Team:Aberdeen Scotland/notebook/lacilatch
From 2009.igem.org
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*Researching the Registry for Biobricks | *Researching the Registry for Biobricks | ||
- | **Identified K182022, K112808, R0062, I712074, I14015 for further | + | **Identified K182022, K112808, R0062, I712074, I14015 for further discussion. |
<br><br> | <br><br> | ||
===Day 2 Tuesday (09/06/09)=== | ===Day 2 Tuesday (09/06/09)=== | ||
*Researching the Registry for Plasmids. | *Researching the Registry for Plasmids. | ||
- | **Identified pSB3T5, pSB3C5, pSB1AC3, pSB1AT3 for further | + | **Identified pSB3T5, pSB3C5, pSB1AC3, pSB1AT3 for further discussion. |
<br><br> | <br><br> | ||
===Day 3 Wednesday (10/06/09)=== | ===Day 3 Wednesday (10/06/09)=== | ||
- | *Literature search for | + | *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 | + | ** Identified the Novagen plasmids pLysS and pLysE as possible solutions. |
<br><br> | <br><br> | ||
*Researching the Registry for LacI Repressor and T7 Polymerase | *Researching the Registry for LacI Repressor and T7 Polymerase | ||
- | ** Identified K145014, K145001 | + | ** Identified K145014, K145001 and I732100 for further discussion |
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===Day 4 Thursday (11/06/09)=== | ===Day 4 Thursday (11/06/09)=== | ||
- | *Parameter search for the | + | *Parameter search for the Modelling effort. |
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*End of week meeting | *End of week meeting | ||
- | *Discussion and planning | + | *Discussion and planning for following week |
<br><br> | <br><br> | ||
+ | |||
==Week 2: BioBrick Rescue (15/06/09 - 19/06/09)== | ==Week 2: BioBrick Rescue (15/06/09 - 19/06/09)== | ||
<br><br> | <br><br> | ||
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===Day 3 Wednesday (17/06/09)=== | ===Day 3 Wednesday (17/06/09)=== | ||
- | * Lambda Digest with HindIII for use as a | + | * Lambda Digest with HindIII for use as a ladder<br><br> |
- | * Gel Electrophoresis of B0034, K145001, J23100 | + | * Gel Electrophoresis of B0034, K145001, J23100 and I732100.<br><br> |
===Day 4 Thursday (18/06/09)=== | ===Day 4 Thursday (18/06/09)=== | ||
- | *Gel Electrophoresis of C0051, R0062, C0040, J37033, K145001 | + | *Gel Electrophoresis of C0051, R0062, C0040, J37033, K145001 and I732100.<br><br> |
- | * | + | *Inoculation of C0051, R0062, J37033, I732100 or miniprep.<br><br> |
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===Day 5 Friday (19/06/09)=== | ===Day 5 Friday (19/06/09)=== | ||
- | *Miniprep of C0051, R0062, J37033 | + | *Miniprep of C0051, R0062, J37033 and I732100.<br><br> |
*End of week meeting<br><br> | *End of week meeting<br><br> | ||
- | ==Week 3: Edinburgh iGEM meeting and BioBrick | + | ==Week 3: Edinburgh iGEM meeting and BioBrick rescuing (22/06/09 - 26/06/09)== |
===Day 1 Monday (22/06/09)=== | ===Day 1 Monday (22/06/09)=== | ||
- | *Rescued BBa_1732820, S03518 | + | *Rescued BBa_1732820, S03518 and B0030<br><br> |
Line 90: | Line 91: | ||
Risat: | Risat: | ||
*Research for Plasmids and J series Promoters<br><br> | *Research for Plasmids and J series Promoters<br><br> | ||
- | *Rescued and Transformed | + | *Rescued and Transformed Psb4K4 and Psb3K5.<br><br> |
- | *Restriction Digest of B0015, R0040, E0840, R0011, K093005, R0051, K081008, J37033 | + | *Restriction Digest of B0015, R0040, E0840, R0011, K093005, R0051, K081008, J37033 and R0062, <br><br> |
Daniel: | Daniel: | ||
