Team:University College London/LabBook/Week4

From 2012.igem.org

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'''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.   
'''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.   
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Step 1 – Thawing Cells: Use W3100 cell line created in Week 2 (Expt 2.1)
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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'''
'''Method'''
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Step 1 – Thawing Cells: Use W3100 cell line created in Week 2 (Expt 2.1)
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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.
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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.
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{| class="wikitable"
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|-
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! colspan="2" |  Samples !! Volume Inoculated !! Antibiotic in Gel (ug/ml)
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|-
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| rowspan="6" |BioBrick ||rowspan="2" | BBa_I13522  || 10ul || rowspan="4" | Ampicillin(50ug/ml)
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|-
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| 90ul
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|-
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| rowspan="2" | BBa_B0034 || 10ul
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|-
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| 90ul
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|-
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| rowspan="2" | BBa_K540000  || 10ul || rowspan="2" | Chloramphenicol (25ug/ml)
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|-
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| 90ul
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|-
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| rowspan="2" | Control || Positive (No BioBrick)|| 36ul || No Antibiotic
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|-
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| Negative (No BioBrick)|| 36ul ||  2x Ampicillin(50ug/ml)
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1x Chloramphenicol (25ug/ml)
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|-
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|}
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== Thursday (5.7.12) ==
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'''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.
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Results: The table below indicates whether or not there was growth on each plate.
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Revision as of 16:09, 1 August 2012

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 (LOGO) Transformation Protocol 1

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.


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