Team:Aberdeen Scotland/notebook/lacilatch

From 2009.igem.org

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      <a href="https://2009.igem.org/Team:Aberdeen_Scotland/notebook/lacilatch/week1"><img src="https://static.igem.org/mediawiki/2009/f/f2/Week_1Aberdeen2009.JPG">
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=LacI-Latch Notebook=
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</html>
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 +
 
 +
==Week 1: Research and Planning (08/06/09 - 12/06/09)==
 +
<br><br>
 +
===Day 1 Monday (08/06/09)===
 +
 
 +
*Researching the Registry for Biobricks
 +
**Identified K182022, K112808, R0062, I712074, I14015 for further discussion.
 +
<br><br>
 +
===Day 2 Tuesday (09/06/09)===
 +
 
 +
*Researching the Registry for Plasmids.
 +
**Identified pSB3T5, pSB3C5, pSB1AC3, pSB1AT3 for further discussion.
 +
 
 +
<br><br>
 +
===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.
 +
 
 +
<br><br>
 +
*Researching the Registry for LacI Repressor and T7 Polymerase
 +
** Identified K145014, K145001 and I732100 for further discussion
 +
 
 +
 
 +
<br><br>
 +
===Day 4 Thursday (11/06/09)===
 +
 
 +
*Parameter search for the Modelling effort.
 +
 
 +
 
 +
<br><br>
 +
===Day 5 Friday (12/06/09)===
 +
 
 +
*End of week meeting
 +
*Discussion and planning for following week
 +
 
 +
<br><br>
 +
 
 +
==Week 2: BioBrick Rescue  (15/06/09 - 19/06/09)==
 +
<br><br>
 +
===Day 1 Monday (15/06/09)===
 +
 
 +
*Prepared LB medium for Lab<br><br>
 +
*Planning of LacI-Latch<br><br>
 +
*Inoculated J23100, K145001, B0034 for Miniprep the following day
 +
 
 +
<br><br>
 +
===Day 2 Tuesday (16/06/09)===
 +
 
 +
*Miniprep of J23100, K145001, B0034<br><br>
 +
*Restriction Digest <br><br>
 +
 
 +
 
 +
 
 +
===Day 3 Wednesday (17/06/09)===
 +
 
 +
* Lambda Digest with HindIII for use as a ladder<br><br>
 +
* Gel Electrophoresis of B0034, K145001, J23100 and I732100.<br><br>
 +
 
 +
 
 +
===Day 4 Thursday (18/06/09)===
 +
 
 +
*Gel Electrophoresis of C0051, R0062, C0040, J37033, K145001 and I732100.<br><br>
 +
*Inoculation of C0051, R0062, J37033, I732100 or miniprep.<br><br>
 +
 
 +
 
 +
 
 +
===Day 5 Friday (19/06/09)===
 +
 
 +
*Miniprep of C0051, R0062, J37033 and I732100.<br><br>
 +
*End of week meeting<br><br>
 +
 
 +
==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<br><br>
 +
 
 +
 
 +
===Day 2 Tuesday (23/06/09)===
 +
 
 +
Risat:
 +
*Research for Plasmids and J series Promoters<br><br>
 +
*Rescued and Transformed Psb4K4 and Psb3K5.<br><br>
 +
*Restriction Digest of B0015, R0040, E0840, R0011, K093005, R0051, K081008, J37033 and R0062,  <br><br>
 +
 
 +
Daniel:
 +
*Edinburgh Igem Meeting<br><br>
 +
 
 +
===Day 3 Wednesday (24/06/09)===
 +
 
 +
Risat:
 +
*Gel Electrophoresis of B0015, R0040, E0840, R0011, K093005, R0051, K081008, J37033 and R0062,  <br><br>
 +
 
