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Team:University College London/LabBook/Week4
From 2012.igem.org
Wednesday (4.7.12)
Aim - Transformation of Key Aggregation Module BioBricks: Expt 4.1 will be transforming the Curli Cluster, Green Fluorescence Protein and Ribosome Binding Site BioBricks into our W3100 competent cell line. Transformed cells will be cultured under the selective pressure of antibiotic, after which the plasmids are purified and the presence of the correct BioBricks will be diagnosed with an Analytical Restriction Enzyme Digest and Gel Electrophoresis.
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.
Method
Step 1 - Addition of BioBrick: To the still frozen competent cells, add 1 - 5 µL of the resuspended DNA to the 2ml tube.
Step 4 - Incubation: Close tube and incubate the cells on ice for 45 minutes.
Step 5 - Heat Shock: Heat shock the cells by immersion in a pre-heated water bath at 37ºC for 10 minutes.
Step 6 - Incubation: Incubate the cells on ice for 2 minutes.
Step 7 - Add media: Add 1.5ml of Lysogeny Broth and transfer to a falcon tube.
Step 8 - Incubation: Incubate the cells at 37ºC for 1 hour at RPM 550.
Step 9 - Transfer: transfer the solution back into a 1.5ml Eppendorf and centrifuge
RPM: 14000
Time: 2 minutes
Temperature (18-25oC)
Step 10 - Resuspend:Remove supernatant and resuspend in 100ul LB
Step 11 - Plating: Spread the resuspended cell solution onto a selective nutrient agar plate. Place the plates in a 37°C static incubator, leave overnight (alternatively a 30°C static incubator over the weekend)
| Function | Module | ||
|---|---|---|---|
| BioBrick | BBa_I13522 | Green Fluorescence Protein(GFP) | Aggregation |
| BBa_B0034 | Ribosome Binding Site (RBS) | All | |
| BBa_K540000 | (rcn-csg BAEFG curli cluster) | Aggregation | |
| Control | No BioBrick | ||
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_I13522 | 10ul | Ampicillin(50ug/ml) |
| 90ul | |||
| BBa_B0034 | 10ul | ||
| 90ul | |||
| BBa_K540000 | 10ul | Chloramphenicol (25ug/ml) | |
| 90ul | |||
| Control | Positive (No BioBrick) | 36ul | No Antibiotic |
| Negative (No BioBrick) | 36ul | 2x Ampicillin(50ug/ml)
1x Chloramphenicol (25ug/ml) | |
Thursday (5.7.12)
Aim 1 - Check results of Transformation. We expect to see growth on plates with transformed BioBricks, as well as the positive control. The negative control should have no growth.
Results: The table below indicates whether or not there was growth on each plate.
| Samples | Volume Inoculated | Colony Formation | |
|---|---|---|---|
| BioBrick | BBa_I13522 | 10ul | Yes |
| 90ul | Yes | ||
| BBa_B0034 | 10ul | No | |
| 90ul | No | ||
| BBa_K54000 | 10ul | Yes | |
| 90ul | Yes | ||
| Control | Positive (No BioBrick) | 36ul | Yes |
| Negative (No BioBrick) | 36ul | Ampicillin– One blank, one contaminated
Chloramphenicol - Blank | |
Conclusion: Transformation has been successful for K540000 and I13522, but B0034 failed. Colonies for K540000 and I13522 will be picked and inoculated into LB media for overnight culture. B0034 transformation will be attempted again.
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 required for each BioBrick.
| Samples | Broth | Antibiotic | |
|---|---|---|---|
| Biobrick | BBa_I13522 | Lysogeny Broth (5ml) | Ampicillin(50ug/ml) |
| BBa_K54000 | |||
Friday (6.7.12)
Aim – Check Results from Colony Picking:
Results: The table below indicates there was growth for each BioBrick.
| Samples | Colony Formation | |
|---|---|---|
| Biobrick | BBa_I13522 | Yes |
| BBa_K54000 | Yes | |
Conclusion: BBa_K540000 and BBa_I13522 are both ready for miniprep and restriction enzyme digest.
Method
Step 2 - Resuspend Cells: Resuspend pelleted bacterial cells in 250ul Buffer P1 and transfer to a microcentrifuge tube
Step 3 - Puncturing Cell Membrane: Add 250ul Buffer P2 and mix thoroughly by inverting the tube 4-6 times until the solution becomes clear. Do not allow the lysis reaction to proceed for more than 5 min.
Step 4 - Neutralising buffer P2: Add 350ul Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times.
Step 5 - Centrifuge:
RPM: 13000
Time:10 minutes
Temperature: 18oC
Step 6 - Centrifuge: Apply the supernatant from step 5 to the QIAprep spin column by decanting or pipetting. Centrifuge for 30-60s and discard the flow-through.
Step 7 - Remove Endonucleases from Sample: Wash the QIAprep spin column by adding 500ul of Buffer PB. Centrifuge for 30-60s and discard flow-through.
Step 8 - Remove salts from sample: Wash the QIAprep spin column by adding 750ul of Buffer PE. Centrifuge for 3-60s and discard flow through.
Step 9 - Centrifuge:
RPM: 13000
Time:1 minute
Temperature: 18oC
Step 10 - Elute DNA: Place the QIAprep column in a clean 1.5ml microcentrifuge tube. To elute DNA, add 50ul Buffer EB to the centre of the spin column, let it stand for 1 min, and centrifuge for 1 min.
Tuesday 10.7.12 for Analytical Digest and Gel Electrophoresis
