Team:Aberdeen Scotland/notebook/lacilatch

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Latest revision as of 16:51, 28 August 2009

University of Aberdeen iGEM 2009

University of Aberdeen - Pico Plumber

iGEM 2009

Contents

1 LacI-Latch Notebook

LacI-Latch Notebook

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

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

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

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

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

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

Week 4: Analysis of BioBricks for cloning (29/06/09 - 03/07/09)

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

Week 5 : Construction of K182001 (06/07/09 - 10/07/09)

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

Week 6 : Construction of K182002 and analysis of clones (13/07/09 - 16/07/09)

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.

Week 7 : Sequencing and Preperation for K182002 assay (20/07/09 - 24/07/09)

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

Week 8 : Fluorescent Microscopy (27/07/09 - 31/07/09)

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

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

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

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

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

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

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

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