Difference between revisions of "Team:TecCEM/Notebook"

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+
<p style="font-family: 'Roboto', sans-serif; text-align: justify-all; font-size: 1.2rem; font-weight: bold">Week overview:</p>
 +
<ul class="des" style="list-style: none; padding: 0; margin: 0; display: flex; flex-direction: column;">
 +
<li style="font-family: 'Roboto', sans-serif; text-align: justify-all; font-size: 1.2rem">Optical density (OD600) for glycerol gradients were measured approximately every 40 minutes for almost 9 hours and graphed.</li>
 +
<li style="font-family: 'Roboto', sans-serif; text-align: justify-all; font-size: 1.2rem">Optical density (OD600) for glycerol gradients were also measured with Lector Híbrido Multi-Modal de Microplacas Synergy H4™️ on the CINVESTAV facilities every 20 minutes for 12 hours.</li>
 +
<li style="font-family: 'Roboto', sans-serif; text-align: justify-all; font-size: 1.2rem">Transformation with laccase biobricks 15 A, 13 I, 15 E, 13 M and PSB1C3.</li>
 +
<li style="font-family: 'Roboto', sans-serif; text-align: justify-all; font-size: 1.2rem">DH5α conservation in LB culture medium and glycerol.</li>
 +
 
 +
                                                        <li style="font-family: 'Roboto', sans-serif; text-align: justify-all; font-size: 1.2rem">BBa_K863010 recovery.</li>
 +
                                                        <li style="font-family: 'Roboto', sans-serif; text-align: justify-all; font-size: 1.2rem">Pictures of glycerol gradient assay results in LB+agar were taken.</li>
 +
</ul>
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</div>
 
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<p style="font-family: 'Roboto', sans-serif; text-align: justify-all; font-size: 1.2rem">Lorem ipsum dolor sit amet, consectetur adipisicing elit. Quisquam rerum quis consequatur voluptatibus praesentium. Ex commodi beatae cum sequi. Impedit repudiandae iste adipisci omnis velit dignissimos ad minus incidunt maiores! Lorem ipsum dolor sit amet, consectetur adipisicing elit. Aut similique, minus et blanditiis aliquid quidem sunt ipsam. Voluptas voluptatem beatae, tempora deleniti qui earum voluptatibus, cumque natus et ut, pariatur.</p>
+
<p style="font-family: 'Roboto', sans-serif; text-align: justify-all; font-size: 1.2rem; font-weight: bold">Week overview:</p>
 +
<ul class="des" style="list-style: none; padding: 0; margin: 0; display: flex; flex-direction: column;">
 +
<li style="font-family: 'Roboto', sans-serif; text-align: justify-all; font-size: 1.2rem">Inoculation of DH5α in petri dishes with glycerol gradients.</li>
 +
<li style="font-family: 'Roboto', sans-serif; text-align: justify-all; font-size: 1.2rem">Inoculation of transformed bacteria with plasmids 13I, 15A, 13M and 15E in petri dishes with pizza format (1 UFC/”slice”).</li>
 +
<li style="font-family: 'Roboto', sans-serif; text-align: justify-all; font-size: 1.2rem">Inoculation of transformed bacteria with plasmids 13I, 15A, 13M and 15E in 10 mL of liquid LB+CAM culture media. 4 tubes per plasmid, 1 UFC/tube.</li>
 +
<li style="font-family: 'Roboto', sans-serif; text-align: justify-all; font-size: 1.2rem">Mini Prep pSB1C3 plasmid extraction.</li>
 +
<li style="font-family: 'Roboto', sans-serif; text-align: justify-all; font-size: 1.2rem">Agarose gel electrophoresis to verify integrity of our extracted plasmid plasmids 13I, 13M, 15E, 15A and psB1C3.</li>
 +
 
 +
<li style="font-family: 'Roboto', sans-serif; text-align: justify-all; font-size: 1.2rem">Transformation by thermal shock of BL21 DE3 with plasmids 13 I, 15A, 13M and 15E.</li>
 +
<li style="font-family: 'Roboto', sans-serif; text-align: justify-all; font-size: 1.2rem">Laccase induction assay with IPTG.</li>
 +
<li style="font-family: 'Roboto', sans-serif; text-align: justify-all; font-size: 1.2rem">psB1C3 enzyme digestion with EcoR1 and Pst1. Agarose gel electrophoresis for digestion products.</li>
 +
 
