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 disccusion.



Day 2 Tuesday (09/06/09)

  • Researching the Registry for Plasmids.
    • Identified pSB3T5, pSB3C5, pSB1AC3, pSB1AT3 for further discusion.



Day 3 Wednesday (10/06/09)

  • Literature search for Parameters of T7 promoter and the Holin system.
    • Conlusion: T7 feedback mechanism of Wiring diagram 1 is unliekly 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, I732100 for further discusion




Day 4 Thursday (11/06/09)

  • Parameter search for the Modeling effort.




Day 5 Friday (12/06/09)

  • End of week meeting
  • Discussion and planning of 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, I732100.


Day 4 Thursday (18/06/09)

  • Gel Electrophoresis of C0051, R0062, C0040, J37033, K145001, I732100.

  • Innoculation of C0051, R0062, J37033, I732100 or miniprep.


Day 5 Friday (19/06/09)

  • Miniprep of C0051, R0062, J37033, 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, 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, R0062,

Daniel:

  • Edinburgh Igem Meeting

Day 3 Wednesday (24/06/09)

Risat:

  • Gel Electrophoresis of B0015, R0040, E0840, R0011, K093005, R0051, K081008, J37033, R0062,

Daniel:

  • Edinburgh Igem Meeting


Day 4 Thursday (25/06/09)

Risat:

  • Gel Electrophoresis of K112808, K112022, 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 BioBrick's for Cloning (29/06/09 - 03/07/09)

Day 1 Monday

  • Run Gel of E0840 ( Testing of digests)


  • Innoculate 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

  • Miniprep
    • I732820 (LacI) R0051 (CI operator) S03518 ( TetR) K112808 ( Holin) PSB3k3, PSB3T5.

  • Planning for Restriction digest
    • R0051, S03518, R0040, PSB3k3, I732820.

  • Restriction Digest
    • R0051, S03518, R0040, PSB3k3, I732820.


  • Gel Electrophoresis
    • R0051, S03518, R0040, PSB3k3, I732820.


  • Transformation (using XL1-Blue)
    • I13600, I13601
      • Result : Failed (no growth)

  • Innoculation for Miniprep
    • PSB4K5, PSB3K3


Day 3 Wednesday

  • Miniprep
    • PSB4K5, PSB3K3


  • Restriction Digest
    • PSB4k5, PSB3K3, S03518, R0040


  • Gel electrophoresis
    • PSB4K5, PSB3K3, S03518, R0040

  • Innoculate for Miniprep
    • PSB1AT3, PSB1AC3


Day 4 Thursday

  • Transformation (using Xl1-Blue)
    • I13600 and I13601
      • Result : Worked

  • Gel Electrophoresis
    • R0040

  • Incoculate for Miniprep
    • PSB3AT3, PSB3AC3


Day 5 Friday

  • Miniprep
    • PSB3AT3, PSB3AC3


  • End of week meeting

Week 5 : Construction of K182001 (05/07/09 - 10/07/09)

Day 1 Monday (06/07/09)

  • Gel Electrophoresis
    • I732820 (LacI), R0040 (Tet O), PSB1AC3, 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 phosphatise treatment of PSB1AC3

  • Plasmid Miniprep
    • I13600 and I13601

  • Pouring of 15 Chlorampenicol plates

  • Ligation
    • PSB1AC3(Plasmid Backbone +RFP -CCDB), I732820 (LacI)(Upstream), R0040 (Tet O)(downstream)

      • The succesful 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 color 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 K182001 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 Chlorampenicol 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 : Succesfull.

  • Cell counts of K182002

  • Transferred 50 colonies of K182002 for color 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 flurescent Microscope
    • Expected Results : I13600 Positive, I13601 Negative (due to LacI repression in XL1-blue
    • Results : I13600 Positive (unambiguous fluorescence observed), I13601 Negative (no flurescence observed)

  • Innoculation 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 to from Ian to Russell and Claudia

  • Maths tutorial for better understanding of Modeling methodes.

Week 7 (20/07/09 - 24/07/09)

Day 1 Monday (20/07/09)

1. Miniprep of K182002 9,10,11 2. Fluorescent Microscope of I13601 + IPTG 3. Digestion of K182001 ( 7,9) K182002 (9,10,11) 4. Gel Electrophoresis of K182001 ( 7,9) K182002 (9,10,11) 5. Miniprep Preperation of K182002 9,10,11

Day 2 Tuesday (21/07/09)

1. Miniprep of K182002 9,10,11 2. Sent Samples K182001 ( 7,9) K182002 (9,10,11) off for sequencing 3. Grow I13601 (1) + I13601 (1) for TSS overnight. 4. Meeting with Sam Miller

Day 3 Wednesday (22/07/09)

1. TSS method of making I13601 (1) competent 2. Restriction digest of K182002 (9,10,11) 3. Gel Electrophoresis of K182002 (9,10,11) 4. Write up

Day 4 Thursday (23/07/09)

1. Transformation of K182002 into I13601 TSS cells. 2. Calculations for K182003 ligation 3. Digestions for K182003 ligation. K182001, K182002 4. Gel Electrophoresis of K182001, K182002. 5. Heat inactivation of enzymes of K182001, K182002. 6. inoculation of Psb3k5

Day 5 Friday (24/07/09)

1. Miniprep of Psb3k5 2. Restriction Digest of Psb3K5 3. Gel Electrophoresis of Psb3k5 4. Friday Meeting

Week 8 (27/07/09 - 31/07/09)

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

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

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

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