Experimental Methodology

For the assemblies constructuion we addopted the following method:

We used the pSB1C3 vector from the Biobrick BBa_J04450. Using this biobrick we added a red/white selection, where the red colonies are those which do not have the assembly, and the white ones are the correct ones.

See our Protocol

Assemblies forms

Assembly form template

This is the template designed to help with the laboratory organization during the assemble of biological parts. If you want to use the assembly method we used you can print the form and complete it for each construction. Hope it helps :)

Life Inside the LAB

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_143012 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_143012 BBa_K081001 BBa_E0840	04/07	05/07	06/07 Inoculum: BBa_143012 BBa_K081001 BBa_E0840
07/07 Miniprep, Quantification and Restriction Analysis: BBa_143012 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_316016 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_316037 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_316037 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_143015 (BBa_143055 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, CIII and KXVI Digestion EXSP	07/10	08/10	09/10	10/10	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

Team:Brasil-SP/Notebook

From 2014.igem.org