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Revision as of 06:39, 18 September 2015

Notebook

Calendar

Table of contents

June

Monday Tuesday Wednesday Thursday Friday Saturday Sunday
01/06 02/06
- iGEM kit arrived! 		03/06 04/06 05/06 06/06 07/06
08/06 09/06
- Transformation:
- B0034
- K1497008
- R0010
- K914003
- R0080
- C0040
- B0015
- R0040
- E0840
- E0040
- P0440
- I13507
 		10/06 11/06
- Inoculum
- B0034
- K1497008
- R0010
- K914003
- R0080
- C0040
- B0015
- R0040
- E0840
- E0040
- P0440
- I13507
 		12/06
- Miniprep and Digestion EXSP
- B0034
- K1497008
- R0010
- K914003
- R0080
- C0040
- B0015
- R0040
- E0840
- E0040
- P0440
- I13507
 		13/06
- Agarose gel (Problems with the buffer! Way too concentrated ><)
- Digestion EXSP again (C0040, B0015, R0040, E0840, E0040, P0440, I13507) [We didn’t have B0034 anymore, so we didn’t prepare the reaction for K1497008, R0010, K914003, R0080] 		14/06
- Agarose gel(C0040, B0015, R0040, E0840, E0040, P0440, I13507)
- Gel purification (C0040, B0015, E0040, P0440)
- Interlab transformations (J23101, J23106, J23117, I13504)
- Ligation Tet_term (C0040 + B0015)
- Ligation GFP_P0440 (E0040 + P0440)
15/06
- Transformation Tet_term (C0040 + B0015)
- Transformation GFP_P0440 (E0040 + P0440)
- Interlab inoculum
- Inoculum (B0034) 		16/06
- Inoculum (Tet_term, GFP_P0440)
- Inoculum (Tet_term, GFP_P0440)
- Interlab minipreps
- Miniprep (B0034)
- Digestion ESXP (B0034, B0034, K1497008, R0010, K914003, R0040, E0840) 		17/06
- Miniprep (Tet_term, GFP_P0440)
- Agarose gel (B0034, B0034, K1497008, R0010, K914003, R0040, E0840)
- Gel purification (B0034, B0034, K1497008, R0010, K914003, R0040, E0840)
- Ligation RBS_hokD (B0034 + K1497008)
- Ligation Pl_RBS (R0010 + B0034)
- Ligation Pt_E0840 (R0040 + E0840)
- Ligation Pt_I13507 (R0040 + I13507) 		18/06
- Transformation (RBS_hokD, Pl_RBS, Pt_E0840, Pt_I13507) 		19/06
- No colonies! Wrong antibiotic!!!!
- New transformation (RBS_hokD, Pl_RBS, Pt_E0840, Pt_I13507) 		20/06
- Inoculum (Pl_RBS, Pt_E0840, Pt_I13507)
- RBS_hokD didn’t grow 		21/06
- Miniprep (Pl_RBS, Pt_E0840, Pt_I13507)
- Digestion NotI (Tet_term, GFP_P0440, Pl_RBS, Pt_E0840, Pt_I13507)
22/06
- Confirmation, agarose gel (Tet_term, GFP_P0440, Pl_RBS, Pt_E0840, Pt_I13507)
- Confirmed (Tet_term, Pl_RBS, Pt_E0480)
- Ligation (GFP_P0040, Pt_I13507, RBS_hokD) 		23/06
- Transformation (GFP_P0040, Pt_I13507, RBS_hokD) 		24/06
- Inoculum [GFP_P0040 (3 colonies), RBS_hokD (3 colonies), I13507, R0080, K914003] 		25/06
- Miniprep [GFP_P0040 (3 colonies), RBS_hokD (3 colonies), I13507, R0080, K914003] 		26/06 27/06
- EXSP digestion (K91403)
- Confirmation digestion (RBS_hokD) 		28/06
- Agarose gel (K91403)
- Agarose gel (RBS_hokD) – positive result \o/
- Gel purification (K91403)
- Ligation Pr_RBS (K91403 + B0034) 22ºC for 4h, 16ºC for 10h, 4º overnight
- EXSP digestion RBS_hokD
- Interlab digestion (from minipreps of June 15th)
- Promoters J23101,J23106 and J23117 - SP enzymes
- GFP part I13504(RBS B0034+GFP E0040+double Term B0010 and B0012 ) - XP enzymes
29/06
- Agarose gel RBS_hokD
- Ligation (Pr_RBS, Pa_RBS) 		30/06
- Inoculum RBS_hokD
- EXSP digestion RBS_hokD

