Team:Aberdeen Scotland/notebook/lacilatch

=LacI-Latch Notebook=

Day 1 Monday (08/06/09)

 * Researching the Registry for Biobricks
 * Identified K182022, K112808, R0062, I712074, I14015 for further discussion.

Day 2 Tuesday (09/06/09)

 * Researching the Registry for Plasmids.
 * Identified pSB3T5, pSB3C5, pSB1AC3, pSB1AT3 for further discussion.

Day 3 Wednesday (10/06/09)

 * Literature search for parameters of T7 promoter and the Holin system.
 * Conclusion: T7 feedback mechanism of wiring diagram 1 is unlikely to be functional without further regulations
 * Identified the Novagen plasmids pLysS and pLysE as possible solutions.


 * Researching the Registry for LacI Repressor and T7 Polymerase
 * Identified K145014, K145001 and I732100 for further discussion

Day 4 Thursday (11/06/09)

 * Parameter search for the Modelling effort.

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 for Lab
 * Planning of LacI-Latch
 * Inoculated J23100, K145001, B0034 for Miniprep the following day

Day 2 Tuesday (16/06/09)

 * Miniprep of J23100, K145001, B0034
 * Restriction Digest

Day 3 Wednesday (17/06/09)

 * Lambda Digest with HindIII for use as a ladder
 * Gel Electrophoresis of B0034, K145001, J23100 and I732100.

Day 4 Thursday (18/06/09)

 * Gel Electrophoresis of C0051, R0062, C0040, J37033, K145001 and I732100.
 * Inoculation of C0051, R0062, J37033, I732100 or miniprep.

Day 5 Friday (19/06/09)

 * Miniprep of C0051, R0062, J37033 and I732100.
 * End of week meeting

Day 1 Monday (22/06/09)

 * Rescued BBa_1732820, S03518 and B0030

Day 2 Tuesday (23/06/09)
Risat:
 * Research for Plasmids and J series Promoters
 * Rescued and Transformed Psb4K4 and Psb3K5.
 * Restriction Digest of B0015, R0040, E0840, R0011, K093005, R0051, K081008, J37033 and R0062,

Daniel:
 * Edinburgh Igem Meeting

Day 3 Wednesday (24/06/09)
Risat:
 * Gel Electrophoresis of B0015, R0040, E0840, R0011, K093005, R0051, K081008, J37033 and R0062,

Daniel:
 * Edinburgh Igem Meeting

Day 4 Thursday (25/06/09)
Risat:
 * Gel Electrophoresis of K112808, K112022 and I732100

Daniel:
 * Edinburgh Igem Meeting

Day 5 Friday (26/06/09)

 * Restriction Digest of E0840
 * Gel Electrophoresis of E0840
 * End of week meeting

Day 1 Monday (29/06/09)

 * Run Gel of E0840 ( Testing of digests)


 * Inoculate for miniprep.
 * I732820 (LacI,) R0051 (CI operator), S03518 ( TetR), K112808 ( Holin), PSB3K3, PSB3T5.


 * Rescue I13600 and I13601
 * Required for testing of constructs

Day 2 Tuesday (30/06/09)

 * Miniprep
 * I732820 (LacI) R0051 (CI operator) S03518 ( TetR) K112808 ( Holin)  PSB3K3, PSB3T5.


 * Planning for Restriction digest
 * R0051, S03518, R0040, PSB3K3 and I732820.


 * Restriction Digest
 * R0051, S03518, R0040, PSB3K3 and I732820.


 * Gel Electrophoresis
 * R0051, S03518, R0040, PSB3K3 and I732820.


 * Transformation (using XL1-Blue)
 * I13600 and I13601
 * Result : Failed (no growth)


 * Inoculation for Miniprep
 * PSB4K5, PSB3K3

Day 3 Wednesday (01/07/09)

 * Miniprep
 * PSB4K5, PSB3K3


 * Restriction Digest
 * PSB4K5, PSB3K3, S03518 and R0040


 * Gel Electrophoresis
 * PSB4K5, PSB3K3, S03518 and R0040


 * Inoculate for Miniprep
 * PSB1AT3, PSB1AC3

Day 4 Thursday (02/07/09)

 * Transformation (using XL1-Blue)
 * I13600 and I13601
 * Result : Worked


 * Gel Electrophoresis
 * R0040


 * Inoculate for Miniprep
 * PSB3AT3 and PSB3AC3

Day 5 Friday (03/07/09)

 * Miniprep
 * PSB3AT3 and PSB3AC3


 * End of week meeting

Day 1 Monday (06/07/09)

 * Gel Electrophoresis
 * I732820 (LacI), R0040 (Tet O), PSB1AC3 and PSB1AT3,


 * Ligation calculation.