 +
Daniel:
 +
*Edinburgh Igem Meeting<br><br>
 +
 
 +
 
 +
===Day 4 Thursday (25/06/09)===
 +
 
 +
Risat:
 +
*Gel Electrophoresis of K112808, K112022 and I732100<br><br>
 +
 
 +
Daniel:
 +
*Edinburgh Igem Meeting<br><br>
 +
 
 +
 
 +
===Day 5 Friday (26/06/09)===
 +
 
 +
*Restriction Digest of E0840<br><br>
 +
*Gel Electrophoresis of E0840<br><br>
 +
*End of week meeting<br><br>
 +
 
 +
==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)<br><br>
 +
 
 +
 
 +
 
 +
*Inoculate for miniprep.  
 +
** I732820 (LacI,) R0051 (CI operator), S03518 ( TetR), K112808 ( Holin), PSB3K3, PSB3T5.<br><br>
 +
 
 +
 
 +
*Rescue I13600 and I13601
 +
** Required for testing of constructs<br><br>
 +
 
 +
 
 +
===Day 2 Tuesday (30/06/09)===
 +
 
 +
*Miniprep
 +
**I732820 (LacI) R0051 (CI operator)  S03518 ( TetR) K112808 ( Holin)  PSB3K3, PSB3T5.<br><br>
 +
 
 +
*Planning for Restriction digest
 +
**R0051, S03518, R0040, PSB3K3 and I732820.<br><br>
 +
 
 +
* Restriction Digest
 +
**R0051, S03518, R0040, PSB3K3 and I732820.<br><br>
 +
 
 +
 
 +
*Gel Electrophoresis
 +
**R0051, S03518, R0040, PSB3K3 and I732820.<br><br>
 +
 
 +
 +
*Transformation (using XL1-Blue)
 +
**I13600 and I13601
 +
*** Result : Failed (no growth)<br><br>
 +
 
 +
*Inoculation for Miniprep
 +
**PSB4K5, PSB3K3<br><br>
 +
 
 +
 
 +
===Day 3 Wednesday (01/07/09)===
 +
 
 +
*Miniprep
 +
**PSB4K5, PSB3K3<br><br>
 +
 
 +
 
 +
 
 +
*Restriction Digest
 +
**PSB4K5, PSB3K3, S03518 and R0040<br><br>
 +
 
 +
 
 +
 
 +
*Gel Electrophoresis
 +
**PSB4K5, PSB3K3, S03518 and R0040<br><br>
 +
 
 +
*Inoculate for Miniprep
 +
**PSB1AT3, PSB1AC3<br><br>
 +
 
 +
 
 +
 
 +
===Day 4 Thursday (02/07/09)===
 +
 
 +
*Transformation (using XL1-Blue)
 +
**I13600 and I13601
 +
*** Result : Worked<br><br>
 +
 
 +
*Gel Electrophoresis
 +
**R0040<br><br>
 +
 
 +
*Inoculate for Miniprep
 +
**PSB3AT3 and PSB3AC3<br><br>
 +
 
 +
 
 +
===Day 5 Friday (03/07/09)===
 +
 +
*Miniprep 
 +
**PSB3AT3 and PSB3AC3<br><br>
 +
 
 +
 
 +
*End of week meeting<br><br>
 +
 
 +
==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,<br><br>
 +
 
 +
*Ligation calculation.<br><br>
 +
 
 +
*Inoculation for Miniprep
 +
** I13600 and I13601<br><br>
 +
 
 +
*Heat inactivation of week 4 Friday digestions. <br><br>
 +
 
 +
*Preparation of LB medium and LB agar medium. <br><br>
 +
 
 +
*Added BioBricks to the Wiki
 +
**BBa_K182001 and BBa_K182002 <br><br>
 +
 
 +
===Day 2 Tuesday (07/07/09)===
 +
 
 +
*Alkaline phosphatase treatment of PSB1AC3<br><br>
 +
*Plasmid Miniprep
 +
**I13600 and I13601<br><br>
 +
*Pouring of 15 Chloramphenicol plates<br><br>
 +
*Ligation
 +
**PSB1AC3(Plasmid Backbone +RFP -CCDB), I732820 (LacI)(upstream), R0040 (Tet O)(downstream)<br><br>
 +
*** The successful ligation would create the intermediate BioBrick K182001<br><br>
 +
*Transforming Ligated plasmid into XL1-Blue.<br><br>
 +
 