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Risat: | Risat: | ||
- | *Gel Electrophoresis of B0015, R0040, E0840, R0011, K093005, R0051, K081008, J37033 | + | *Gel Electrophoresis of B0015, R0040, E0840, R0011, K093005, R0051, K081008, J37033 and R0062, <br><br> |
Daniel: | Daniel: | ||
Line 108: | Line 109: | ||
Risat: | Risat: | ||
- | *Gel Electrophoresis of K112808, K112022 | + | *Gel Electrophoresis of K112808, K112022 and I732100<br><br> |
Daniel: | Daniel: | ||
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*End of week meeting<br><br> | *End of week meeting<br><br> | ||
- | ==Week 4: Analysis of | + | ==Week 4: Analysis of BioBricks for cloning (29/06/09 - 03/07/09)== |
- | ===Day 1 Monday=== | + | ===Day 1 Monday (29/06/09)=== |
*Run Gel of E0840 ( Testing of digests)<br><br> | *Run Gel of E0840 ( Testing of digests)<br><br> | ||
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- | * | + | *Inoculate for miniprep. |
- | ** I732820 (LacI,) R0051 (CI operator), S03518 ( TetR), K112808 ( Holin), | + | ** I732820 (LacI,) R0051 (CI operator), S03518 ( TetR), K112808 ( Holin), PSB3K3, PSB3T5.<br><br> |
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- | ===Day 2 Tuesday=== | + | ===Day 2 Tuesday (30/06/09)=== |
*Miniprep | *Miniprep | ||
- | **I732820 (LacI) R0051 (CI operator) S03518 ( TetR) K112808 ( Holin) | + | **I732820 (LacI) R0051 (CI operator) S03518 ( TetR) K112808 ( Holin) PSB3K3, PSB3T5.<br><br> |
*Planning for Restriction digest | *Planning for Restriction digest | ||
- | **R0051, S03518, R0040, | + | **R0051, S03518, R0040, PSB3K3 and I732820.<br><br> |
* Restriction Digest | * Restriction Digest | ||
- | **R0051, S03518, R0040, | + | **R0051, S03518, R0040, PSB3K3 and I732820.<br><br> |
*Gel Electrophoresis | *Gel Electrophoresis | ||
- | **R0051, S03518, R0040, | + | **R0051, S03518, R0040, PSB3K3 and I732820.<br><br> |
*Transformation (using XL1-Blue) | *Transformation (using XL1-Blue) | ||
- | **I13600 | + | **I13600 and I13601 |
*** Result : Failed (no growth)<br><br> | *** Result : Failed (no growth)<br><br> | ||
- | * | + | *Inoculation for Miniprep |
**PSB4K5, PSB3K3<br><br> | **PSB4K5, PSB3K3<br><br> | ||
- | ===Day 3 Wednesday=== | + | ===Day 3 Wednesday (01/07/09)=== |
*Miniprep | *Miniprep | ||
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*Restriction Digest | *Restriction Digest | ||
- | ** | + | **PSB4K5, PSB3K3, S03518 and R0040<br><br> |
- | *Gel | + | *Gel Electrophoresis |
- | **PSB4K5, PSB3K3, S03518 | + | **PSB4K5, PSB3K3, S03518 and R0040<br><br> |
- | * | + | *Inoculate for Miniprep |
**PSB1AT3, PSB1AC3<br><br> | **PSB1AT3, PSB1AC3<br><br> | ||
- | ===Day 4 Thursday=== | + | ===Day 4 Thursday (02/07/09)=== |
- | *Transformation (using | + | *Transformation (using XL1-Blue) |
**I13600 and I13601 | **I13600 and I13601 | ||
*** Result : Worked<br><br> | *** Result : Worked<br><br> | ||
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**R0040<br><br> | **R0040<br><br> | ||
- | * | + | *Inoculate for Miniprep |
- | **PSB3AT3 | + | **PSB3AT3 and PSB3AC3<br><br> |
- | ===Day 5 Friday=== | + | ===Day 5 Friday (03/07/09)=== |
*Miniprep | *Miniprep | ||
- | **PSB3AT3 | + | **PSB3AT3 and PSB3AC3<br><br> |
*End of week meeting<br><br> | *End of week meeting<br><br> | ||
- | ==Week 5 : Construction of K182001 ( | + | ==Week 5 : Construction of K182001 (06/07/09 - 10/07/09)== |
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*Gel Electrophoresis | *Gel Electrophoresis | ||