 * Inoculation for Miniprep
 * I13600 and I13601


 * Heat inactivation of week 4 Friday digestions.


 * Preparation of LB medium and LB agar medium.


 * Added BioBricks to the Wiki
 * BBa_K182001 and BBa_K182002

Day 2 Tuesday (07/07/09)

 * Alkaline phosphatase treatment of PSB1AC3
 * Plasmid Miniprep
 * I13600 and I13601
 * Pouring of 15 Chloramphenicol plates
 * Ligation
 * PSB1AC3(Plasmid Backbone +RFP -CCDB), I732820 (LacI)(upstream), R0040 (Tet O)(downstream)
 * The successful ligation would create the intermediate BioBrick K182001
 * Transforming Ligated plasmid into XL1-Blue.

Day 3 Wednesday(08/07/09)

 * Restriction digest
 * S03518, K112808, I13600 and I13601.
 * Gel Electrophoresis
 * S03518, K112808, I13600 and I13601
 * Made colony counts for K182001.
 * Transferred 50 colonies of K182001 for colour screening

Day 4 Thursday (09/07/09)

 * Digestion for ligating of K182002
 * S03518 and R0051
 * Gel Electrophoresis for S03518 and R0051

Day 5 Friday (10/07/09)

 * Preparation for ligation.
 * Preperation for PCR
 * Write Up
 * Friday Meeting

Day 1 Monday (13/07/09)

 * Heat Inactivation
 * Digestions of S03518, R0051


 * Alkaline phosphatase treatment
 * 20ul pSB1AC
 * Heat inactivation of Alkaline phosphatase


 * Pouring of 15 Chloramphenicol plates


 * Construction of K182002
 * Ligation
 * pSB1AC3 (plasmid Backbone) + S03518 (downstream), R0051 (upstream)
 * Transformation into XL1-Blue.


 * Colony PCR
 * K182001 (16 colonies)
 * Gel Electrophoresis
 * Result : Failed (no bands on gel) (possibly due to problem with tubes used)


 * Requested Parts from the registry
 * BBa_J37032 and pSB3K5.

Day 2 Tuesday (14/07/09)

 * Colony PCR (Stansfield lab PCR machine)
 * K182001 (16 colonies)
 * Gel Electrophoresis of PCR
 * Result : Successful.


 * Cell counts of K182002


 * Transferred 50 colonies of K182002 for colour screening

Day 3 Wednesday (15/07/09)

 * Write up + organisation.


 * Colony PCR (Stansfield lab PCR machine)
 * K182002 (16 colonies)


 * Took pictures of I13600, I13601 under fluorescent microscope
 * Expected Results : I13600 Positive, I13601 Negative (due to LacI repression in XL1-blue)
 * Results : I13600 Positive (unambiguous fluorescence observed), I13601 Negative (no fluorescence observed)


 * Inoculation for Miniprep
 * K182001


 * Research for Digestion sites in K182001 and K182002 to confirm inserts.

Day 4 Thursday (16/07/09)

 * Miniprep
 * K182001 (Colonies 6,7 and 9)
 * Gel electrophoresis
 * PCR from K182002


 * Restriction Digest
 * K182001 (Colonies 6,7,9,10) and K182002 (Colonies 7,9,10,11)


 * Restriction Digest for 1ml Lambda ladder


 * 1600ml (4x400ml) of Lb+Agar and 400ml of LB


 * Gel Electrophoresis
 * K182001 (Colonies 6,7,9,10) and K182002 (Colonies 7,9,10,11)

Day 5 Friday (17/07/09)

 * Friday Meeting
 * Handover from Ian to Russell and Claudia
 * Maths tutorial for better understanding of Modelling methods.

Day 1 Monday (20/07/09)

 * Miniprep
 * K182002 Colonies 9,10,11
 * Fluorescent Microscope
 * I13601 + IPTG
 * Result : Positive ( fluorescence was induced as expected.)


 * Restriction Digest
 * K182001 (Colonies 7,9) K182002 (Colonies 9,10,11)


 * Gel Electrophoresis
 * K182001 (Colonies 7,9) K182002 (Colonies 9,10,11)


 * Inoculation for Miniprep
 * K182002 colonies 9,10,11

Day 2 Tuesday (21/07/09)

 * Miniprep
 * K182002 colonies 9,10,11
 * Sent samples for sequencing
 * K182001 (Colonies 7,9) K182002 (Colonies 9,10,11)
 * Inoculate for making competent using TSS method
 * 2 samples of I13601 (Colony 1)
 * Meeting with Sam Miller
 * discuss possible Motility Assays

Day 3 Wednesday (22/07/09)

