Team:University College London/LabBook/Week4

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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

Transformation Protocol 1

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

(LOGO) Miniprep Protocol 1 – Qiagen.

Tuesday 10.7.12 for Analytical Digest and Gel Electrophoresis


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