- | **I732820 (LacI), R0040 (Tet O), PSB1AC3 | + | **I732820 (LacI), R0040 (Tet O), PSB1AC3 and PSB1AT3,<br><br> |
*Ligation calculation.<br><br> | *Ligation calculation.<br><br> | ||
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** I13600 and I13601<br><br> | ** I13600 and I13601<br><br> | ||
- | *Heat inactivation of week 4 Friday digestions. | + | *Heat inactivation of week 4 Friday digestions. <br><br> |
- | + | ||
*Preparation of LB medium and LB agar medium. <br><br> | *Preparation of LB medium and LB agar medium. <br><br> | ||
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===Day 2 Tuesday (07/07/09)=== | ===Day 2 Tuesday (07/07/09)=== | ||
- | *Alkaline | + | *Alkaline phosphatase treatment of PSB1AC3<br><br> |
*Plasmid Miniprep | *Plasmid Miniprep | ||
**I13600 and I13601<br><br> | **I13600 and I13601<br><br> | ||
- | *Pouring of 15 | + | *Pouring of 15 Chloramphenicol plates<br><br> |
*Ligation | *Ligation | ||
- | **PSB1AC3(Plasmid Backbone +RFP -CCDB), I732820 (LacI)( | + | **PSB1AC3(Plasmid Backbone +RFP -CCDB), I732820 (LacI)(upstream), R0040 (Tet O)(downstream)<br><br> |
- | *** The | + | *** The successful ligation would create the intermediate BioBrick K182001<br><br> |
*Transforming Ligated plasmid into XL1-Blue.<br><br> | *Transforming Ligated plasmid into XL1-Blue.<br><br> | ||
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*Gel Electrophoresis | *Gel Electrophoresis | ||
** S03518, K112808, I13600 and I13601 | ** S03518, K112808, I13600 and I13601 | ||
- | *Made | + | *Made colony counts for K182001. <br><br> |
- | *Transferred 50 colonies of K182001 for | + | *Transferred 50 colonies of K182001 for colour screening<br><br> |
===Day 4 Thursday (09/07/09)=== | ===Day 4 Thursday (09/07/09)=== | ||
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*Friday Meeting <br><br> | *Friday Meeting <br><br> | ||
- | ==Week 6 : Construction of | + | ==Week 6 : Construction of K182002 and analysis of clones (13/07/09 - 16/07/09)== |
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***Heat inactivation of Alkaline phosphatase<br><br> | ***Heat inactivation of Alkaline phosphatase<br><br> | ||
- | *Pouring of 15 | + | *Pouring of 15 Chloramphenicol plates<br><br> |
*Construction of K182002 | *Construction of K182002 | ||
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** K182001 (16 colonies) | ** K182001 (16 colonies) | ||
**Gel Electrophoresis | **Gel Electrophoresis | ||
- | *** Result : Failed (no bands on gel) (possibly due to problem with tubes used | + | *** Result : Failed (no bands on gel) (possibly due to problem with tubes used) |
* Requested Parts from the registry | * Requested Parts from the registry | ||
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**K182001 (16 colonies) | **K182001 (16 colonies) | ||
**Gel Electrophoresis of PCR | **Gel Electrophoresis of PCR | ||
- | *** Result : | + | *** Result : Successful.<br><br> |
*Cell counts of K182002<br><br> | *Cell counts of K182002<br><br> | ||
- | *Transferred 50 colonies of K182002 for | + | *Transferred 50 colonies of K182002 for colour screening<br><br> |
Line 301: | Line 301: | ||
**K182002 (16 colonies)<br><br> | **K182002 (16 colonies)<br><br> | ||
- | *Took pictures of I13600, I13601 under | + | *Took pictures of I13600, I13601 under fluorescent microscope |
- | **Expected Results : I13600 Positive, I13601 Negative (due to LacI repression in XL1-blue | + | **Expected Results : I13600 Positive, I13601 Negative (due to LacI repression in XL1-blue) |