 +
 
 +
</ul>
 +
 
</div>
 
</div>
 
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+
<p style="font-family: 'Roboto', sans-serif; text-align: justify-all; font-size: 1.2rem; font-weight: bold">Week overview:</p>
 +
<ul class="des" style="list-style: none; padding: 0; margin: 0; display: flex; flex-direction: column;">
 +
<li style="font-family: 'Roboto', sans-serif; text-align: justify-all; font-size: 1.2rem">Re-inoculation of BL21 strain with each laccase plasmid (15E, 15A, 13M and 13 I) in LB+agar+CAM and LB+CAM culture media and induction with IPTG.</li>
 +
<li style="font-family: 'Roboto', sans-serif; text-align: justify-all; font-size: 1.2rem">Laccase induction assay with colorants, acetone and guayacol. SDS-PAGE.</li>
 +
<li style="font-family: 'Roboto', sans-serif; text-align: justify-all; font-size: 1.2rem">psB1C3 enzyme digestion with EcoR1 and Pst1 was repeated. Agarose gel electrophoresis for digestion products.</li>
 +
 
 +
</ul>
 +
 
</div>
 
</div>
 
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<p style="font-family: 'Roboto', sans-serif; text-align: justify-all; font-size: 1.2rem">Lorem ipsum dolor sit amet, consectetur adipisicing elit. Quisquam rerum quis consequatur voluptatibus praesentium. Ex commodi beatae cum sequi. Impedit repudiandae iste adipisci omnis velit dignissimos ad minus incidunt maiores! Lorem ipsum dolor sit amet, consectetur adipisicing elit. Aut similique, minus et blanditiis aliquid quidem sunt ipsam. Voluptas voluptatem beatae, tempora deleniti qui earum voluptatibus, cumque natus et ut, pariatur.</p>
+
<p style="font-family: 'Roboto', sans-serif; text-align: justify-all; font-size: 1.2rem; font-weight: bold">Week overview:</p>
 +
<ul class="des" style="list-style: none; padding: 0; margin: 0; display: flex; flex-direction: column;">
 +
<li style="font-family: 'Roboto', sans-serif; text-align: justify-all; font-size: 1.2rem">Propagation of competent cells and strains with laccase 15 A, 15 E, 13 M and 13 I.</li>
 +
<li style="font-family: 'Roboto', sans-serif; text-align: justify-all; font-size: 1.2rem">Laccase precipitation assay. 2 SDS-PAGE to verify laccase concentration.</li>
 +
<li style="font-family: 'Roboto', sans-serif; text-align: justify-all; font-size: 1.2rem">Microplastic determination assay with DLS (Dynamic Light Scattering). The assay was not successful because the equipment didn’t work properly.</li>
 +
 
 +
<li style="font-family: 'Roboto', sans-serif; text-align: justify-all; font-size: 1.2rem">psB1C3 enzyme digestion with EcoR1 and Pst1. Agarose gel electrophoresis for digestion products.</li>
 +
<li style="font-family: 'Roboto', sans-serif; text-align: justify-all; font-size: 1.2rem">We received the sequences that we sent to IDT for the enterprise to synthesize them for us (GBlocks ESR1 and EmGFP), along with the primers (M13 Fwd and M13 Rev) for us to make several copies of them. Amplification of received sequences.</li>
 +
 