 +
===Day 3 Wednesday(08/07/09)===
 +
 
 +
*Restriction digest
 +
**S03518, K112808, I13600 and I13601.<br><br>
 +
*Gel Electrophoresis
 +
** S03518, K112808, I13600 and I13601
 +
*Made colony counts for K182001. <br><br>
 +
*Transferred 50 colonies of K182001 for colour screening<br><br>
 +
 
 +
===Day 4 Thursday (09/07/09)===
 +
 
 +
*Digestion for ligating of K182002
 +
**S03518 and R0051<br><br>
 +
*Gel Electrophoresis for  S03518 and R0051<br><br>
 +
 
 +
===Day 5 Friday (10/07/09)===
 +
 
 +
*Preparation for ligation.
 +
*Preperation for PCR
 +
*Write Up
 +
*Friday Meeting <br><br>
 +
 
 +
==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 <br><br>
 +
 
 +
*Alkaline phosphatase treatment
 +
**20ul pSB1AC
 +
***Heat inactivation of  Alkaline phosphatase<br><br>
 +
 
 +
*Pouring of 15 Chloramphenicol plates<br><br>
 +
 
 +
*Construction of K182002
 +
**Ligation
 +
***pSB1AC3 (plasmid Backbone) + S03518 (downstream) , R0051 (upstream)
 +
** Transformation into XL1-Blue.<br><br>
 +
 
 +
*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. <br><br>
 +
 
 +
===Day 2 Tuesday (14/07/09)===
 +
 
 +
*Colony PCR  (Stansfield lab PCR machine)
 +
**K182001 (16 colonies)
 +
**Gel Electrophoresis of PCR
 +
*** Result : Successful.<br><br>
 +
 
 +
*Cell counts of K182002<br><br>
 +
 
 +
*Transferred 50 colonies of K182002 for colour screening<br><br>
 +
 
 +
 
 +
===Day 3 Wednesday (15/07/09)===
 +
 
 +
*Write up + organisation.<br><br>
 +
 
 +
*Colony PCR  (Stansfield lab PCR machine)
 +
**K182002 (16 colonies)<br><br>
 +
 
 +
*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)  <br><br>
 +
 
 +
*Inoculation for Miniprep
 +
**K182001<br><br>
 +
 
 +
*Research for Digestion sites in K182001 and K182002 to confirm inserts.<br><br>
 +
 
 +
 
 +
===Day 4 Thursday (16/07/09)===
 +
 
 +
*Miniprep
 +
** K182001 (Colonies 6,7 and 9)<br><br>
 +
*Gel electrophoresis
 +
** PCR from K182002<br><br>
 +
 
 +
*Restriction Digest
 +
** K182001 (Colonies 6,7,9,10) and K182002 (Colonies 7,9,10,11)<br><br>
 +
 
 +
*Restriction Digest for 1ml Lambda ladder<br><br>
 +
 
 +
*1600ml (4x400ml) of Lb+Agar and 400ml of LB<br><br>
 +
 
 +
*Gel Electrophoresis
 +
** K182001 (Colonies 6,7,9,10) and K182002 (Colonies 7,9,10,11)<br><br>
 +
 
 +
 
 +
===Day 5 Friday (17/07/09)===
 +
 
 +
* Friday Meeting <br><br>
 +
* Handover from Ian to Russell and Claudia<br><br>
 +
* Maths tutorial for better understanding of Modelling methods.<br><br>
 +
 
