Team:Brasil-USP/Notebook/Calendar

Calendar

Notebook

July

Monday	Tuesday	Wednesday	Thursday	Friday	Saturday	Sunday
30/06 PCR lasR (BBa_C0079). The purpose of this PCR was to add the RBS (BBa_K143021), through addition of the sequence in the primer foward, and also to remove the LVA tag. In our primers we only added the restriction sites X and S, so we still need to put it in the pSB1C3 for the Biobrick standard completion. When standardized this part will be called BBa_K1521001.	01/07 Gel Purification of lasR PCR product	02/07 Transformation of the Biobricks: BBa_K143012 BBa_K081001 BBa_E0840 Inoculum of the Biobricks sent by iGEM HQ: BBa_K316037 BBa_K316018 BBa_K316015	03/07 Transformation of the Biobricks sent by the Imperial College Team: BBa_K316016 BBa_K143031 Miniprep, Quantification of the plasmidial DNA samples and Restriction Analysis: BBa_K316037 BBa_K316018 BBa_K316015	04/07	05/07	06/07 Inoculum: BBa_K143012 BBa_K081001 BBa_E0840
07/07 Miniprep, Quantification and Restriction Analysis: BBa_K143012 BBa_K081001 BBa_E0840 Transformation: BBa_B0015	08/07	09/07 Inoculum: BBa_B0015	10/07 Miniprep, Quantification and Restriction Analysis: BBa_B0015 (restriction analysis fail, repeat) PCR the qteE gene for amplification. We also added the RBS (BBa_K143021,) using the foward primer. In our primers we only added the restriction sites X and S, so we still need to put it in the pSB1C3 for the Biobrick standard completion. When standardized this biobrick will be called BBa_K1521000. Ligation of RBS+lasR in PTZ57R/T vector (instaclone kit)	11/07 Prepare 40% glycerol solution Transformation: RBS + lasR (BBa_C0079) Gel Purification: RBS + qteE	12/07	13/07
14/07 Assembly:AIII Digestion EXSP Prepare X-gal Prepare more LB medium (solid + liquid)	15/07 Assembly:AIII Quantification of digestion EXSP products Replate RBS+lasR Ligation of PCR qteE in pGEM T-easy vector Transformation: BBa_J04450 Restriction analysis: BBa_B0015	16/07 Transformation: BBa_K823003 RBS+qteE Electrophoresis analysis: BBa_B0015	17/07 Glycerol Stock and Miniprep: BBa_J04450 RBS+lasR BBa_B0015 Digestion: BBa_J04450 (EP) BBa_J04450 (XS) Inoculum: BBa_K823003 Transformation BBa_K143055	18/07 Glycerol Stock and Miniprep: BBa_J04450 BBa_B0015 Gel Purification: BBa_J04450 (EP) BBa_J04450 (XS) Restriction analysis: BBa_K823003	19/07	20/07
21/07 Transformation: BBa_K316016 Inoculum: BBa_K143055 RBS+qteE Assembly KI: Digestion EXSP	22/07 Inoculum: BBa_K316016 Miniprep, Restriction analysis and Digestion: RBS+qteE Assemblies AIII and KI: Gel Purification Ligation	23/07 Assemblies AIII and KI: Transformation Prepare more competent cells Prepare LB medium (solid and liquid) Inoculum BBa_K316016 Prepare RBS+lasR and RBS+qteE for sequencing	24/07 Assemblies AIII and KI: Inoculum Miniprep: BBa_K316016	25/07 Assemblies AIII and KI: Glycerol Stock Miniprep Restriction analysis	26/07	27/07
28/07 Assembly AIII: Restriction analysis (+NdeI enzyme) OBS: This analysis was reapeted because we had problems confirming the assembly. The issue was that both the construction and the pSB1C3 vector had similar size. Assembly AII, AV and AVI: Digestion EXSP	29/07 >Assembly AII, AV and AVI: Gel Purification Ligation	30/07 >Assembly AII, AV and AVI: Transformation	31/07 Assembly AII, AV and AVI: Inoculum PCR BBa_K143055 for RBS removal OBS: This was one of the parts sent by the Imperial College Team. Because of a comunication failure we assumed that this part was comming without the RBS, so we included a RBS in the parts we were going to use with this Lac promotor :) PCR of Plac BBa_K143055 was followed by a Gel Purification and Ligation in the PUC19 vector

