Nootebook

Week 1 (07/23/2023 - 07/29/2023) Week 2 (07/30/2023 - 08/05/2023) Week 3 (08/06/2023 - 08/12/2023) Week 4 (08/13/2023 - 08/19/2023) Week 5 (08/20/2023 - 08/26/2023) Week 6 (08/27/2023 - 09/02/2023) Week 7 (09/03/2023 - 09/09/2023) Week 8 (09/10/2023 - 09/16/2023) Week 9 (09/17/2023 - 09/23/2023) Week 10 (09/24/2023 - 09/30/2023) Week 11 (10/01/2023 - 10/06/2023)

Week 1 (07/23/2023 - 07/29/2023)

Objective of the week: Reactivating competent cells with the RFP plasmid...

  • 07/28

    Testing the quimiocompetence INOUE method with DH5a cells

    Reactivation of DH10B cells that had the pSB1K3 plasmid

    Responsible: Jamily Brito Dorado and Bernie Reyes

Week 2 (07/30/2023 - 08/05/2023)

Objective of the week: Testing our available competent cells for future clonings...

  • 08/01

    Inoculation of reactivated DH10B_pSB1K3-RFP cells

    Cultivation of inoculated DH10B_pSB1K3-RFP cells

    Responsible: Jamily Brito Dorado, Lucas Ovelar, Cristian Rojas, and Bernie Reyes

  • 08/02

    Cultivation of INOUE_DH5a cells

    Centrifugation of eppendofs with aliquots from the culture of DH10B_pSB1K3-RFP cells...

    Responsible: Jamily Brito Dorado, Lucas Ovelar, Cristian Rojas, and Bernie Reyes

Week 3 (08/06/2023 - 08/12/2023)

Objective of the week: Design primers, continue the transformation process while monitoring...

  • 08/06

    Designing primers for colony PCR

    Responsible: Jamily Brito Dorado, Lucas Ovelar

  • 08/07

    Verify the cell growth in petri dishes

    Purification of the minipreps made in 08/03

    Responsible: Jamily Brito Dorado, Matheus Araújo, and Bernie Reyes

  • 08/08

    Continuation of the purification process

    Nanodrop readings

    Responsible: Jamily Brito Dorado, Lucas Ovelar, Bernie Reyes, and Cristian Rojas

  • 08/09

    NanoDrop readings of the minipreps

    New inoculum culture for new miniprep assay

    Responsible: Jamily Brito Dorado, Lucas Ovelar, Bernie Reyes, and Cristian Rojas

  • 08/10

    Miniprep using homemade kit

    Miniprep using NEB kit

    NanoDrop readings

    Responsible: Jamily Brito Dorado, Lucas Ovelar, Matheus Araújo, and Bernie Reyes

  • 08/11

    Preparing transformation plates

    Preparing minipreps for transformation

    NanoDrop readings

    Responsible: Jamily Brito Dorado and Bernie Reyes

Week 4 (08/13/2023 - 08/19/2023)

Objective of the week: Evaluation of the transformations based on the transformation of chemically competent cells...

  • 08/14

    Aliquoting the plasmid pSB1K3

    Resuspension of the plasmid pJP32PHL7

    NanoDrop readings of the pJP32PHL7 resuspension

    Chemically competent cells transformation with pSB1K3 using the heat shock method

    Chemically competent cells transformation with pJP32PHL7 using the heat shock method

    Responsible: Jamily Brito Dorado, Lucas Ovelar, Bernie Reyes, and Cristian Rojas

  • 08/15

    Evaluating transformation plates

    Preparing plates for culturing

    Preparing tubes for inoculum

    Antibiotic quotation (Zeocin/Bleomicin)

    Responsible: Jamily Brito Dorado and Bernie Reyes

  • 08/16

    Inoculum of the transformed cells (pJP32PHL7)

    Responsible: Jamily Brito Dorado

  • 08/17

    pJP32PHL7 miniprep

    Aliquoting and storing the miniprep

    Responsible: Jamily Brito Dorado, Thaynara Castro, and Bernie Reyes

  • 08/18

    We received the strains CC-400, CC-1690, and CC-1691, kindly donated by professor Lívia Seno...

    Identifying restriction regions of the pJP32PHL7 plasmid

    Responsible: Jamily Brito Dorado, Lucas Ovelar, and Bernie Reyes

Week 5 (08/20/2023 - 08/26/2023)

Objective of the week: Preparing the TAP media for culturing our donated Chlamydomonas reinhardtii strains and continuing to verify the pJP32PHL7 resuspension.

