Team:University College London/LabBook/Week4

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== 4.1 ==
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== Wednesday (4.7.12)==
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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 W3110 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 W3110 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.
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'''Method'''
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<html><div class="protocol protocol-Transformation">Transformation Protocol 1</div><div class="protocolContent"></html>{{:Team:University_College_London/Protocols/Transformation1}}<html></div></html>
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{| class="wikitable"
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|-
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!  colspan="2" |  !! Function !! Module
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|-
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| rowspan="3" |BioBrick|| BBa_I13522  || Green Fluorescence Protein(GFP) || Aggregation
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|-
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| BBa_B0034  || Ribosome Binding Site (RBS)|| All
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|-
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| BBa_K540000  || (rcn-csg BAEFG curli cluster) || Aggregation
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|-
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| Control || No BioBrick|| ||
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|}
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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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{| 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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{| class="wikitable"
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|-
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! colspan="2" |  Samples !! Volume Inoculated !! Colony Formation
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|-
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| rowspan="6" |BioBrick ||rowspan="2" | BBa_I13522  || 10ul || Yes
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|-
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| 90ul || Yes
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|-
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| rowspan="2" | BBa_B0034  || 10ul || No
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|-
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| 90ul || No
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|-
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| rowspan="2" | BBa_K540000  || 10ul || Yes
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|-
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| 90ul || Yes
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|-
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| rowspan="2" | Control || Positive (No BioBrick)|| 36ul || Yes
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|-
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| Negative (No BioBrick)|| 36ul || Ampicillin– One blank, one contaminated
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Chloramphenicol - Blank
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|-
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|}
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'''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.
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'''Method'''
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<html><div class="protocol protocol-ColPic">Picking Colonies Protocol</div><div class="protocolContent"></html>{{:Team:University_College_London/Protocols/ColPic}}<html></div></html>
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'''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.
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{| class="wikitable"
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|-
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! colspan="2" |  Samples  !! Broth !! Antibiotic
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|-
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|rowspan="6" |Biobrick || BBa_I13522 || rowspan="6" | Lysogeny Broth (5ml) || rowspan="6" |Ampicillin(50ug/ml)
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|-
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| BBa_K540000
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|-
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|}
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 +
== Friday (6.7.12) ==
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'''Aim – Check Results from Colony Picking:'''
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 +
'''Results:''' The table below indicates there was growth for each BioBrick.
 +
 
 +
{| class="wikitable"
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|-
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! colspan="2" |  Samples  !! Colony Formation
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|-
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|rowspan="6" |Biobrick || BBa_I13522 || Yes
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|-
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| BBa_K540000 || Yes
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|-
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|}
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'''Conclusion:''' BBa_K540000 and BBa_I13522 are both ready for miniprep and restriction enzyme digest.
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'''Method'''
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<html><div class="protocol protocol-Miniprep">Miniprep Protocol 1 (Qiagen)</div><div class="protocolContent"></html>{{:Team:University_College_London/Protocols/Miniprep1}}<html></div></html>
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Tuesday 10.7.12 for Analytical Digest and Gel Electrophoresis
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<html></div>
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{{:Team:University_College_London/templates/foot}}
{{:Team:University_College_London/templates/foot}}

Latest revision as of 01:04, 27 September 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 W3110 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 W3110 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_K540000 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

Picking Colonies Protocol
Step 1 – Creating culture media: In a sterile environment, set up X numbers of falcons, each with 5mls of media.


Step 2 - Inoculating Colonies into a Selective Broth:: Add Yul of antibiotic to reach desired antibiotic concentration.


(For Ampicillin this is 50ug/ml, For Kanamycin it is 25ug/ml, for Tetracycline it is 15ug/ml, and for Chloramphenicol it is 25ug/ml)


Step 4 – Selecting a Colony: Select a clear, isolated colony and using an inoculation hoop scoop up a colony onto the tip. Deposit in the falcon tube


Step 5 - Culture: Culture your falcon tubes overnight at a temperature of 37 oC. Leave for no longer than 16 hours.

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_K540000


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_K540000 Yes

Conclusion: BBa_K540000 and BBa_I13522 are both ready for miniprep and restriction enzyme digest.

Method

Miniprep Protocol 1 (Qiagen)
Step 1 - Pellet Cells: Pellet 1-5ml bacterial overnight culture by centrifugation at >8000rpm (68800 x g) for 3 min at room temperature (15-25oC)

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


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