 +
==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<br><br>
 +
*Fluorescent Microscope
 +
**I13601 + IPTG
 +
*** Result : Positive ( fluorescence was induced as expected.)<br><br>
 +
 
 +
* Restriction Digest
 +
** K182001 (Colonies 7,9)  K182002 (Colonies 9,10,11)<br><br>
 +
 
 +
*Gel Electrophoresis
 +
** K182001 (Colonies 7,9)  K182002 (Colonies 9,10,11)<br><br>
 +
 
 +
*Inoculation for Miniprep
 +
** K182002 colonies 9,10,11 <br><br>
 +
 
 +
===Day 2 Tuesday (21/07/09)===
 +
 
 +
*Miniprep
 +
** K182002 colonies 9,10,11<br><br>
 +
*Sent samples for sequencing
 +
**K182001 (Colonies 7,9)  K182002 (Colonies 9,10,11)<br><br>
 +
*Inoculate for making competent using TSS method
 +
**2 samples of I13601 (Colony 1) <br><br>
 +
*Meeting with Sam Miller
 +
** discuss possible Motility Assays <br><br>
 +
 
 +
===Day 3 Wednesday (22/07/09)===
 +
 
 +
*TSS method for making cells competent
 +
** I13601<br><br>
 +
*Restriction digest
 +
** K182002 (Colonies 9,10,11)
 +
**Gel Electrophoresis of K182002 (Colonies 9,10,11)<br><br>
 +
*Write up<br><br>
 +
 
 +
===Day 4 Thursday (23/07/09)===
 +
 
 +
*Transformation
 +
**K182002 into I13601 TSS cells.<br><br>
 +
*Calculations for K182003 ligation<br><br>
 +
*Restriction Digest for construction of K182003 from K182002 and K182001
 +
** K182001 (downstream)and K182002 (upstream)<br><br>
 +
*Gel Electrophoresis
 +
**K182001, K182002.<br><br>
 +
*Heat inactivation of enzymes of K182001 and K182002.<br><br>
 +
*Inoculation for Miniprep
 +
**pSB3K5<br><br>
 +
 
 +
===Day 5 Friday (24/07/09)===
 +
 
 +
*Miniprep of Psb3K5
 +
**Restriction Digest of Psb3K5
 +
***Gel Electrophoresis of Psb3K5<br><br>
 +
*Friday Meeting<br><br>
 +
 
 +
==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. <br><br>
 +
*Get sequencing from Russell
 +
** Analyse Sequencing<br><br>
 +
*Pour Plates <br><br>
 +
*Discuss Model with Prof. McGlynn<br><br>
 +
*Inoculation for miniprep
 +
**K182002 (colony 4,5) <br><br>
 +
 
 +
===Day 2 Tuesday (28/07/09)===
 +
 
 +
*Ligation Maths <br><br>
 +
*Phosphatase Treatment
 +
** Psb1AT3 <br><br>
 +
*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)<br><br>
 +
*Miniprep
 +
**K182002 Colony 4 and 5
 +
**Estimate DNA in miniprep using spectrometer.<br><br> 
 +
*Send off for sequencing
 +
**(Colony 4,5 and 9)<br><br>
 +
*Colony PCR
 +
** K182002 (18 colonies)<br><br>
 +
 
 +
===Day 3 Wednesday (29/07/09)===
 +
 
 +
*Colony PCR
 +
** K182003 (Colony 9) {17 colonies}
 +
** K182003 (Colony 10) {17 colonies}<br><br>
 +
*Restriction Digest
 +
** Colony PCR of K182002  <br><br> 
 +
*Gel Electrophoresis
 +
**K182002 (digested + undigested)
 +
**K182003 (9,10) (undigested) <br><br>
 +
 