- | ** Results : I13600 Positive (unambiguous fluorescence observed), I13601 Negative (no | + | ** Results : I13600 Positive (unambiguous fluorescence observed), I13601 Negative (no fluorescence observed) <br><br> |
- | * | + | *Inoculation for Miniprep |
**K182001<br><br> | **K182001<br><br> | ||
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** K182001 (Colonies 6,7,9,10) and K182002 (Colonies 7,9,10,11)<br><br> | ** K182001 (Colonies 6,7,9,10) and K182002 (Colonies 7,9,10,11)<br><br> | ||
- | *Restriction Digest for 1ml Lambda | + | *Restriction Digest for 1ml Lambda ladder<br><br> |
*1600ml (4x400ml) of Lb+Agar and 400ml of LB<br><br> | *1600ml (4x400ml) of Lb+Agar and 400ml of LB<br><br> | ||
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* Friday Meeting <br><br> | * Friday Meeting <br><br> | ||
- | * Handover | + | * Handover from Ian to Russell and Claudia<br><br> |
- | * Maths tutorial for better understanding of | + | * Maths tutorial for better understanding of Modelling methods.<br><br> |
- | ==Week 7 (20/07/09 - 24/07/09)== | + | ==Week 7 : Sequencing and Preperation for K182002 assay (20/07/09 - 24/07/09)== |
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*Fluorescent Microscope | *Fluorescent Microscope | ||
**I13601 + IPTG | **I13601 + IPTG | ||
- | *** Result : Positive ( | + | *** Result : Positive ( fluorescence was induced as expected.)<br><br> |
* Restriction Digest | * Restriction Digest | ||
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** K182001 (Colonies 7,9) K182002 (Colonies 9,10,11)<br><br> | ** K182001 (Colonies 7,9) K182002 (Colonies 9,10,11)<br><br> | ||
- | * | + | *Inoculation for Miniprep |
** K182002 colonies 9,10,11 <br><br> | ** K182002 colonies 9,10,11 <br><br> | ||
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*Miniprep | *Miniprep | ||
** K182002 colonies 9,10,11<br><br> | ** K182002 colonies 9,10,11<br><br> | ||
- | *Sent | + | *Sent samples for sequencing |
**K182001 (Colonies 7,9) K182002 (Colonies 9,10,11)<br><br> | **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> | **2 samples of I13601 (Colony 1) <br><br> | ||
*Meeting with Sam Miller | *Meeting with Sam Miller | ||
- | ** discuss possible Motility | + | ** discuss possible Motility Assays <br><br> |
===Day 3 Wednesday (22/07/09)=== | ===Day 3 Wednesday (22/07/09)=== | ||
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*Calculations for K182003 ligation<br><br> | *Calculations for K182003 ligation<br><br> | ||
*Restriction Digest for construction of K182003 from K182002 and K182001 | *Restriction Digest for construction of K182003 from K182002 and K182001 | ||
- | ** K182001 (downstream) | + | ** K182001 (downstream)and K182002 (upstream)<br><br> |
*Gel Electrophoresis | *Gel Electrophoresis | ||
**K182001, K182002.<br><br> | **K182001, K182002.<br><br> | ||
- | *Heat inactivation of enzymes of K182001 | + | *Heat inactivation of enzymes of K182001 and K182002.<br><br> |
- | * | + | *Inoculation for Miniprep |
- | ** | + | **pSB3K5<br><br> |
===Day 5 Friday (24/07/09)=== | ===Day 5 Friday (24/07/09)=== | ||
- | *Miniprep of | + | *Miniprep of Psb3K5 |
**Restriction Digest of Psb3K5 | **Restriction Digest of Psb3K5 | ||
- | ***Gel Electrophoresis of | + | ***Gel Electrophoresis of Psb3K5<br><br> |
*Friday Meeting<br><br> | *Friday Meeting<br><br> | ||
