Team:Brasil-USP/Notebook/Calendar

Notebook

Calendar

July

Monday Tuesday Wednesday Thursday Friday Saturday Sunday
01/06
- Agarose gel (RBS_hokD)

- Gel purification (RBS_hokD)

- Ligation 2X_RBS_hokD (RBS_hokD + RBS_hokD)

- EXSP Digestion (P0040, Pl_RBS, GFP_R0440)

- Agarose gel (P0040)

- Gel purification (P0040)

- Ligation Pt_I13507(P0040 + I13507)

- Interlab gel purification and ligation


02/06
- Transformation Ptet_I13507

- Agarose gel (Pl_RBS, GFP_P0440)

- Gel purification (Pl_RBS, GFP_P0440)

- Ligation Pl_P0440 (Pl_RBS + GFP_P0440)

- Interlab transformation (with wrong antibiotic ampicillin - is possible found in registry the same parts in another backbone BBa_J61002)


 03/06
- Transformation (Pr_RBS, 2X_RBS_hokD)

- EXSP digestion (P0440, R0040)

- Transformation Pl_R0440 (Pl_RBS + GFP_P0440)

- Inoculum (Pt_I13507)


 04/06
- Agarose gel (P0440, R0040)

- Gel purification (P0440, R0040)

- Ligation tet_Ptet (P0440 + R0040)

- Inoculum (2X_RBS_hokD)

- Miniprep (Pt_I13507)


 05/06
- Inoculum (R0080, K91403)

- EXSP digestion (tet_Term, Pt_E0840)

- Miniprep (2X_RBS_hokD)
06/06
- NotI digestion (2X_RBS_hokD, Pr_RBS, Pa_RBS)

- Transformation (tet_Ptet)

- Agarose gel (2X_RBS_hokD) – positive results

- Agarose gel (tet_Term, Pt_E0840)

- EXSP digestion (Pt_E0480)

- Inoculum (Pl_P0440)

- NotI digestion (Pt_I13507) – positive results


07/06
- Agarose gel (Pr_RBS, Pa_RBS, Pt_E0480) – positive results for Pr_RBS and Pt_E0840)

- EXSP digestion (2X_RBS_hokD, B0015)

- Ligation Term_E0840 (Pt_E0840 + tet_Term)

- Inoculum (tet_Ptet)

- Transformation (Term_E0840)

- Miniprep (Pl_P0440)

- Not digestion (Pl_P0440) – positive results

- EXSP digestion (Pl_P0440, Pt_I13507)

- Inoculum (GFP_P0440)


 08/06
- Agarose gel (2X_RBS_hokD, B0015) – B0015 didn’t digest

- Gel purification (2X_RBS_hokD)

- EXSP digestion (B0015)

- Miniprep (tet_Ptet)

- NotI digestion (tet_Ptet)

- Agarose gel (tet_Ptet, B0015) – positive results

- Gel digestion (B0015)

- Ligation hokD_Term (2X_RBS_hokD + B0015)

- Ligation Pl_I13507 (Pl_P0440 + Pt_I13507)

- Miniprep (GFP_P0440)

- EXSP digestion (GFP_P0440)


 09/06
- Agarose gel and gel purification (GFP_P0440)

- Ligation GFP_I13507 (GFP_P0440 + Pt_I13507)

- Transformation (Pl_I13507)


 10/06
- Transformation (hokD_Term)

- Inoculum (Pl_I13507)

- Transformation (GFP_I13507)


 11/06
- Miniprep (Pl_I13507)


 12/06
13/06
- Inoculum (GFP_I13507)

- NotI digestion (Pl_I13507)

- Agarose gel (Pl_I13507) – negative results


 14/06
- Miniprep (GFP_I13507)

- NotI digestion (GFP_I13507) – negative results

- EXSP digestion (Pl_P0440, Pt_I13507)

- Inoculum (GFP_P0440, Pt_I13507)


 15/06
- Agarose gel (Pl_P0440, Pt_I13507)

- Gel purification (Pl_P0440, Pt_I13507)

- Ligation Pl_I13507 (Pl_P0440 + Pt_I13507)

- Miniprep (GFP_P0440, Pt_I13507)

- EXSP digestion (GFP_P0440, Pt_I13507)


 16/06
- Agarose gel and gel purification (GFP_P0440, Pt_I13507)

- Ligation GFP_I13507 (GFP_P0440 + Pt_I13507)

- Transformation (Pl_I13507)


 17/06
- Transformation (GFP_I13507)

- Inoculum (Pt_Term, Pl_I1350)