 +
</ul>
 +
 
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<p style="font-family: 'Roboto', sans-serif; text-align: justify-all; font-size: 1.2rem">Lorem ipsum dolor sit amet, consectetur adipisicing elit. Quisquam rerum quis consequatur voluptatibus praesentium. Ex commodi beatae cum sequi. Impedit repudiandae iste adipisci omnis velit dignissimos ad minus incidunt maiores! Lorem ipsum dolor sit amet, consectetur adipisicing elit. Aut similique, minus et blanditiis aliquid quidem sunt ipsam. Voluptas voluptatem beatae, tempora deleniti qui earum voluptatibus, cumque natus et ut, pariatur.</p>
+
<p style="font-family: 'Roboto', sans-serif; text-align: justify-all; font-size: 1.2rem; font-weight: bold">Week overview:</p>
 +
<ul class="des" style="list-style: none; padding: 0; margin: 0; display: flex; flex-direction: column;">
 +
<li style="font-family: 'Roboto', sans-serif; text-align: justify-all; font-size: 1.2rem">Laccase induction assay with colorants.</li>
 +
<li style="font-family: 'Roboto', sans-serif; text-align: justify-all; font-size: 1.2rem">PCR for ESR1 and emRFP: Agarose gel electrophoresis for amplicons and DNA extraction from agarose gel with GenElute™ Gel Extraction Kit.</li>
 +
<li style="font-family: 'Roboto', sans-serif; text-align: justify-all; font-size: 1.2rem">Biobrick BBa_B0010 extraction.</li>
 +
 
 +
<li style="font-family: 'Roboto', sans-serif; text-align: justify-all; font-size: 1.2rem">Enzyme digestion for pSB1C3 with EcoR1 and Pst1.</li>
 +
<li style="font-family: 'Roboto', sans-serif; text-align: justify-all; font-size: 1.2rem">Chemocompetent BL21 preparation protocol.</li>
 +
 
 +
 
 +
 
 +
<li style="font-family: 'Roboto', sans-serif; text-align: justify-all; font-size: 1.2rem">Laccase extraction assay with Nickel and repetition.</li>
 +
<li style="font-family: 'Roboto', sans-serif; text-align: justify-all; font-size: 1.2rem">Inoculation of J3100-17D in LB+agar+AMP culture media. No growth observed.</li>
 +
<li style="font-family: 'Roboto', sans-serif; text-align: justify-all; font-size: 1.2rem">Transformation by thermal shock of DH5α with psIJ8.</li>
 +
<li style="font-family: 'Roboto', sans-serif; text-align: justify-all; font-size: 1.2rem">BBa_180005 propagation (promoter and RBS) in chemocompetent DH5α with LB + CAM culture medium.</li>
 +
<li style="font-family: 'Roboto', sans-serif; text-align: justify-all; font-size: 1.2rem">Transformation by thermal shock of DH5a with pSIJ8 and BBa_K081005.</li>
 +
 
 +
 
 +
<li style="font-family: 'Roboto', sans-serif; text-align: justify-all; font-size: 1.2rem">KatE and PUL-ori-MCS ligation. They were cut with NcoI and HindIII with buffer 2.1.</li>
 +
<li style="font-family: 'Roboto', sans-serif; text-align: justify-all; font-size: 1.2rem">Ligation of GFP, ESR1 and pSB1C3.</li>
 +
<li style="font-family: 'Roboto', sans-serif; text-align: justify-all; font-size: 1.2rem">Ligation of KatE, PuDHT, AldoO and mRFP in backbone.</li>
 +
<li style="font-family: 'Roboto', sans-serif; text-align: justify-all; font-size: 1.2rem">pSB1C3 plasmid extraction.</li>
 +
<li style="font-family: 'Roboto', sans-serif; text-align: justify-all; font-size: 1.2rem">pSB1C digestion with EcoRI and PstI and agarose gel electrophoresis.</li>
 +
</ul>
 +
 
</div>
 
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Revision as of 05:34, 14 October 2021

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Notebook lab

Week 1

Week overview:

  • Cleansing and organization of our drawers.
  • Sterilization of water, flasks, microtubes, conic tubes and micropipette tips.
  • Preparation of antibiotic stock solutions.
  • Preparation and sterilization of LB+agar, LB+agar+CAM, LB+CAM and LB culture media (the media with antibiotic were sterilized before adding the antibiotic).
  • Preparation and sterilization of cell preservation glycerol solutions.
  • Propagation of E. coli strains in LB+agar culture medium.
  • Chemocompetent cells preparation for Sure and C43 strains.
  • Transformation of BL21 chemocompetent cells with laccase BBa_K729006.
  • Electrocompetent cells preparation for SURE and C43.
  • Cell preservation in glycerol of Sure and C43 strains.

