Team:TecCEM/Notebook

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Protocols

Laboratory protocols:


This document is a compilation of the protocols we followed all along our experimentation and work inside the laboratory. It contains the detailed procedure of 27 molecular biology, microbiology, and DLS protocols, as well as the recipes for 10 buffers and solutions. It also contains an index so that the desired protocol could be found in a quick and easy manner.

Through our lab notebook you will find which protocols we carried out (they are highlighted in color blue), but the procedure that we followed is not detailed. We decided to create this document, so that you know exactly how each and every one of our protocols were carried out. The purpose of this document was also for us to follow a standardized procedure for not having variations in our results due to differences in the way we carried out those 27 protocols.

We are also aiming to share this document with the Bioengineering Department of our school, for future use of iGEM teams or students in general. Having all molecular biology and microbiology protocols in one document makes the learning process so much easier.

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

Week overview:

  • Inoculation of E. coli 10B cells with plasmid pSIJ8.
  • Inoculation of E. coli DH5a cells with BBa_K081005.
  • Plasmid extraction from 10B-pSIJ8 and DH5a-BBa_K081005 with PureLink® Quick Plasmid Mini Prep Kit and agarose gel electrophoresis.
  • Laccase induction assay with colorants.
  • Agarose gel electrophoresis to verify ligations from Week7 and DNA elution from agarose gel of each well, except marker, J23100 and CFP, with Gen Elute™ Gel Extraction Kit.
  • Transformation by thermal shock of SoluBL21 with the laccases C- 15A and D - 13I. IPTG induction of BL21-15E culture.
  • 4 SDS-PAGE from Week 8 laccase induction assays samples.
  • Ligation of pUCori and pSB1C3 with different inserts (ESR1, PuDHT, AldO, mRFP and KatE).
  • Transformation by thermal shock with ligation product. Competent DH5a and pSBIC3 + GFP (10 mL).
  • pSIJ8 digestion with NcoI and HindIII.
  • BBa_K863010 (15A) induction with IPTG.

Week 10

Week overview:

  • Two SDS-PAGE for pellet and media samples of laccase 15A in E. coli SoluBL21, obtained through laccase induction with IPTG in Week 9.
  • E. coli 10β was transformed by thermal shock with the ligation products from Week 9.
  • Nickel interaction assay with the soluble fraction and the media of our induced cultures with laccase 15 A
  • Digestions for BBa_K081005, J23100, KatE and PucOri.
  • Laccase induction asay with colorants.
  • Ligation of genes in pSB1C3.
  • Transformation by thermal shock of DH5a with ligation products.
  • Colony PCR of emGFP.
  • J23100 enzyme digestion with PstI and SpeI and agarose gel electrophoresis.
  • Laccase interaction assay with Nickel and SDS-PAGE.
  • Electrotransformation of C43 with laccase 15A.

Week 11

Week overview:

  • Re-inoculation of E.coli with pSB1C3+ESR1.
  • Repetition of Electrotransformation of C43 with laccase 15A.
  • Plasmid extraction (pSB1C3-ESR1) using Pure Link® Quick Plasmid Miniprep Kit.
  • PCR of ESR1.
  • DH5a Transformation by thermal shock of with the following ligation products
    • pSB1CE + KatE
    • pSB1C3 + aldo
    • pUCori + KatE
    • pSB1C3 + PuDAT
    • pSB1C3 + mRFP
  • Preparation of chemocompetent E. coli C43.
  • BBa_K081005 (laccase 15 E) extraction
  • Digestion of J23100, BBa_K081005 and pSB1C3-ESR1, agarose gel electrophoresis of digestion products and elution of fragments J23100 S+N+P and ESR1 X+P.
  • Plasmid extraction of DH5a pSIJ8, PucOri+KatE, pSB1C3+PucOri, pSB1C3+ESR1 and SoluBL21 laccase 15E (BBa_K081005) using Pure Link® Quick Plasmid Miniprep Kit.
  • Transformation by thermal shock of competent DH5a with BBa_K081005.
  • Cloning of AldO, PuDHT, mRFP, pUCori, KatE and ESR1 ¿ in cloning plasmid TA: TOPO-Ligation.
  • Agarose gel electrophoresis of PCR and digestion products of ERS1, J23100 and pSB1C3+ESR1 and elution of J23100 S+N+P from electrophoresis using Gen Elute™ Gel Extraction Kit.
  • Transformation by thermal shock of:
    • BB for UPIBI: CAM BBa_K325909.
    • J251 - CAM.
    • J2CFF - CAM.
    • PCR products: ALDO, PUDHT, mRFP, pUC.ori, KatE, ESR1.