 +
===Day 4 Thursday (30/07/09)===
 +
 
 +
*Sequence Analysis. <br><br>
 +
*Plan Anhydrotetracycline assay. <br><br>
 +
*make up 1.25 ml of Anhydrotetracycline stock.<br><br>
 +
*Fluorescent microscopy
 +
**I13600/K182002 (Colony 10)<br><br>
 +
*Colony PCR
 +
** K182002 + K182003 (Colony 10)<br><br>
 +
*Plated K182002 (10) for AHT assay (with 20 ul AHT)<br><br>
 +
 
 +
===Day 5 Friday (31/07/09)===
 +
 
 +
* Fluorescent Microscopy <br><br>
 +
 
 +
==Week 9 (03/08/09 - 07/08/09)==
 +
 
 +
===Day 1 Monday (03/08/09)===
 +
 
 +
 
 +
*Gel Electrophoresis
 +
** K182003 and K182002<br><br>
 +
*Inoculation for Miniprep
 +
**K182003 and K182002<br><br>
 +
 
 +
 
 +
===Day 2 Tuesday (04/08/09)===
 +
 
 +
*Miniprep
 +
** K182002 and K182003<br><br>
 +
 
 +
*Restriction digestion
 +
** K182002 and K182003<br><br>
 +
 
 +
*Inoculation of CFP constructs for AHT Assay<br><br>
 +
 
 +
===Day 3 Wednesday (05/08/09)===
 +
 
 +
 
 +
*Gel Electrophoresis
 +
** K182003
 +
*Inoculation of CFP constructs for AHT Assays<br><br>
 +
 
 +
 
 +
===Day 4 Thursday (06/08/09)===
 +
 
 +
*Made up 800ul LB<br><br>
 +
 
 +
*Fluorescence Assay (Fluorometer)
 +
*Fluorescence Assay (Microscopy)<br><br>
 +
 
 +
 
 +
 
 +
===Day 5 Friday (07/08/09)===
 +
 
 +
*Write up<br><br>
 +
 
 +
==Week 10 (10/08/09 - 14/08/09)==
 +
 
 +
===Day 1 Monday (10/08/09)===
 +
 
 +
*Wrote Notebook for Wiki<br><br>
 +
*Wrote reviews for biobricks used<br><br>
 +
 
 +
===Day 2 Tuesday (11/08/09)===
 +
 
 +
*Worked on updating the wiki<br><br>
 +
*Wrote reviews for biobricks used<br><br>
 +
 
 +
===Day 3 Wednesday (12/08/09)===
 +
 
 +
*Got Fluorescent pictures from Standsfield Computer<br><br>
 +
*Chassed up people for the “Our Team” section of the Wiki<br><br>
 +
*General Work on the Wiki<br><br>
 +
 
 +
===Day 4 Thursday (13/08/09)===
 +
 
 +
*Working on the Wiki <br><br>
 +
*Planning Quantitative Assay for K182005<br><br>
 +
*Innoculating Cells for K182005 Assay<br><br>
 +
 
 +
===Day 5 Friday (14/08/09)===
 +
 
 +
*Working on the Wiki <br><br>
 +
*Quantitative Assay for K182005<br><br>
 +
 
 +
==Week 11 (17/08/09 - 21/08/09)==
 +
 
 +
===Day 1 Monday (17/08/09)===
 +
 
 +
*Working on the Wiki and Poster<br><br>
 +
*Planning 2nd Quantitative Assay for K182005<br><br>
 +
*Inoculating Cells for K182005 Assay<br><br>
 +
 
 +
===Day 2 Tuesday (18/08/09)===
 +
 
 +
*Quantitative Assay for K182005<br><br>
 +
 
 +
===Day 3 Wednesday (19/08/09)===
 +
 
 +
*Working on Wiki<br><br>
 +
 
 +
 
 +
===Day 4 Thursday (20/08/09)===
 +
 
 +
*Working on Wiki<br><br>
 +
 
 +
===Day 5 Friday (21/08/09)===
 +
 
 +
*Cake<br><br>
 +
 
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Latest revision as of 16:51, 28 August 2009

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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