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Team:University College London/LabBook/Week5
From 2012.igem.org
Contents |
Monday (9.7.12)
Aim: Transformation of Key BioBricks
Method
(LOGO) Transformation Protocol 2
Step 1 – Thawing Cells: Use W3100 cell line created in Week 2 (Expt 2.1) Step 3 – Addition of BioBrick: To each 2ml eppendorf, add 1ul of the following BioBricks. Include an extra tube as a control, with no BioBrick added.
| Function | Module | ||
|---|---|---|---|
| BioBrick | BBa_K123003 | Oestrogen Receptor | Detection |
| BBa_K123002 | Oestrogen Response Element | Detection | |
| BBa_K398108 | Salt Tolerance Cluster | Salt Tolerance | |
| BBa_J23100 | Constitutive Promoter | ||
| BBa_J23107 | Constitutive Promoter | ||
| BBa_R0040 | TetR Repressible Promoter | Buoyancy | |
| BBa_J23106 | Constitutive Promoter | ||
| BBa_B0034 | Ribosome Binding Site (RBS) | All | |
| Control | (No BioBricks) | ||
Step 9 – Plating samples on Agar Plates: The table below indicates the chosen inoculation volume (two for each BioBrick) and the correct gel antibiotic concentration for all samples.
| Samples | Volume Inoculated | Antibiotic in Gel (ug/ml) | |
|---|---|---|---|
| BioBrick | BBa_ K123003 | 20ul | Ampicillin(50ug/ml) |
| 200ul | |||
| BBa_ K123002 | 20ul | ||
| 200ul | |||
| BBa_B0034 | 20ul | ||
| 200ul | |||
| BBa_J23100 | 20ul | ||
| 200ul | |||
| BBa_J23107 | 20ul | ||
| 200ul | |||
| BBa_R0040 | 20ul | ||
| 200ul | |||
| BBa_J23106 | 20ul | ||
| 200ul | |||
| BBa_ K398108 | 20ul | Chloramphenicol (25ug/ml) | |
| 200ul | |||
| Control | Positive (No BioBrick) | 36ul | No Antibiotic |
| Negative (No BioBrick) | 36ul | 2x Ampicillin(50ug/ml)
1x Chloramphenicol (25ug/ml) | |
Tuesday (10.7.12)
Aim - Check results from Transformation
Results: The table below indicates whether or not there was growth on each plate. Included is an image demonstrating the growth noted for BBa_K123003, BBa_K398108, and the Positive Control
| Samples | Volume Inoculated | Growth/No Growth | |
|---|---|---|---|
| BioBrick | BBa_ K123003 | 20ul | Growth |
| 200ul | Growth | ||
| BBa_ K123002 | 20ul | No Growth | |
| 200ul | No Growth | ||
| BBa_B0034 | 20ul | No Growth | |
| 200ul | No Growth | ||
| BBa_J23100 | 20ul | No Growth | |
| 200ul | No Growth | ||
| BBa_J23107 | 10ul | No Growth | |
| 2000ul | No Growth | ||
| BBa_R0040 | 20ul | No Growth | |
| 200ul | No Growth | ||
| BBa_J23106 | 20ul | No Growth | |
| 200ul | No Growth | ||
| BBa_ K398108 | 20ul | No Growth | |
| 200ul | Growth | ||
| Control | Positive (No BioBrick) | 36ul | Growth |
| Negative (No BioBrick) | 36ul | No Growth | |
Conclusion: Only BBa_K398108 and BBa_K123003 had successfully transformed. This is a serious setback, which suggests our cell competency is not as high as we first anticipated. Method
(LOGO) Picking Colonies
Step 2 - Inoculating Colonies into a Selective Broth: The table below indicates the volume of broth and the concentration of antibiotic used for the BioBrick.
4.1
5.3
5.2