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)

    September

    Monday Tuesday Wednesday Thursday Friday Saturday Sunday
    01/09
    - miniprep (roxA)

    - Inoculum (Lcp_Term I)

    - Ligation (Lcp_Term II)


    		 02/09
    - miniprep (Lcp_Term I)

    - digestion (Lcp_Term I, roxA))

    - Transformation (Lcp_Term II)


    		 03/09
    - Transformation (Pr_RBS, 2X_RBS_hokDLigation Pl_hokD (Pl + 2xhokD))

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


    		 04/09
    - Transformation (Pl_2xhokD)

    -Sample preparation of test promoters circuits (plate reader)

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


    		 05/09
    - Inoculum (Pl_2xhokD)


    		 06/09
    - Transformation in Bl21(DE3) (Pl_I13507, Pa_I13507, Pr_I13507, Pl_Term, Pa_Term, Pr_Term)
    07/09
    - Inoculum (Pl_I13507, Pa_I13507, Pr_I13507, Pl_Term, Pa_Term, Pr_Term, in BL21(DE3))


    		 08/09
    - Miniprep (Pl_I13507, Pa_I13507, Pr_I13507, Pl_Term, Pa_Term, Pr_Term, in BL21(DE3))

    - Digestion with SacI (OmpA_Lcp, OmpA_roxA, TAT_Lcp, TAT_roxA)

    - Digestion with SacI and XbaI (OmpA, signalTAT, Lcp, RoxA)

    - EXSP digestion (R0010, R0080, K914003)

    -NotI digestion (Pl_I13507, Pa_I13507, Pr_I13507, Pl_Term, Pa_Term, Pr_Term, in BL21(DE3))

    - Agarose gel (OmpA_Lcp, OmpA_roxA, TAT_Lcp, TAT_roxA, OmpA, signalTAT, Lcp, RoxA, Pl_I13507, Pa_I13507, Pr_I13507, Pl_Term, Pa_Term, Pr_Term) - Positive results for Pl_I13507 and Pr_I13507)

    - Ligation OmpA_Lcp, OmpA_roxA, TAT_Lcp, TAT_roxA, Pl_I13507II (R0010 + GFP_I13507), Pl_TermII (R0010 + tet_Term), Pa_I13507II (R0080 + GFP_I13507), Pa_TermII (R0080 + tet_Term), Pr_I13507II (K914003 + GFP_I13507), Pr_TermII (K914003 + tet_Term),

    -Transformation (OmpA_Lcp, OmpA_roxA, TAT_Lcp, TAT_roxA, Pl_I13507II, Pa_I13507II, Pr_I13507II, Pl_TermII, Pa_TermII, Pr_TermII)


    		 09/09
    -Western blot to see if LCP was been expressed in pETSUMO

    - inoculum (Pl_I13507II, Pa_I13507II, Pl_TermII, Pa_TermII, Pr_TermII, TAT_Lcp_RBS, OmpA_Lcp_RBS, OmpA_roxA_Pt)

    - Prepare plates for characterization (Pl_I13507 and Pr_I13507 in BL21(DE3)) 10th


    		 10/09
    - Miniprep (Pl_I13507II, Pa_I13507II, Pl_TermII, Pa_TermII, Pr_TermII, TAT_Lcp_RBS, OmpA_Lcp_RBS, OmpA_roxA_Pt)

    -NotI digestion (Pl_I13507II, Pe_I13507II, Pl_TermII, Pa_TermII, Pr_TermII)

    - SacI digestion again (OmpA_Lcp, OmpA_roxA, TAT_Lcp, TAT_roxA, TAT_Lcp_RBS, OmpA_Lcp_RBS, OmpA_roxA_Pt)

    -Agarose gel (Pl_I13507II, Pe_I13507II, Pl_TermII, Pa_TermII, Pr_TermII, OmpA_Lcp, OmpA_roxA, TAT_Lcp, TAT_roxA, TAT_Lcp_RBS, OmpA_Lcp_RBS, OmpA_roxA_Pt)