  • 08/21

    NanoDrop readings (pJP32PHL7)

    Enzyme digestion protocol (NEB) familiarization

    Organizing and obtaining the ingredients for TAP medium preparation

    Responsible: Jamily Brito Dorado, Lucas Ovelar, Ghadir Wissam Rizk, Bernie Reyes, and Cristian Rojas

  • 08/22

    NanoDrop readings (pJP32PHL7)

    Enzyme digestion protocol execution

    Agarose gel electrophoresis with the digestion result

    IDT sequences order

    Electrophoresis gel results analysis

    Responsible: Jamily Brito Dorado, Lucas Ovelar, Ghadir Wissam Rizk, Bernie Reyes, and Cristian Rojas

  • 08/23

    TAP medium ingredients were obtained and separated

    Agarose gel electrophoresis with the digestion result

    Responsible: Cristian Rojas

  • 08/24

    Preparation of TAP medium stock solutions

    Responsible: Jamily Brito Dorado, Lucas Ovelar, Ivan Mateo Espitia, Thaynara Castro, Bernie Reyes, and Cristian Rojas

  • 08/25

    Electrophoresis gel results analysis

    TAP medium stock solutions pH adjustments

    New streaking plates with pHP32PHL7 E. coli colonies

    Responsible: Jamily Brito Dorado, Lucas Ovelar, Ivan Mateo Espitia, and Cristian Rojas

  • 08/26

    We kindly received a donation of the antibiotic zeocin from Wesley Luzetti Fotoran, a colleague USP student

    Responsible: Lucas Ovelar

Week 6 (08/27/2023 - 09/02/2023)

Objective of the week: Preparing the TAP media for culturing our donated Chlamydomonas reinhardtii strains and continuing to verify the pJP32PHL7 resuspension.

  • 08/28

    pJP32PHL7 streak plates evaluation

    New solid medium plates preparation

    1L TAP medium preparation

    Responsible: Jamily Brito Dorado, Lucas Ovelar, and Bernie Reyes

  • 08/29

    C. reinhardtii CC-400, CC-1690 and CC1691 inoculum with TAP medium

    DH10B cells with pJP32PHL7 inoculum

    LB liquid medium preparation

    LB solid medium preparation

    Responsible: Jamily Brito Dorado, Lucas Ovelar, and Bernie Reyes

  • 08/30

    C. reinhardtii strains inoculum growth verification

    Responsible: Jamily Brito Dorado

  • 08/31

    Alkaline lysis miniprep execution

    Homemade miniprep execution

    Miniprep solutions preparation

    NanoDrop readings

    Responsible: Jamily Brito Dorado, Lucas Ovelar, and Bernie Reyes

  • 09/01

    C. reinhardtii strains inoculum growth verification

    Responsible: Jamily Brito Dorado

Week 7 (09/03/2023 - 09/09/2023)

Objective of the week: Purify our minipreps to confirm that we obtained the pJP32PHL7 plasmid, isolate the Chlamydomonas reinhardtii cells from contaminated culture and results analysis.

  • 09/04

    Ethanol precipitation of RNA/DNA protocol execution

    C. reinhardtii strains inoculum growth verification: contaminated

    New C. reinhardtii inoculums with TAP medium

    Miniprep NanoDrop readings

    Responsible: Jamily Brito Dorado, Lucas Ovelar, and Bernie Reyes

  • 09/05

    Agarose gel electrophoresis preparation

    Contaminated crops were placed next to a lamp so that C. reinhardtii cells migrated towards the light and isolated

    Responsible: Jamily Brito Dorado, Lucas Ovelar, Bernie Reyes, and Cristian Rojas

  • 09/06

    Agarose gel electrophoresis

    Responsible: Jamily Brito Dorado, Lucas Ovelar, and Bernie Reyes

  • 09/07

    Agarose gel results analysis

    C. reinhardtii culture results analysis

    Responsible: Jamily Brito Dorado, Lucas Ovelar, and Bernie Reyes

Week 8 (09/10/2023 - 09/16/2023)

Objective of the week: Culturing our donated Chlamydomonas reinhardtii strains, evaluating the dye reagents we had available to verify their functioning, as well as verifying the success of the minipreps made throughout the weeks using samples already known as a comparison parameter.

  • 09/11

    pJP32PHL7 transformation assay

    Agarose gel electrophoresis to verify the samples and dyes

    C. reinhardtii culture verification

    C. reinhardtii cell counting

    Degradation test plates reagents organization and quotation

    Responsible: Jamily Brito Dorado, Lucas Ovelar, Bernie Reyes, and Cristian Rojas

  • 09/12

    pJP32PHL7 cloning assay

    Miniprep protocol evaluation

    Agarose gel dyes results evaluation

    Responsible: Jamily Brito Dorado, Lucas Ovelar, and Bernie Reyes

  • 09/13

    Agarose gel electrophoresis with different concentrations of dye and successful DNA samples

    Agarose gel results analysis

    Microplastic dispersion research

    Responsible: Jamily Brito Dorado and Cristian Rojas

  • 09/14

    pJP32PHL7 transformation using the heat shock method with donated competent cells from PTI and our available DH5a cells

    Agarose gel electrophoresis with different concentrations of Diamond Dye and TAE buffer

    Agarose gel analysis by transluminator results

    TAP medium preparation

    Responsible: Jamily Brito Dorado, Thaynara Castro, Lucas Ovelar, and Bernie Reyes

  • 09/15

    Agarose gel electrophoresis with different 1:100 concentration of Diamond Dye and TBE buffer

    Agarose gel analysis by transluminator results

    Antifungal plate with TAP medium preparation

    Structuring the Chlamy Manual

    Responsible: Jamily Brito Dorado, Lucas Ovelar, and Bernie Reyes

Week 9 (09/17/2023 - 09/23/2023)

Objective of the week: Culturing our donated Chlamydomonas reinhardtii strains, continuing to verify the pJP32PHL7 resuspension, and executing a collaboration protocol with other iGEM teams.