 * TSS method for making cells competent
 * I13601
 * Restriction digest
 * K182002 (Colonies 9,10,11)
 * Gel Electrophoresis of K182002 (Colonies 9,10,11)
 * Write up

Day 4 Thursday (23/07/09)

 * Transformation
 * K182002 into I13601 TSS cells.
 * Calculations for K182003 ligation
 * Restriction Digest for construction of K182003 from K182002 and K182001
 * K182001 (downstream)and K182002 (upstream)
 * Gel Electrophoresis
 * K182001, K182002.
 * Heat inactivation of enzymes of K182001 and K182002.
 * Inoculation for Miniprep
 * pSB3K5

Day 5 Friday (24/07/09)

 * Miniprep of Psb3K5
 * Restriction Digest of Psb3K5
 * Gel Electrophoresis of Psb3K5
 * Friday Meeting

Day 1 Monday (27/07/09)

 * Preperation
 * 7 Eppendorf tubes (x2) 12 Chl + 2 Amp plates
 * Put LB agar into Autoclave.
 * Get sequencing from Russell
 * Analyse Sequencing
 * Pour Plates
 * Discuss Model with Prof. McGlynn
 * Inoculation for miniprep
 * K182002 (colony 4,5)

Day 2 Tuesday (28/07/09)

 * Ligation Maths
 * Phosphatase Treatment
 * Psb1AT3
 * Ligation
 * K182001 {Colony 7}(downstream), K182002 {Colony 9}(upstream), Psb1AT3 (plasmid Backbone)
 * K182001 {Colony 7}(downstream), K182002 {Colony 10}(upstream), Psb1AT3 (plasmid Backbone)
 * Transformation of ligations (SCS1 supercompetent Cells)
 * Miniprep
 * K182002 Colony 4 and 5
 * Estimate DNA in miniprep using spectrometer.
 * Send off for sequencing
 * (Colony 4,5 and 9)
 * Colony PCR
 * K182002 (18 colonies)

Day 3 Wednesday (29/07/09)

 * Colony PCR
 * K182003 (Colony 9) {17 colonies}
 * K182003 (Colony 10) {17 colonies}
 * Restriction Digest
 * Colony PCR of K182002
 * Gel Electrophoresis
 * K182002 (digested + undigested)
 * K182003 (9,10) (undigested)

Day 4 Thursday (30/07/09)

 * Sequence Analysis.
 * Plan Anhydrotetracycline assay.
 * make up 1.25 ml of Anhydrotetracycline stock.
 * Fluorescent microscopy
 * I13600/K182002 (Colony 10)
 * Colony PCR
 * K182002 + K182003 (Colony 10)
 * Plated K182002 (10) for AHT assay (with 20 ul AHT)

Day 5 Friday (31/07/09)

 * Fluorescent Microscopy

Day 1 Monday (03/08/09)

 * Gel Electrophoresis
 * K182003 and K182002
 * Inoculation for Miniprep
 * K182003 and K182002

Day 2 Tuesday (04/08/09)

 * Miniprep
 * K182002 and K182003


 * Restriction digestion
 * K182002 and K182003


 * Inoculation of CFP constructs for AHT Assay

Day 3 Wednesday (05/08/09)

 * Gel Electrophoresis
 * K182003
 * Inoculation of CFP constructs for AHT Assays

Day 4 Thursday (06/08/09)

 * Made up 800ul LB


 * Fluorescence Assay (Fluorometer)
 * Fluorescence Assay (Microscopy)

Day 5 Friday (07/08/09)

 * Write up

Day 1 Monday (10/08/09)

 * Wrote Notebook for Wiki
 * Wrote reviews for biobricks used

Day 2 Tuesday (11/08/09)

 * Worked on updating the wiki
 * Wrote reviews for biobricks used

Day 3 Wednesday (12/08/09)

 * Got Fluorescent pictures from Standsfield Computer
 * Chassed up people for the “Our Team” section of the Wiki
 * General Work on the Wiki

Day 4 Thursday (13/08/09)

 * Working on the Wiki
 * Planning Quantitative Assay for K182005
 * Innoculating Cells for K182005 Assay

Day 5 Friday (14/08/09)

 * Working on the Wiki
 * Quantitative Assay for K182005

Day 1 Monday (17/08/09)

 * Working on the Wiki and Poster
 * Planning 2nd Quantitative Assay for K182005
 * Inoculating Cells for K182005 Assay

Day 2 Tuesday (18/08/09)

 * Quantitative Assay for K182005

Day 3 Wednesday (19/08/09)

 * Working on Wiki

Day 4 Thursday (20/08/09)

 * Working on Wiki

Day 5 Friday (21/08/09)

 * Cake