 18/06
- Miniprep (Pt_Term, Pl_I13507)

- NotI digestion (Pt_Term, Pl_I13507)

- Agarose gel (Pt_Term, Pl_I13507) – positive results for Pt_Term


 19/06
20/06
  • iGEM kit arrived!
    		•  21/06
    - Inoculum (GFP_I13507, Pl_I13507)

    - New interlab transformations


    		 22/06
    - Miniprep (GFP_I13507, Pl_I13507)

    - NotI digestion (GFP_I13507, Pl_I13507)

    - EXSP digestion (Pr_RBS, R0080)

    - Agarose gel (GFP_I13507, Pl_I13507, Pr_RBS)

    - Gel purification (Pr_RBS)

    - Inoculum (GFP_I13507, Pl_I13507)

    - Interlab inocula


    		 23/06
    - Agarose gel (R0080)

    - Miniprep (GFP_I13507, Pl_I13507)

    - NotI digestion (GFP_I13507, Pl_I13507)

    - Agarose gel (GFP_I13507, Pl_I13507) – Positive results for GFP_I13507

    - EXSP digestion (GFP_I13507)

    - Interlab miniprep and restriction analysis


    		 24/06
    - Miniprep (B0015, Pt_P0440)

    - EXSP digestion (2X_RBS_hokD, B0015)

    - Transformation (Pa_RBS)

    - Agarose gel of restriction analysis: devices confirmed


    		 25/06
    - agarose gel (2X_RBS_hokD, B0015) - B0015 did not digest


    		 26/06
    - Agarose gel and gel purification (GFP_I13507)

    - Ligation Pr_I13507 (Pr_RBS + GFP_I13507)


    27/06
    - EXSP digestion (Pl_RBS, Pt_Term)

    - Transformation (Pr_I13507, Pa_RBS)


    		 28/06
    - Agarose gel (Pl_RBS, Pt_Term)

    ligation Pr_I13507(Pr_RBS + Pt_Term)


    		 29/06
    - EXSP digestion (B0015, 2X_RBS_hokD)

    - agarose gel (B0015, 2X_RBS_hokD) - 2X_RBS_hokD didn’t digest

    - EXSP digestion overnight (2X_RBS_hokD)

    - transformation (Pr_Term, Pl_RBS)


    		 30/06
    - inoculum (Pl_RBS, Pa_RBS)

    - agarose gel (B0015, 2X_RBS_hokD) - Digestion worked!

    - ligation 2X_hokD_Term (B0015 + 2X_RBS_hokD) P0440_hokD(P0440_term + 2X_RBS_hokD)


    		 31/06

    August

    Monday Tuesday Wednesday Thursday Friday Saturday Sunday
    01/08 02/08
    03/08
    - EXSP digestion (R0010, R0080, B0034, Pt_I13507)

    - NotI digestion (Pl_I13507)

    - Ligation Pa_RBS (R0080 + B0034) and Pl_RBS (R0010 + B0034)


    		 04/08
    - NotI digestion (Pr_I13507)

    - Agarose gel (Pr_I13507) - Positive results!!

    - EXSP digestion (Pt_I13507)

    - Transformation (Pl_RBS and Pa_RBS)


    		 05/08
    - agarose gel (Pl_I13507, Pt_I13507)- negative results for Pl_13507 :(

    - Agarose gel and gel purification (Pt_I13507)

    - ligation Pl_I13507 (Pl_P0440 + Pt_I13507)

    - inoculum (Pl_RBS, Pa_RBS)


    		 06/08
    - Transformation (Pl_I13507, Pr_I13507)

    - miniprep (Pl_RBS, Pa_RBS)

    - NotI digestion (Pl_RBS, Pa_RBS)


    		 07/08
    - Agarose gel (Pl_RBS, Pa_RBS) - Only Pa_RBS was positive

    - EXSP digestion (Pa_RBS, Pt_Term)

    - Ligation Pa_Term(Pa_RBS + Pt_Term) Pa_I13507 (Pa_RBS + GFP_I13507)


    		 08/08
    - Ligation (2X_RBS_hokD + Term)

    - Transformation (Pa_I13507, Pa_Term)


    		09/06
    - Inoculum (Pa_I13507,Pl_I13507,Pa_Term)
    10/08
    - miniprep (Pa_I13507, Pl_I13507, Pa_Term)

    - NotI digestion (Pa_I13507, Pa_Term)

    - Agarose gel (Pa_I13507, Pl_I13507, Pa_Term, Pl_RBS) - Positive results for Pa_I13507 and Pa_Term

