Team:Aberdeen Scotland/notebook/andgate

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Latest revision as of 09:01, 20 August 2009

University of Aberdeen iGEM 2009

University of Aberdeen - Pico Plumber

iGEM 2009

Contents

1 AND Gate Notebook

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 The End is Nigh! (17/08/09 - 21/08/09)

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

Day 5 Friday (21/08/09)

Back to Modeling Conclusions

Continue to Notebook, LacI-Latch Section

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