Week 2

Week overview:

  • First Antibiogram-like assay with glycerol gradients 0-25% and DH5α strains.
  • Inoculation and propagation of E. coli strains with their respective antibiotics.
  • Electrotransformation of Sure and C43 with laccase, at 2000 and 2500 V
  • Transformation of BL21 DE3 by heat shock with laccase plasmid.
  • First glycerol gradient assay in LB culture medium.
  • Induction with 1 M IPTG of electrotransformed C43 cells.
  • Sure and C43 competent cells preservation in glycerol.
  • Glycerol gradient assay was repeated.

Week 3

Week overview:

  • Electrocompetent preparation of E. coli strains.
  • BL21-laccase induction with IPTG and SDS-PAGE of induced samples and Semi-dry membrane transference.
  • Measurement of optical density (OD600) of glycerol gradient assay cultures.
  • Optical density measurement for competent cell cultures.

Week 4

Week overview:

  • Optical density (OD600) for glycerol gradients were measured approximately every 40 minutes for almost 9 hours and graphed.
  • Optical density (OD600) for glycerol gradients were also measured with Lector Híbrido Multi-Modal de Microplacas Synergy H4™️ on the CINVESTAV facilities every 20 minutes for 12 hours.
  • Transformation with laccase biobricks 15 A, 13 I, 15 E, 13 M and PSB1C3.
  • DH5α conservation in LB culture medium and glycerol.
  • BBa_K863010 recovery.
  • Pictures of glycerol gradient assay results in LB+agar were taken.

Week 5

Week overview:

  • Inoculation of DH5α in petri dishes with glycerol gradients.
  • Inoculation of transformed bacteria with plasmids 13I, 15A, 13M and 15E in petri dishes with pizza format (1 UFC/”slice”).
  • Inoculation of transformed bacteria with plasmids 13I, 15A, 13M and 15E in 10 mL of liquid LB+CAM culture media. 4 tubes per plasmid, 1 UFC/tube.
  • Mini Prep pSB1C3 plasmid extraction.
  • Agarose gel electrophoresis to verify integrity of our extracted plasmid plasmids 13I, 13M, 15E, 15A and psB1C3.
  • Transformation by thermal shock of BL21 DE3 with plasmids 13 I, 15A, 13M and 15E.
  • Laccase induction assay with IPTG.
  • psB1C3 enzyme digestion with EcoR1 and Pst1. Agarose gel electrophoresis for digestion products.

Week 6

Week overview:

  • Re-inoculation of BL21 strain with each laccase plasmid (15E, 15A, 13M and 13 I) in LB+agar+CAM and LB+CAM culture media and induction with IPTG.
  • Laccase induction assay with colorants, acetone and guayacol. SDS-PAGE.
  • psB1C3 enzyme digestion with EcoR1 and Pst1 was repeated. Agarose gel electrophoresis for digestion products.

Week 7

Week overview:

  • Propagation of competent cells and strains with laccase 15 A, 15 E, 13 M and 13 I.
  • Laccase precipitation assay. 2 SDS-PAGE to verify laccase concentration.
  • Microplastic determination assay with DLS (Dynamic Light Scattering). The assay was not successful because the equipment didn’t work properly.
  • psB1C3 enzyme digestion with EcoR1 and Pst1. Agarose gel electrophoresis for digestion products.
  • We received the sequences that we sent to IDT for the enterprise to synthesize them for us (GBlocks ESR1 and EmGFP), along with the primers (M13 Fwd and M13 Rev) for us to make several copies of them. Amplification of received sequences.