    - Pre-inoculum of interlab study devices, BBa_K876004, and controls


    		 11/08
    - Vector exchange digestion (Pr_RBS, GFP_I13507, GFP_P0440, Tet_Term)

    - Ligation with pSB1C3 (Pr_RBS, GFP_I13507, GFP_P0440, Tet_Term)

    - EXSP digestion (Pl_RBS, GFP_I13507, Pt_Term)

    - ligation (Pl_I13507, Pl_Term)

    - Transformation OmpA

    - Sample preparation of Interlab Study devices and controls (plate reader)

    - End-point measurement of Interlab Study devices and controls

    - Sample preparation of BBa_K876004 part (plate reader)

    - Kinetic measurement of BBa_K876004 (14 hours).


    		 12/08
    - Transformation (Pr_RBS, GFP_I13507, GFP_P0440, Tet_Term)

    - Inoculum OmpA

    - Pre-inoculum of interlab study devices, BBa_K876004, and controls


    		13/08
    - Inoculum (Pr_RBS, GFP_I13507, GFP_P0440, Tet_Term)

    - miniprep OmpA

    - EXSP digestion (B0034, OmpA)

    - agarose gel (B0034, OmpA)

    - Ligation RBS_OmpA (RBS + OmpA)

    - Sample preparation of Interlab Study devices and controls (flow cytometry)

    - Measurement of Interlab Study devices and controls

    - Sample preparation of BBa_K876004 part (flow cytometry)

    - Flow cytometry measurement of BBa_K876004 (14 hours).


    		 14/08
    - miniprep (Pr_RBS, GFP_P0440(C3), Pl_I13507) - Positive results for Pl_I13507

    - and GFP_P0440

    - Transformation RBS_OmpA


    		 15/08
    - Inoculum (RBS_OmpA, GFP_I13507(C3)


    		 16/08
    - miniprep (RBS_OmpA, GFP_I13507(C3))

    - NotI digestion (RBS_OmpA, GFP_I13507(C3))

    - Agarose gel (RBS_OmpA, GFP_I13507(C3))

    - Ligation Pl_Term(Pl_RBS + Pl_Term)


    17/08
    - Inoculum (RBS_OmpA)

    - Transformation (Pl_Term)


    		 18/08
    - IDT primers and gBlocks arrived

    - miniprep (RBS_OmpA)

    - NotI digestion (RBS_OmpA)

    - Agarose gel (RBS_OmpA) - Postive

    - Vector exchange digestion (RBS_hokD, Pr_RBS)

    - Ligation RBS_hokD(C3), Pr_RBS(C3)

    - Inoculum (Pl_Term)


    		 19/08
    - Transformation (RBS_hokD(C3), Pr_RBS(C3))

    - NotI digestion (Pl_Term)

    - Agarose gel (Pl_Term)- positive!!!


    		 20/08
    - gBlocks and Primers resuspension and quantification

    - EP digestion (Pl_I13507, Pa_I13507, Pr_I13507, Pl_term, Pa_Term, Pr_Term)

    - Ligation Pl_I13507, Pa_I13507, Pr_I13507, Pl_term, Pa_Term, Pr_Term with pBS1C3

    - Inoculum (RBS_hokD(C3), Pr_RBS(C3))


    		 21/08
    - PCR for mutation of EcoRI roxA site

    - Agarose gel for amplification confirmation and purification of roxA PCR mutation

    - DpnI digestion of purificated PCR and DH5a transformation using the DNA digested

    - PCR for insert amplification of Lcp inserting Biobrick sites

    - PCR for insert amplification of Lcp inserting pETSUMO restriction sites (Lcp heterologous expression)

    - Agarose gel for amplification confirmation and purification of lcp PCRs

    - Digestion of Lcp PCR biobrick and pSB1C3 with EP

    - Digestion of Lcp PCR for pETSUMO and pETSUMO with restriction enzymes BamHI and HindIII

    - miniprep (RBS_hokD(C3), Pr_RBS(C3))

    - NotI digestion (RBS_hokD(C3), Pr_RBS(C3))

    - Agarose gel (RBS_hokD(C3), Pr_RBS(C3)) - All negative

    - Transformation (Pl_I13507, Pa_I13507, Pr_I13507, Pl_term, Pa_Term, Pr_Term)

    - Pre-inoculum (Pl_I13507, Pa_I13507, Pr_I13507, interlab study devices and controls)