    -Ligation (OmpA_Lcp, OmpA_roxA, TAT_Lcp, TAT_roxA, TAT_Lcp_RBS, OmpA_Lcp_RBS, OmpA_roxA_Pt, GFP_TermA (J23101 + linkerGFP_Term), GFP_TermB (J23117 + linkerGFP_Term), GFP_TermC (J23106 + linkerGFP_Term),

    -Transformation (OmpA_Lcp, OmpA_roxA, TAT_Lcp, TAT_roxA, TAT_Lcp_RBS, OmpA_Lcp_RBS, OmpA_roxA_Pt, GFP_TermA, GFP_TermB, GFP_TermC


    		 11/09
    - Transformation (TAT_ lcp_RBS, TAT_ lcp, TAT_ roxA, OmpA_roxA_Pt)

    -Inoculum (GFP_TermA, GFP_TermB, GFP_TermC)


    		 12/09
    - Miniprep (GFP_TermA, GFP_TermB, GFP_TermC)

    - Not digestion (GFP_TermA, GFP_TermB, GFP_TermC)

    - Xbal and NotI digestion (RoxA, signalTAT and Lcp)

    -Agarose gel (GFP_TermA, GFP_TermB, GFP_TermC, RoxA, signalTAT and Lcp)

    -Ligation TAT_Lcp (signalTAT + Lcp), TAT_RoxA (signalTAT + roxA)


    		 13/09
    - Transformation (TAT_Lcp, TAT_roxA)
    14/09
    - EP digestion to change the vector (Pa_Term, signalTAT, OmpA, Lcp, RoxA)

    - SacI and EcoRI digestion (signalTat, OmpA)

    - SacI and PstI digestion (Lcp, RoxA)

    - Agarose gel ( Pa_Term, signalTAT, OmpA, Lcp, RoxA)

    -Ligation (pSB1A2 + RoxA, pSB1A2 + Lcp, pSB1A2 + OmpA and pSB1A2 + SignalTAT)

    -3A assembly (TAT_RoxA(pSB1A2 +signalTA+RoxA), TAT_Lcp(pSB1A2 +signalTA+Lcp), OmpA_RoxA(pSB1A2 +OmpA+RoxA),OmpA_Lcp( pSB1A2 +OmpA+Lcp))

    -Transformation (RoxA, Lcp, OmpA, SignalTAT, TAT_RoxA, TAT_Lcp, OmpA_RoxA, OmpA_Lcp)

    - Inoculum (linkerGFP_J23117, linkerGFP_J23106)


    		 15/09
    - Ligation again (pSB1A2 + RoxA, pSB1A2 + Lcp, pSB1A2 + OmpA and pSB1A2 + SignalTAT)

    - 3A assembly again (TAT_RoxA(pSB1A2 +signalTA+RoxA), TAT_Lcp(pSB1A2 +signalTA+Lcp), OmpA_RoxA(pSB1A2 +OmpA+RoxA),OmpA_Lcp( pSB1A2 +OmpA+Lcp))

    - Sample preparation (linkerGFP_J23117, linkerGFP_J23106)

    - Measurements (linkerGFP_J23117, linkerGFP_J23106)


    		 16/09
    - Transformation (RoxA, Lcp, OmpA, SignalTAT, TAT_RoxA, TAT_Lcp, OmpA_RoxA, OmpA_Lcp)

    - Prepare the plates to submit the new parts

    - Lcp Expression using pETSUMO in Bl21(DE3)


    		 17/09
    - Inoculum (RoxA, Lcp, OmpA, SignalTAT, TAT_RoxA, TAT_Lcp, OmpA_RoxA, OmpA_Lcp)

    - Cell lyses

    - Western blot (Lcp)


    		 18/09
    - Miniprep (RoxA, Lcp, OmpA, SignalTAT, TAT_RoxA, TAT_Lcp, OmpA_RoxA, OmpA_Lcp)

    - NotI digestion (RoxA, Lcp, OmpA, SignalTAT, TAT_RoxA, TAT_Lcp, OmpA_RoxA, OmpA_Lcp)

    - Agarose gel (RoxA, Lcp, OmpA, SignalTAT, TAT_RoxA, TAT_Lcp, OmpA_RoxA, OmpA_Lcp)

    - Western blot (Lcp)


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