Week 8

Week overview:

  • Laccase induction assay with colorants.
  • PCR for ESR1 and emRFP: Agarose gel electrophoresis for amplicons and DNA extraction from agarose gel with GenElute™ Gel Extraction Kit.
  • Biobrick BBa_B0010 extraction.
  • Enzyme digestion for pSB1C3 with EcoR1 and Pst1.
  • Chemocompetent BL21 preparation protocol.
  • Laccase extraction assay with Nickel and repetition.
  • Inoculation of J3100-17D in LB+agar+AMP culture media. No growth observed.
  • Transformation by thermal shock of DH5α with psIJ8.
  • BBa_180005 propagation (promoter and RBS) in chemocompetent DH5α with LB + CAM culture medium.
  • Transformation by thermal shock of DH5a with pSIJ8 and BBa_K081005.
  • KatE and PUL-ori-MCS ligation. They were cut with NcoI and HindIII with buffer 2.1.
  • Ligation of GFP, ESR1 and pSB1C3.
  • Ligation of KatE, PuDHT, AldoO and mRFP in backbone.
  • pSB1C3 plasmid extraction.
  • pSB1C digestion with EcoRI and PstI and agarose gel electrophoresis.

Week 9

Lorem ipsum dolor sit amet, consectetur adipisicing elit. Quisquam rerum quis consequatur voluptatibus praesentium. Ex commodi beatae cum sequi. Impedit repudiandae iste adipisci omnis velit dignissimos ad minus incidunt maiores! Lorem ipsum dolor sit amet, consectetur adipisicing elit. Aut similique, minus et blanditiis aliquid quidem sunt ipsam. Voluptas voluptatem beatae, tempora deleniti qui earum voluptatibus, cumque natus et ut, pariatur.

Week 10

Lorem ipsum dolor sit amet, consectetur adipisicing elit. Quisquam rerum quis consequatur voluptatibus praesentium. Ex commodi beatae cum sequi. Impedit repudiandae iste adipisci omnis velit dignissimos ad minus incidunt maiores! Lorem ipsum dolor sit amet, consectetur adipisicing elit. Aut similique, minus et blanditiis aliquid quidem sunt ipsam. Voluptas voluptatem beatae, tempora deleniti qui earum voluptatibus, cumque natus et ut, pariatur.

Week 11

Lorem ipsum dolor sit amet, consectetur adipisicing elit. Quisquam rerum quis consequatur voluptatibus praesentium. Ex commodi beatae cum sequi. Impedit repudiandae iste adipisci omnis velit dignissimos ad minus incidunt maiores! Lorem ipsum dolor sit amet, consectetur adipisicing elit. Aut similique, minus et blanditiis aliquid quidem sunt ipsam. Voluptas voluptatem beatae, tempora deleniti qui earum voluptatibus, cumque natus et ut, pariatur.

Week 12

Lorem ipsum dolor sit amet, consectetur adipisicing elit. Quisquam rerum quis consequatur voluptatibus praesentium. Ex commodi beatae cum sequi. Impedit repudiandae iste adipisci omnis velit dignissimos ad minus incidunt maiores! Lorem ipsum dolor sit amet, consectetur adipisicing elit. Aut similique, minus et blanditiis aliquid quidem sunt ipsam. Voluptas voluptatem beatae, tempora deleniti qui earum voluptatibus, cumque natus et ut, pariatur.

Week 13

Lorem ipsum dolor sit amet, consectetur adipisicing elit. Quisquam rerum quis consequatur voluptatibus praesentium. Ex commodi beatae cum sequi. Impedit repudiandae iste adipisci omnis velit dignissimos ad minus incidunt maiores! Lorem ipsum dolor sit amet, consectetur adipisicing elit. Aut similique, minus et blanditiis aliquid quidem sunt ipsam. Voluptas voluptatem beatae, tempora deleniti qui earum voluptatibus, cumque natus et ut, pariatur.

Week 14

Lorem ipsum dolor sit amet, consectetur adipisicing elit. Quisquam rerum quis consequatur voluptatibus praesentium. Ex commodi beatae cum sequi. Impedit repudiandae iste adipisci omnis velit dignissimos ad minus incidunt maiores! Lorem ipsum dolor sit amet, consectetur adipisicing elit. Aut similique, minus et blanditiis aliquid quidem sunt ipsam. Voluptas voluptatem beatae, tempora deleniti qui earum voluptatibus, cumque natus et ut, pariatur.

Protocols

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