Team:Aberdeen Scotland/notebook/andgate

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University of Aberdeen iGEM 2009


Contents

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
  • End of week meeting



Week 6 Identifying K182100 (13/07/09 - 17/07/09)

Day 1 Monday (13/07/09)

  • The results of transformation of XL-1 Blue competent cells with pSB1AC3, C0051, E0840, look positive.
  • Preparation of grid plates (with 40 colonies picked from overnight culturem and 5 control colonies from Vector and Vector+Ligase cultures).
  • Preparation for PCR colony



Day 2 Tuesday (14/07/09)

  • Preparation, calculation for PCR colony
  • PCR colony



Day 3 Wednesday (15/07/09)

  • Calculation for double digest of PCR product
  • Double digest of PCR product with HindIII and NdeI to hopefully show insert containing part of E0840 and C0051



Day 4 Thursday (16/07/09)

  • Mini prep of good colonies
  • Double digest of the positive transformants with EcoRi-HF + PstI and HindIII + NdeI



Day 1 Friday (17/07/09)

  • Weekly meeting, cake and planning ahead



Week 7 2nd Cloning Step (20/07/09 - 24/07/09)

Day 1 Monday (20/07/09)

  • Fusion PCR for K182102 was prepared
  • K182100 sent for sequencing



Day 2 Tuesday (21/07/09)

  • Fusion PCR for K182102 was repeated
  • Gel electrophoresis of PCR product
  • Gel DNA extraction and purification



Day 3 Wednesday (22/07/09)

  • Double digest of PCR product and K182100 with XbaI + PstI
  • Gel electrophoresis of previous double digests



Day 4 Thursday (23/07/09)

  • Calculation for ligation
  • Ligation and transformation of SCS1 supercompetent cells



Day 5 Friday (24/07/09)

  • The results form the ligation were obtained, though they showed a poor Vector+Inserts+Ligase ratio to Vector+Ligase.
    • Preparation of a new fusion PCR
    • Gel electrophoresis of PCR product
    • DNA gel extraction and purification



Week 8 Déjà vu (27/07/09 - 31/07/09)

Day 1 Monday (27/07/09)

  • Double digest of PCR product with EcoRI-HF + PstI
  • Double digest of PCR product with EcoRI + SpeI



Day 2 Tuesday (28/07/09)

  • Heat inactivation of restriction enzymes
    • Dephosphorylation of pSB3T5 vector
    • Ligation of pSB3T5 + K182103
  • Motility assay
  • Chemotaxis experiment on minimal media with 1%, 2% and 4% aspartate



Day 3 Wednesday (29/07/09)

  • The results of chemotaxis experiment (unpromising)
  • The results of ligation (unpromising)
  • Preparation of IPTG
  • Preparation of grid plates



Day 4 Thursday (30/07/09)

  • Colony PCR
  • Double digest of pSB3T5 with EcoRI-HF and PstI



Day 5 Friday (31/07/09)

  • Gel electrophoresis of the pSB3T5 double digest
  • End of week meeting



Week 9 Motility focus (03/08/09 - 07/08/09)

Day 1 Monday (03/08/09)

  • New fusion PCR
    • Purification of PCR product
    • Double digest and gel electrophoresis
  • Motility assay



Day 2 Tuesday (04/08/09)

  • Calculation for ligation
  • Ligation and transformation of SCS-1 supercompetent cells
  • Motiliy experiment



Day 3 Wednesday (05/08/09)

  • The results of ligation and transformation
  • Preparation of mini prep
  • Motility assay
  • Preparation of grid plates



Day 4 Thursday (06/08/09)

  • The results of motility assay were obtained
  • PCR colony of K182103
  • Gel electrophoresis of the PCR product



Day 5 Friday (07/08/09)

  • Colony PCR of the K182102
  • Gel electrophoresis of the PCR product
  • Double digest of the mini preps prepared



Week 10 Wrapping Up (10/08/09 - 14/08/09)

Day 1 Monday (10/08/09)

  • Double digest of the K182100 and potential K182103
  • Estimation of the concentration (ng/ul) in our mini preps to be send for sequencing
  • Gel electrophoresis of the double digest



Day 2 Tuesday (11/08/09)

  • Preparation for the sequencing
  • Potential K182102 and K182103 sent for sequencing
  • Working on the team wiki



Day 3 Wednesday (12/08/09)

  • Overnight incubation of the motile strain MG1655
  • Working on the team wiki



Day 4 Thursday (13/08/09)

  • Motility and chemotaxis assay, though it failed to yield suitable/usable results
  • Results from sequencing K182102, sequencing was satisfactory



Day 5 Friday (14/08/09)

  • Capillary chemotaxis assay
  • Weekly meeting and team photos taken



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

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