Team:Aberdeen Scotland/notebook/lacilatch

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University of Aberdeen iGEM 2009


Contents

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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