- | ==Week 8 (27/07/09 - 31/07/09)== | + | ==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)== | ==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)== | ==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)== | ==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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<tr> | <tr> | ||
<td> | <td> | ||
- | <a href="https://2009.igem.org/Team:Aberdeen_Scotland/notebook/andgate"><img src="https://static.igem.org/mediawiki/2009/e/ed/Aberdeen_Left_arrow.png"> AND Gate</a> | + | <a href="https://2009.igem.org/Team:Aberdeen_Scotland/notebook/andgate"><img src="https://static.igem.org/mediawiki/2009/e/ed/Aberdeen_Left_arrow.png"> Back to Notebook, AND Gate Section</a> |
</td> | </td> | ||
<td align="right"> | <td align="right"> | ||
- | <a href="https://2009.igem.org/Team:Aberdeen_Scotland/notebook/quorumsensing">Quorum Sensing <img src="https://static.igem.org/mediawiki/2009/4/4c/Aberdeen_Right_arrow.png"></a> | + | <a href="https://2009.igem.org/Team:Aberdeen_Scotland/notebook/quorumsensing">Continue to Notebook, Quorum Sensing Section <img src="https://static.igem.org/mediawiki/2009/4/4c/Aberdeen_Right_arrow.png"></a> |
</td> | </td> | ||
</tr> | </tr> |
Latest revision as of 16:51, 28 August 2009
University of Aberdeen - Pico Plumber
iGEM 2009
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.
- I732820 (LacI,) R0051 (CI operator), S03518 ( TetR), K112808 ( Holin), PSB3K3, PSB3T5.
- Rescue I13600 and I13601
- Required for testing of constructs
- Required for testing of constructs
Day 2 Tuesday (30/06/09)
- Miniprep
- I732820 (LacI) R0051 (CI operator) S03518 ( TetR) K112808 ( Holin) PSB3K3, PSB3T5.
- I732820 (LacI) R0051 (CI operator) S03518 ( TetR) K112808 ( Holin) PSB3K3, PSB3T5.
- Planning for Restriction digest
- R0051, S03518, R0040, PSB3K3 and I732820.
- R0051, S03518, R0040, PSB3K3 and I732820.
- Restriction Digest
- R0051, S03518, R0040, PSB3K3 and I732820.
- R0051, S03518, R0040, PSB3K3 and I732820.
- Gel Electrophoresis
- R0051, S03518, R0040, PSB3K3 and I732820.
- R0051, S03518, R0040, PSB3K3 and I732820.
- Transformation (using XL1-Blue)
- I13600 and I13601
- Result : Failed (no growth)
- Result : Failed (no growth)
- I13600 and I13601
- Inoculation for Miniprep
- PSB4K5, PSB3K3
- PSB4K5, PSB3K3
Day 3 Wednesday (01/07/09)
- Miniprep
- PSB4K5, PSB3K3
- PSB4K5, PSB3K3
- Restriction Digest
- PSB4K5, PSB3K3, S03518 and R0040
- PSB4K5, PSB3K3, S03518 and R0040
- Gel Electrophoresis
- PSB4K5, PSB3K3, S03518 and R0040
- PSB4K5, PSB3K3, S03518 and R0040
- Inoculate for Miniprep
- PSB1AT3, PSB1AC3
- PSB1AT3, PSB1AC3
Day 4 Thursday (02/07/09)
- Transformation (using XL1-Blue)
- I13600 and I13601
- Result : Worked
- Result : Worked
- I13600 and I13601
- Gel Electrophoresis
- R0040
- R0040
- Inoculate for Miniprep
- PSB3AT3 and PSB3AC3
- PSB3AT3 and PSB3AC3
Day 5 Friday (03/07/09)
- Miniprep
- PSB3AT3 and PSB3AC3
- 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,
- I732820 (LacI), R0040 (Tet O), PSB1AC3 and PSB1AT3,
- Ligation calculation.
- Inoculation for Miniprep
- I13600 and I13601
- 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
- BBa_K182001 and BBa_K182002
Day 2 Tuesday (07/07/09)
- Alkaline phosphatase treatment of PSB1AC3
- Plasmid Miniprep
- I13600 and I13601
- 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
- The successful ligation would create the intermediate BioBrick K182001
- PSB1AC3(Plasmid Backbone +RFP -CCDB), I732820 (LacI)(upstream), R0040 (Tet O)(downstream)
- Transforming Ligated plasmid into XL1-Blue.
