Team:Aberdeen Scotland/notebook/andgate

=AND Gate Notebook=

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

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

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

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

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

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

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

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

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

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

Day 1 Monday (17/08/09)

 * Updated wiki notebook
 * Added several translations of front page summary to wiki

Day 2 Tuesday (18/08/09)

 * Zuzana - Motility Assay for modelling photographs
 * Calum - Had a 30cm plate removed from my right arm and several areas of my shoulderbone cut off

Day 3 Wednesday (19/08/09)

 * Zuzana - Updated wiki Wetlab section
 * Calum - Bled on things

Day 4 Thursday (20/08/09)

 * Editing and tidying of wiki and devouring of cake