Week 12

Week overview:

  • BBa_K081005 Plasmid extraction using Pure Link® Quick Plasmid Miniprep Kit.
  • Transformation by thermal shock of Biobricks and GBlocks.
  • BBa_K081005 Plasmid extraction of E. coli C43 with 15A laccase.
  • IPTG induction of transformed products and BL21 DE3 + 15A.
  • BBa_K081005 in pSB1C3

    digestion

    with SpeI and PstI.
  • Protein precipitation assay on SoluBL21 with 15A laccase culture.
  • Transformation by thermal shock of DH5a with BBa_K325409, for UPIBI.
  • Digestion of ESR1 with XbaI, PstI and NcoI.
  • Digestion of emRFP with XbaI and PstI.
  • Digestion of BBa_K081005 with SpeI and PstI.
  • Agarose gel electrophoresis for digestion products and elution of fragments from previous gel using Gen Elute™ Gel Extraction Kit.
  • SDS-PAGE for laccase induction assay samples (before and after induction, culture media and soluble fraction).
  • Transformation by thermal shock of DH5a with BBa_K325909.
  • Propagation of pSB1C3-emGFP-DH5a in LB+CAM culture medium.
  • Propagation of pSB1C3-ESR1-DH5a in LB+CAM culture medium.
  • Propagation of BBa_K081005-DH5a in LB+CAM culture medium.

Week 13

Week overview:

  • Zetasizer protocol was carried out to quantify endocrine disruptive compounds on different water brands at different conditions.
  • ESR1 plasmid extraction using Pure Link® Quick Plasmid Miniprep Kit, agarose gel electrophoresis and elution of DNA.
  • BBa_K081005 and GFP plasmid extraction using Pure Link® Quick Plasmid Miniprep Kit.
  • IPTG induction of transformed products and BL21 DE3 + 15A.
  • Ligation of ESR1 and GFP in BBa_K081005, using New England Biolabs® kit.
  • PCR PolyA of emRFP.
  • PCR of aldO and PuDHT, agarose gel electrophoresis and elution of DNA.
  • Topo ligation of mRFP for transformation of DH5a and agarose gel electrophoresis.
  • Laccase induction assay with Nickel to purify laccase and measurement of absorbance at 280 nm of purification fractions.
  • Genetic edition assay to DH5a (knockout).
  • Transformation by thermal shock of DH5a ligation with ESR1 and GFP.

Week 14

Week overview:

  • SDS-PAGE for laccase induction and purification assay carried out last week.
  • Colony PCR of colonies with ERS1 and GFP to verify inserts on BBa_K081005 and agarose gel electrophoresis for PCR products.
  • Plasmid extraction of GFP+K081005 and ESR1+BBa_K08005 ligation candidates, and agarose gel electrophoresis.
  • Samples from laccase induction assay with colorants were analyzed with spectrophotometer: typical curves of each colorant were obtained as well as absorbance vs time curves.
  • Protein quantification with BCA assay.
  • Colony PCR of 30 different DH5a-pSB1C3+ESR1 colonies and agarose gel electrophoresis to verify ESR1 insert of Colony PCR products.
  • AldO and puDHT PCR products precipitation with glycogen and ethanol, for genetic edition assay, agarose gel electrophoresis, and elution from gel.
  • Plasmid extraction of DH5a pSB1C3-ESR1 colonies numbered as 8, 13 and 18, and agarose gel electrophoresis.
  • Transformation of BL21 with pSB1C3-ESR1 candidates 8, 13 and 18.
  • Transformation of DH5a with pSIJ8 and Knockout with pUCOri+KatE.
  • Transformation of BL21 with BBa_K081005-ESR1.
  • Plasmid extraction of DH5a pSB1C3-ESR1 colonies numbered as 21, 23 and 26.
  • Transformation of BL21 with pSB1C3-ESR1 candidates 21, 23 and 26.
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