➡ 08/31/2014

  • RelA (RA2) and Mesh1 (MS8) were assembled with a Lac promoter and the reference RBS (BBa_B0034) to characterize parts. 3A protocol was implemented to assemble the upstream part with downstream part and with the destination plasmid. Tg1 competent cells were transformed with these ligation products and spread on Kan glucose dish to prevent RelA and Mesh1 expression that could slow cells growth. The cloning plan is presented below.

    Part Number Description Upstream Part Downstream Part Destination Plasmid
    x Promoter with RBS and RelA with stop codon PiGEM_01.08 Cm PiGEM_02.41 Amp PiGEM_02.28 Kan
    y Promoter with RBS and Mesh1 with stop codon PiGEM_01.08 Cm PiGEM_02.48 Amp PiGEM_02.28 Kan

  • We realize day 9 of Lambda Red protocol on W3110 sdaBC- strain, and day 2 of Lambda Red protocol on W3110 sdaBC- strain with #294 PCR product

  • Sequencing results of plasmids PiGEM_02.65, PiGEM_02.66, PiGEM_02.67 and PiGEM_02.68 were received. PiGEM_02.67 and PiGEM_02.68 had the right construction but PiGEM_02.65 and PiGEM_02.66 had not. Thus, RBS BBa_B0034 was correct. There was no reason for the colonies containing bricks 200 and 202 to be green or red without promoter sequence.

  • Several control PCR were realized to verify whether RelA and Mesh1 cloning with their promoter and RBS worked. (SH3)2 new construction were also tested. Taq polymerase was used.

    # DNA Template Primers Numbers Primers Names Weight of amplified DNA
    AS43 BBa_J04500 with RelA
    Clone 1    		 43 - 44 Prefix_up
    Suffix_down    		 2480 bp
    AS44 BBa_J04500 with RelA
    Clone 2    		 43 - 44 Prefix_up
    Suffix_down    		 2480 bp
    AS45 BBa_J04500 with RelA
    Clone 3    		 43 - 44 Prefix_up
    Suffix_down    		 2480 bp
    AS46 BBa_J04500 with RelA
    Clone 4    		 43 - 44 Prefix_up
    Suffix_down    		 2480 bp
    AS47 BBa_J04500 with RelA
    Clone 5    		 43 - 44 Prefix_up
    Suffix_down    		 2480 bp
    AS48 BBa_J04500 with RelA
    Clone 6    		 43 - 44 Prefix_up
    Suffix_down    		 2480 bp
    AS49 BBa_J04500 with RelA
    Clone 7    		 43 - 44 Prefix_up
    Suffix_down    		 2480 bp
    AS50 BBa_J04500 with RelA
    Clone 8    		 43 - 44 Prefix_up
    Suffix_down    		 2480 bp
    AS51 BBa_J04500 with RelA
    Clone 9    		 43 - 44 Prefix_up
    Suffix_down    		 2480 bp
    AS52 BBa_J04500 with RelA
    Clone 10    		 43 - 44 Prefix_up
    Suffix_down    		 2480 bp
    AS53 BBa_J04500 with Mesh1
    Clone 1    		 43 - 44 Prefix_up
    Suffix_down    		 779 bp
    AS54 BBa_J04500 with Mesh1
    Clone 2    		 43 - 44 Prefix_up
    Suffix_down    		 779 bp
    AS55 BBa_J04500 with Mesh1
    Clone 3    		 43 - 44 Prefix_up
    Suffix_down    		 779 bp
    AS56 BBa_J04500 with Mesh1
    Clone 4    		 43 - 44 Prefix_up
    Suffix_down    		 779 bp
    AS57 BBa_J04500 with Mesh1
    Clone 5    		 43 - 44 Prefix_up
    Suffix_down    		 779 bp
    AS58 BBa_J04500 with Mesh1
    Clone 6    		 43 - 44 Prefix_up
    Suffix_down    		 779 bp
    AS59 BBa_J04500 with Mesh1
    Clone 7    		 43 - 44 Prefix_up
    Suffix_down    		 779 bp
    AS60 BBa_J04500 with Mesh1
    Clone 8    		 43 - 44 Prefix_up
    Suffix_down    		 779 bp
    AS61 BBa_J04500 with Mesh1
    Clone 9    		 43 - 44 Prefix_up
    Suffix_down    		 779 bp
    AS62 BBa_J04500 with Mesh1
    Clone 10    		 43 - 44 Prefix_up
    Suffix_down    		 779 bp
    AS63 (SH3)2
    Clone 1    		 43 - 44 Prefix_up
    Suffix_down    		 500 bp
    AS64 (SH3)2
    Clone 2    		 43 - 44 Prefix_up
    Suffix_down    		 500 bp
    AS65 (SH3)2
    Clone 3    		 43 - 44 Prefix_up
    Suffix_down    		 500 bp
    AS66 (SH3)2
    Clone 4    		 43 - 44 Prefix_up
    Suffix_down    		 500 bp
    AS61 (SH3)2
    Clone 5    		 43 - 44 Prefix_up
    Suffix_down    		 500 bp

    The efficiency of the amplification was tested thanks to agarose gel electrophoresis. Results are presented below.