  • 09/18

    Transformation plates with pJP32PHL7 and PTI competent cells were evaluated and with good results

    Inoculum preparation for miniprep

    C. reinhardtii culture verification - contaminated

    New streak plates on TAP solid medium with all the C. reinhardtii strains available

    Streaking C. reinhardtii in plates prepared previously with antifungal and TAP medium

    Responsible: Jamily Brito Dorado, Lucas Ovelar, Bernie Reyes, and Cristian Rojas

  • 09/19

    Miniprep protocol execution

    Check plates with pJP32 PTI transformed cells

    LB medium plates preparation for G0 plasmid transformation

    Polyurethane dispersion quotation

    iGEM Stockholm x iGEM VIT Vellore Collaboration protocol execution

    Responsible: Jamily Brito Dorado, Lucas Ovelar, Bernie Reyes, and Cristian Rojas

  • 09/20

    NanoDrop readings

    Results evaluation

    Next steps analysis

    Responsible: Lucas Ovelar and Bernie Reyes

  • 09/21

    TE buffer preparation

    EDTA solution preparation

    Agarose gel electrophoresis

    Chemically competent cells quotation

    Responsible: Jamily Brito Dorado, Lucas Ovelar, Bernie Reyes, and Cristian Rojas

  • 09/22

    Reviewing all transformation assays and analyzing results

    Lab next steps organization and planning

    Agarose gel electrophoresis

    Agarose gel electrophoresis results evaluation

    Responsible: Jamily Brito Dorado, Lucas Ovelar, and Bernie Reyes

Week 10 (09/24/2023 - 09/30/2023)

Objective of the week: Reactivation and linearization of G0 and pJP32PHL7 plasmids, resuspending IDT primers, optimizing lab work, and planning next steps based on our meeting with João Molino (Advisor).

  • 09/25

    TE 10X pH verification

    Setting a meeting with our advisor João Molino

    Lambda digestion with PstI

    IDT primers resuspensions

    G0 reactivation

    Responsible: Lucas Ovelar, Bernie Reyes, and Cristian Rojas

  • 09/26

    Organization of the week

    Polystyrene dispersion quotation

    Responsible: Lucas Ovelar, Bernie Reyes, and Cristian Rojas

  • 09/27

    pJP32PHL7 linearization PCR

    Agarose gel electrophoresis

    C. reinhardtii new plates re-streak

    Responsible: Jamily Brito Dorado, Lucas Ovelar, Bernie Reyes, and Cristian Rojas

  • 09/28

    Agarose gel plasmid extraction

    Streaking CC-400 strain to carbanzandin plates with different concentrations

    Culturing CC-400 strain into new liquid TAP medium

    Inoue method testing assay

    Responsible: Jamily Brito Dorado, Lucas Ovelar, Bernie Reyes, and Cristian Rojas

  • 09/29

    Inoue method execution

    Advisor meeting

    Policaprolactone purchase

    Preparation of materials for culturing CC-400 strain in sewage with different concentrations and mediums

    TAP tris minimal preparation

    Responsible: Jamily Brito Dorado

  • 09/30

    Policaprolactone purchase

    Preparation of materials for culturing CC-400 strain in sewage with different concentrations and mediums

    TAP tris minimal preparation

    Responsible: Jamily Brito Dorado

Week 11 (10/01/2023 - 10/06/2023)

Objective of the week: Culturing the strain CC-400 in different concentrations of sewage and medium, verifying growth in the carbandanzin plates with CC-400 strain, and linearizing the pJP32PHL7 and G0 plasmids.

  • 10/02

    Autoclaving materials for sewage culture

    pJP32PHL7 linearization PCR with different polymerases

    Agarose gel electrophoresis

    CC-400 growth verification in carbandanzin plates

    CC-400 inoculum in cultures with different concentrations of sewage

    Responsible: Jamily Brito Dorado, Cristian Rojas, and Bernie Reyes

  • 10/03

    Agarose gel electrophoresis with PCR products and different samples for comparison

    Agarose gel results analysis

    Microplastic dispersion research

    Responsible: Jamily Brito Dorado and Cristian Rojas

  • 10/04

    G0 linearization PCR

    LB with kanamycin plates preparation

    Agarose gel electrophoresis preparation

    Responsible: Jamily Brito Dorado

  • 10/05

    Agarose gel electrophoresis with linearization results

    Results analysis - negative

    G0 linearization PCR

    Agarose gel electrophoresis with linearization results

    Responsible: Jamily Brito Dorado