    		 22/08
    - Agarose gel purification for Lcp ligations

    - Transformation of Lcp for biobrick and Lcp for pETSUMO using insert and vector purified

    - Inoculum of 6 roxA mutant colonies

    - Inoculum (Pl_I13507(C3))

    - Sample preparation (Pl_I13507, Pa_I13507, Pr_I13507, interlab study devices and controls) (plate reader) - five 96-well plates

    - Measurement of Pl_I13507, Pa_I13507, Pr_I13507, interlab study devices and controls (12 hours - plate reader)

    - Pre-inoculum (Pl_Term, Pa_Term, Pr_Term, BBa_K876004 and controls) (codes))


    		 23/08
    - gBlock reactions and transformations (linker-GFP, 6XHis, signalTAT)

    - Miniprep roxA mutants

    - Digestion of EcoRI and HindIII of roxA mutants and plasmid non-mutated (control)

    - Agarose gel of the restriction analysis

    - Sample preparation (Pl_Term, Pa_Term, Pr_Term, BBa_K876004 and controls) (plate reader) - five 96-well plates

    - Measurement of Pl_Term, Pa_Term, Pr_Term, Pl_I13507, Pa_I13507, Pr_I13507 and BBa_K876004 (12 hours - plate reader)

    - miniprep (Pl_I13507(C3))

    - NotI digestion (Pl_I13507(C3))

    - Agarose gel (Pl_I13507(C3)) - posite results


    24/08
    - Inoculum (linker-GFP, 6XHis, signalTAT, Lcp(pETSUMO), Lcp(pSB1C3))

    - miniprep (Pa_Term, Pl_Term)

    - EP digestion (Pa_Term, Pr_Term, Pl_Term, 2xHokD, B0015)

    - PCR of mutant roxA for biobrick ligation


    		 25/08
    - miniprep (OmpA_RBS, Pr_RBS, Pr_I13507, linker-GFP, signalTAT, 6xHis, Lcp(pETSUMO), Lcp(pSB1C3))

    - NotI and EXSP digestions ( (OmpA_RBS, Pr_RBS, Pr_I13507, linker-GFP, signalTAT, 6xHis, Lcp(pETSUMO), Lcp(pSB1C3))

    - Agarose gel ((OmpA_RBS, Pr_RBS, Pr_I13507, linker-GFP, signalTAT, 6xHis, Lcp(pETSUMO), Lcp(pSB1C3)) - Positive results for OmpA_RBS ,linker-GFP, signalTAT, 6xHis, Lcp(pETSUMO), Lcp(pSB1C3)

    - Ligation 6xHis_Term (linker6xHis +B0015), GFP_Term (linkerGFP + B0015), OmpA_Lcp (OmpA + Lcp(pSB1C3)), Tat_Lcp (signalTAT + Lcp(pSB1C3)

    - Ligation (Pa_Term(C3), Pl_Term(C3), Pr_Term(C3)

    - Agarose gel (mutant RoxA)


    		 26/08
    - Transformation 6xHis_Term, GFP_Term, OmpA_Lcp, Tat_Lcp


    		 27/08
    - Inoculum (6xHis_Term, GFP_Term, mpA_Lcp, Tat_Lcp)

    - Transformation (2xhokD_Term)

    - PCR of mutant roxA for biobrick ligation

    - Agarose gel of mutant roxA PCR product


    		28/08
    - roxA PCR product purification

    - roxA PCR product digestion with EP

    - miniprep (6xHis_Term, GFP_Term, OmpA_Lcp, Tat_Lcp)

    - NotI and EXSP digestions (6xHis_Term, GFP_Term, OmpA_Lcp, Tat_Lcp) - Only 6xHis_Term was negative

    - ligation Lcp_Term I (Tat_Lcp + GFP_Term), Lcp_Term II (OmpA_Lcp + GFP_Term)

    - Pre-inoculum (Pl_Term, Pa_Term, Pr_Term, Pl_I13507, Pa_I13507, Pr_I13507)


    		 29/08
    - Agarose gel (roxA PCR product)

    - Ligation roxA(mutant) +pSB1C3

    - Sample preparation (Pl_Term, Pa_Term, Pr_Term, Pl_I13507, Pa_I13507, Pr_I13507) (plate reader) - seven 96-well plates

    - Measurement of test promoters (Pl_Term, Pa_Term, Pr_Term, Pl_I13507, Pa_I13507, Pr_I13507)


    		 30/08
    - Transformation (roxA)


    31/08
    - Inoculum (roxA)

    - Transformation (Lcp_Term I, Lcp_Term II)

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