    Some clones seemed to present the right construction. Clones 1 and 2 of BBa_J04500 with RelA were chosen to continue the part characterization. In the mean time, to go on the part characterization, clones 2, 5, 6 and 8 of BBa_J04500 with Mesh1 were selected. Clones 2 and 5 were going to be used as negative controls (empty plasmids) and clones 6 and 8 were going to be used as functional parts.

  • We realize day 3 of Lambda Red protocol on W3110 sdaBC- strain CheA- KanR.

  • Check the construction 200(2) (RBS B0034 + RFP E1010) by digestion.

    7µL E1010 (control) (500ng)
    36µL H2O
    5µ NEB Buffer 2.1
    1µL XbaI
    1µL EcoRI


    2µL 200(2) (500ng)
    36µL H2O
    5µ NEB Buffer 2.1
    1µL XbaI
    1µL EcoRI

    Incubation 53' at 37°C. Then, inactivation of enzymes 25' at 80°C.

    It is difficult to see if 200(2) possesses the RBS. The plasmid 200(2) is sent to sequencing and it's OK.

  • Clean-up of PCR product number 82 and 83 from 8-8-14 (50µL)

  • SOE reaction (= overlap PCR):

    Mix:

    • PCR product 82= 3µL
    • PCR product 83 = 3µL
    • dNTP 10mM = 2µL
    • Q5 buffer = 8µL
    • Q5 polymerase = 0,5µL
    • H2O qsp 40µL


    Program:

    1. 98°C 2’
    2. 98°C 20”
    3. 45°C 25”
    4. 72°C 1’
    5. 72°C 10’
    Repeat the steps 3-4-5, 10 times.

  • Two new cells strain were selected to characterize RelA and Mesh1 parts. The first one is EB425 (ppGpp°) which does not produce ppGpp any more. The second one is EB544 (ppGpp++) which produces ppGpp with greater stability. These Escherichia coli strains were used to prepare two samples of competent cells. EB425 were going to be complemented by RelA whereas EB544 were going to be complemented by Mesh1.

  • We realize day 4 of Lambda Red protocol on W3110 sdaBC- strain CheA- KanR. strain.

  • PCR2: Second PCR round to amplification of serA from the SOE product (26-08-14) with primers 5 and 6, using the iGEM PCR protocol with Q5 polymerase. The temperature of annealing is 60°C

    Result: the amplification of serA is good. Clean-up of serA (PCR2) and elution with 45µL of water.

  • Digestion of pSB1A3 and pSB1C3 during 1h30 at 37°C. Then, inactivation of enzymes during 20’ at 80°C

    Mix:

    • 10µL of plasmid
    • 1µL EcoRI
    • 1µL PstI
    • 5µL Buffer 2.1
    • 33 µL H2O

  • Digestion of the insert (serA) during 1h at 37°C. Then inactivation of enzymes during 20’ at 80°C

    Mix:

    • 20µL of serA
    • 1µL EcoRI
    • 1µL PstI
    • 5µL Buffer 2.1
    • 13 µL H2O

  • NEB ligation:

    • 2µL of digested plasmid
    • 2µL of digested serA insert
    • 2µL ligase buffer
    • 1µL ligase (NEB)
    • 13 µL H2O


    Incubation overnight at 16°C

  • Several plasmids were purified thanks to Macherey Nagel MiniPrep Kit :

    • J04500 with RelA (RA2) Clones 1 and 2 ⇒ PiGEM_02.69 and PiGEM_02.70
    • J04500 Mesh1 (MS8) Clones 2 and 5 ⇒ PiGEM_02.71 and PiGEM_02.72
    • Empty plasmids Clones 6 and 8 ⇒ PiGEM_02.73 and PiGEM_02.74

  • To characterize RelA and Mesh1 parts, growth rate would be evaluated. At first, EB425 competent cells (ppGpp°) were transformed with PiGEM_02.05 (pT18 RelA, positive control), with PiGEM_02.74 (empty plasmid, negative control) and with PiGEM_02.69 and PiGEM_02.70 (pLac RelA, built part). Then, EB544 competent cells (ppGpp++) were transformed with PiGEM_02.06 (pBAD Mesh1, positive control), with PiGEM_02.74 (empty plasmid, negative control) and with PiGEM_02.71 and PiGEM_02.72 (pLac Mesh1, built part). Cells were spread over Kan glucose dish to prevent RelA and Mesh1 expression. Some colonies were going to be spread later on selective dish to observe any difference in growth rate.

  • We realize day 5 of Lambda Red protocol on W3110 sdaBC- CheA- KanR strain.

  • Transformation of 90µL of DH5α super competent cells with 10µL of ligation (pSB1C3-SerA or pSB1A2-SerA). The selective medium is LB + Ampicillin (Ap) 100µg/mL or LB + Chloramphenicol (Cm) 30µg/mL

    pSB1C3-SerA-1 seemed good. It was sent to sequencing. It’s OK

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Retrieved from "http://2014.igem.org/Team:Aix-Marseille/Notebook_08"