August

Monday	Tuesday	Wednesday	Thursday	Friday	Saturday	Sunday
				01/08 Transformation: BBa_K143055 Assembly AII, AV and AVI: Glycerol Stock Miniprep Restriction analysis	02/08	03/08
04/08 Inoculum E. coli DH5-alpha KI Plac BBa_K143055 Transformation BBa_P0312 Assembly AV: Prepare more inoculum Repeat Restriction analysis	05//08 Prepare assemblies KI for cytometry characterization Miniprep: Plac BBa_K143055 Inoculum BBa_P0312 Assembly AV: Miniprep And once again! Restriction analysis (this BBa_K316016 is tough)	06/08	07/08 Miniprep and Restriction analysis: BBa_P0312	08/08	09/08	10/08
11/08 Prepare Plac BBa_K143055 for sequencing Analyse the RBS+qteE and RBS+lasR sequencing file	12/08 Digestion: RBS+qteE RBS+lasR OBS: this digestion was performed so we clone this parts in the pSB1C3 vector to put them in Biobrick standard	13/08 Assemblies BII and BIII: Digestion EXSP	14/08 Gel Purification and Ligation in pSB1C3 for BBiobrick standard: RBS+qteE RBS+lasR Assemblies BII and BIII: Gel Purification OBS: we had some problems here with the purification and we lost all our digestions, so we're repeating the digestion	15/08	16/08 Assemblies BII and BIII: Digestion EXSP	17/08 Assemblies BII and BIII: Gel Purification
18/08 Assemblies BII and BIII: Ligation Prepare LB medium (liquid and solid)	19/08 Assemblies BII and BIII: Transformation	20/08 RBS+qteE, RBS+lasR and Assemblies BII and BIII: Inoculum	21/08 RBS+qteE, RBS+lasR and Assemblies BII and BIII: Glycerol Stock Miniprep Restriction analysis	22/08 Prepare LB medium (liquid and solid) Assembly BII: Repeat restriction analysis adding the enzyme NdeI.	23/08	24/08
25/08 Inoculum BBa_J04450 BBa_K143055 PCR BBa_K143055 with higher melting temperature.	26/08 Assemblies AI, CII, KIII, KIV and KVI Digestion EXSP	27/08 Assemblies AI, CII, KIII, KIV and KVI Gel Purification	28/08	29/08	30/08	31/08

September

Monday	Tuesday	Wednesday	Thursday	Friday	Saturday	Sunday
01/09 Assembly CII Digestion of the part BII with EcoRV. Assemblies AI, CII, KIII, KIV and KVI: Ligation	02/09 Assemblies AI, CII, KIII, KIV and KVI: Transformation	03/09 Assemblies AI, CII, KIII, KIV and KVI: Inoculum	04/09 Assemblies AI, CII, KIII, KIV and KVI: Glycerol Stock Miniprep Restriction analysis	05/09	06/09	07/09
08/09 QuickChange PCR to mutate the cleavage site of the BBa_K316037 to a Cathepsin S recognition site Transformation of the QuickCHange PCR product	09/09	10/09 The QuickChange protocol didn't work well. So try again with more attention! QuickChange PCR to mutate the cleavage site of the BBa_K316037 to a Cathepsin S recognition site Transformation of the QuickCHange PCR product Assemblies DI, KVIII, KX and KXI: Digestion EXSP	11/09 Prepare inoculum of the mutated BBa_K316037 Assemblies DI, KVIII, KX and KXI: Gel Purification Ligation	12/09 Assemblies DI, KVIII, KX and KXI: Transformation We had far to many difficulties trying to remove the RBS from the BBa_K143055, so we decided to synthesize it using PCR. PCR for synthesis of the BBa_K143015 (BBa_K143055 without RBS)	13/09 Assembly KX Inoculum Assemblies DI, KVIII and KXI: Repeat Ligation Prepare LB medium (solid and liquid)	14/09 Assembly KX Glycerol Stock Miniprep Assemblies DI, KVIII and KXI: Transformation
15/09 Assembly KX Restriction analysis Assemblies DI, KVIII and KXI: Inoculum Gel Purification of the BBa_K143015	16/09 Prepare 40% glycerol solution Assemblies DI, KVIII and KXI: Glycerol Stock Miniprep Restriction analysis	17/09 Ligation of the synthesized BBa_K143015 in TOPO vector followed by Transformation Measurement Interlab Study - Samples growth biobrick devices 1, 2 and 3	18/09 Replate BBa_K143015. We forgot the X-gal :( Measurement Interlab Study - Sample preparation and characterization by Flow Cytometry and Fluorometry	19/09	20/09	21/09
22/09 Inoculum of the BBa_K143015 Assembly KXIV: Digestion EXSP	23/09 Assembly KXIV: Digestion EXSP Miniprep and Restriction Analysis of the BBa_K143015	24/09 Assembly KXIV: Gel Purification Ligation Transformation	25/09 Assembly KXIV: Inoculum	26/09 Assembly KXIV: Glycerol Stock Miniprep Restriction analysis Assemnlies KII, KV and AIV: Digestion EXSP	27/09	28/09
29/09 Assemnlies KII, KV and AIV: Gel Purification Ligation Transformation	30/09 Assemnlies KII, KV and AIV: Inoculum

October

Monday	Tuesday	Wednesday	Thursday	Friday	Saturday	Sunday
		01/10 Assemnlies KII, KV and AIV: Glycerol Stock Miniprep	02/10	03/10	04/10	05/10
06/10 Assemnlies KII, KV and AIV: Restriction analysis Assemblies KIX, BIV and KXVI Digestion EXSP	07/10 Assemblies KIX, BIV and KXVI Gel Purification Ligation	08/10 Assemblies KIX, BIV and KXVI Transformation	09/10 Assemblies KIX, BIV and KXVI Inoculum	10/10 Assemblies KIX, BIV and KXVI Restriction analysis	11/10	12/10
13/10	14/10	15/10	16/10	17/10	18/10	19/10
20/10	21/10	22/10	23/10	24/10	25/10	26/10
27/10	28/10	29/10	30/10	31/10

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