Contents 1 AND Gate Notebook 1.1 Week 1: Research and Planning (08/06/09 - 12/06/09) 1.1.1 Day 1 Monday (08/06/09) 1.1.2 Day 2 Tuesday (09/06/09) 1.1.3 Day 3 Wednesday (10/06/09) 1.1.4 Day 4 Thursday (11/06/09) 1.1.5 Day 5 Friday (12/06/09) 1.2 Week 2: BioBrick Rescue (15/06/09 - 19/06/09) 1.2.1 Day 1 Monday (15/06/09) 1.2.2 Day 2 Tuesday (16/06/09) 1.2.3 Day 3 Wednesday (17/06/09) 1.2.4 Day 4 Thursday (18/06/09) 1.2.5 Day 5 Friday (19/06/09) 1.3 Week 3: Edinburgh iGEM meeting and Digestions(22/06/09 - 26/06/09) 1.3.1 Day 1 Monday (22/06/09) 1.3.2 Day 2 Tuesday (23/06/09) 1.3.3 Day 3 Wednesday (24/06/09) 1.3.4 Day 4 Thursday (25/06/09) 1.3.5 Day 5 Friday (26/06/09) 1.4 Week 4:Digestions Galore(29/06/09 - 03/07/09) 1.4.1 Day 1 Monday (29/06/09) 1.4.2 Day 2 Tuesday (30/06/09) 1.4.3 Day 3 Wednesday (01/07/09) 1.4.4 Day 4 Thursday (02/07/09) 1.4.5 Day 5 Friday (03/07/09) 1.5 Week 5 Preperation of K182100 (06/07/09 - 11/07/09) 1.5.1 Day 1 Monday (06/07/09) 1.5.2 Day 2 Tuesday (07/07/09) 1.5.3 Day 3 Wednesday (08/07/09) 1.5.4 Day 4 Thursday (09/07/09) 1.5.5 Day 5 Friday (10/07/09) 1.6 Week 6 13/07/09 - 16/07/09 1.7 Week 7 20/07/09 - 24/07/09 1.8 Week 8 27/07/09 - 31/07/09 1.9 Week 9 03/08/09 - 07/08/09 1.10 Week 10 10/08/09 - 14/08/09 1.11 Week 11 17/08/09 - 21/08/09

AND Gate Notebook

Week 1: Research and Planning (08/06/09 - 12/06/09)

Day 1 Monday (08/06/09)

• Researching the Registry for Biobricks
  • Focus on T7 parts (I712074, I719005, K113011, K113012, J34814, K103021, R0085, R0180, R0182, Z0251 and Z0252) for possible use in our project.
  • Other BioBricks identified for other sub-projects include R0062, J40001, J37015 and R0063

Day 2 Tuesday (09/06/09)

• Researching literature for T7 promoter strength and degradation rate
  • Useful article - Imburgio, D., Rong, M., Ma, K., and W. T. McAllister. (2000) "Studies of Promoter Recognition and Start Site Selection by T7 RNA Polymerase Using a Comprehensive Collection of Promoter Variants" Biochemistry.39:10419-10430

Day 3 Wednesday (10/06/09)

• Researching literature for parameters of LuxR, LacO, TetO and cIO
  • Useful article - Braff, J. C., Conboy, C. C., and D.E. Endy. Promoter Characterization Experiments. (found at openwetware.org/images/3/30/PromoterChar_Report_Revised.doc)
• Identified plasmids that potential Biobricks were on and prepared for rescue.
  • Also indentified LVA tags and any other anomalies on selected Biobricks.

Day 4 Thursday (11/06/09)

• PoPs to LacZ alpha repoter identified (E0435) and potenially useful Input Output sensor (pSB1A10)
  • Other possible reporters include LacZ alpha fragments and Enhanced stable YFP.
• Possibility of intentional SNP generated within T7 to alter promoter strength was deemed unworkable in the scale of project.