Day 3 Wednesday(08/07/09)
- Restriction digest
- S03518, K112808, I13600 and I13601.
- 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
- 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
- Digestions of S03518 , R0051
- Alkaline phosphatase treatment
- 20ul pSB1AC
- Heat inactivation of Alkaline phosphatase
- Heat inactivation of Alkaline phosphatase
- 20ul pSB1AC
- Pouring of 15 Chloramphenicol plates
- Construction of K182002
- Ligation
- pSB1AC3 (plasmid Backbone) + S03518 (downstream) , R0051 (upstream)
- Transformation into XL1-Blue.
- Ligation
- 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.
- 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.
- 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)
- 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
- 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)
- K182001 (Colonies 6,7 and 9)
- Gel electrophoresis
- PCR from K182002
- PCR from K182002
- Restriction Digest
- K182001 (Colonies 6,7,9,10) and K182002 (Colonies 7,9,10,11)
- 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)
- 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
- K182002 Colonies 9,10,11
- Fluorescent Microscope
- I13601 + IPTG
- Result : Positive ( fluorescence was induced as expected.)
- Result : Positive ( fluorescence was induced as expected.)
- I13601 + IPTG
- Restriction Digest
- K182001 (Colonies 7,9) K182002 (Colonies 9,10,11)
- K182001 (Colonies 7,9) K182002 (Colonies 9,10,11)
- Gel Electrophoresis
- K182001 (Colonies 7,9) K182002 (Colonies 9,10,11)
- K182001 (Colonies 7,9) K182002 (Colonies 9,10,11)
- Inoculation for Miniprep
- K182002 colonies 9,10,11
- K182002 colonies 9,10,11
Day 2 Tuesday (21/07/09)
- Miniprep
- K182002 colonies 9,10,11
- K182002 colonies 9,10,11
- Sent samples for sequencing
- K182001 (Colonies 7,9) K182002 (Colonies 9,10,11)
- K182001 (Colonies 7,9) K182002 (Colonies 9,10,11)
- Inoculate for making competent using TSS method
- 2 samples of I13601 (Colony 1)
- 2 samples of I13601 (Colony 1)
- Meeting with Sam Miller
- discuss possible Motility Assays
- discuss possible Motility Assays
Day 3 Wednesday (22/07/09)
- TSS method for making cells competent
- I13601
- 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.
- K182002 into I13601 TSS cells.
- Calculations for K182003 ligation
- Restriction Digest for construction of K182003 from K182002 and K182001
- K182001 (downstream)and K182002 (upstream)
- K182001 (downstream)and K182002 (upstream)
- Gel Electrophoresis
- K182001, K182002.
- K182001, K182002.
- Heat inactivation of enzymes of K182001 and K182002.
- Inoculation for Miniprep
- pSB3K5
- pSB3K5
Day 5 Friday (24/07/09)
- Miniprep of Psb3K5
- Restriction Digest of Psb3K5
- Gel Electrophoresis of Psb3K5
- Gel Electrophoresis of Psb3K5
- Restriction Digest 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
- Analyse Sequencing
- Pour Plates
- Discuss Model with Prof. McGlynn
- Inoculation for miniprep
- K182002 (colony 4,5)
- K182002 (colony 4,5)
Day 2 Tuesday (28/07/09)
- Ligation Maths
- Phosphatase Treatment
- Psb1AT3
- 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 4,5 and 9)
- Colony PCR
- K182002 (18 colonies)
- 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
- 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)
- I13600/K182002 (Colony 10)
- Colony PCR
- K182002 + K182003 (Colony 10)
- 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
- K182003 and K182002
- Inoculation for Miniprep
- K182003 and K182002
- K182003 and K182002
Day 2 Tuesday (04/08/09)
- Miniprep
- K182002 and K182003
- K182002 and K182003
- Restriction digestion
- K182002 and K182003
- 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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