Day 5 Friday (12/06/09)

• End of week meeting
• Discussion and planning for following week

Week 2: BioBrick Rescue (15/06/09 - 19/06/09)

Day 1 Monday (15/06/09)

• Prepared LB medium and LB+Amp plates
• Preperation of Kanamycin stock (30mg ml-1)
• Inoculated C0040, R0062, J37033 and C0051 for miniprep the following day

Day 2 Tuesday (16/06/09)

• Miniprep of above Biobricks
• Restriction digest of C0040, R0062, J37033 and C0051 with EcoRI-HF + SpeI and gel electrophoreis of these.

Day 3 Wednesday (17/06/09)

• Rescue of a range of vectors (pSB1AT3, pSB1AC3, pSB3T5, pSB3C5, pSB4T5, pSB4C5)
• Preperation of Tet and Chlo agar plates for the above
• Transformation of DB3.1 cells (ccdB gene resistant)

Day 4 Thursday (18/06/09)

• Preparation of more Chlo and Tet plates.
• Sub-cultivation of transformants

Day 5 Friday (19/06/09)

• End of week meeting to present weeks work and plan ahead

Week 3: Edinburgh iGEM meeting and Digestions(22/06/09 - 26/06/09)

Day 1 Monday (22/06/09)

• Preperation for cloning and selection of desired biobricks from earlier rescues

Day 2 Tuesday (23/06/09)

• Edinburgh Igem Meeting

Day 3 Wednesday (24/06/09)

• Edinburgh Igem Meeting

Day 4 Thursday (25/06/09)

• Double digest of the biobricks B0030, C0051, I0462, J23105, J23107, J23115, with EcoRI-HF + Spe I
• Double digest of the biobricks S03518, E0840 with XbaI + PstI
• Double digest of the biobricks pSB3K3, pSB4K5, pSB3T5, pSB1AC3, pSB1AT3

Day 5 Friday (26/06/09)

• DB3.1 cells transformed with pSB1AK3
• Gel Electrophoresis of above digests
• End of week meeting

Week 4:Digestions Galore(29/06/09 - 03/07/09)

Day 1 Monday (29/06/09)

• Dilution double digest of the biobrick E0840 with XbaI + PstI

Day 2 Tuesday (30/06/09)

• Double digest of the biobrick C0051 with EcoRI-HF + SpeI

Day 3 Wednesday (01/07/09)

• Dilution double digest of pSB3T5 with EcoRI-HF + PstI
• Dilution double digest of E0840 with XbaI + SpeI
• Transformation of DB3.1 competent cells with pSB3K5

Day 4 Thursday (02/07/09)

• Electrophoresis of the digests form the previous day on 0.8% (E0840) and 1% (C0051) agar

Day 5 Friday (03/07/09)

• Repeated dilution double digests for biobricks E0840, C0051 and pSB3T5
• End of week meeting

Week 5 Preperation of K182100 (06/07/09 - 11/07/09)

Day 1 Monday (06/07/09)

• Planning and calculations for ligation
• Electrophoresis of the dilution double digests

Day 2 Tuesday (07/07/09)

• Double digest of the biobrick pSB3K5
• Dephosphorylation of the pSB3T5

Day 3 Wednesday (08/07/09)

• Electrophoresis of the pSB3K5 double digest
• Electrophoresis of the double digests of J series promoters (J23107, J23105, J23115)

Day 4 Thursday (09/07/09)

• Electrophoresis of the double digest – pSB1AC3
  • Ligation , Cloning and Transformation
  • Transformation of XL1 Blue competent cells
• Preparation of IPTG stock
• Preparation of Chlo agar plates

Day 5 Friday (10/07/09)

• The results of the transformation
• New transformation of the XL1- Blue competent cells with C0051, E0840 and pSB1AC3

Week 6 13/07/09 - 16/07/09

Week 7 20/07/09 - 24/07/09

Week 8 27/07/09 - 31/07/09

Week 9 03/08/09 - 07/08/09

Week 10 10/08/09 - 14/08/09

Week 11 17/08/09 - 21/08/09

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