Difference between revisions of "Team:Manchester/Notebook"

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<button type="button" class="week5button">Week 5 (25<sup>th</sup> to 29<sup>th</sup> of July)</button>
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    <h1 class="subnote">Pilot Experiment</h1>
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        <li>To increase the brightness of the colour change we repeated the experiment we performed on 22/07/2016. However this time we doubled the concentration of each reagent that made up the master mix <br />(table 3).</li>
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            <th>Final reagent concentration (μg/ml)</th>
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            <th>Glucose oxidase</th>
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            <td>120</td>
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            <td>HRP</td>
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            <td>20</td>
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            <td>ABTS</td>
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            <td>200</td>
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        <b>Table 3. Master mix </b>
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    <h1 class="subnote">Inducible Gene Switch</h1>
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Revision as of 09:53, 14 October 2016

Manchester iGEM 2016

Notebook

Inducible Gene Switch

  • Re-suspended DNA (constitutive promoters) from iGEM Distribution kit and transformed them into DH5α strain.
  • Growth was found on the negative control plates so re-transformed DNA
  • Inoculated of transformed cells.

Pilot Experiment

  • Made stocks of our reagents Glucose Stock for 0.005 g/mL, Glucose Oxidase Stock for 0.0025 g/mL, HRP Stock for 250 μL/mL, ABTS for 0.0025 g/mL

Inducible Gene Switch

  • Performed miniprep for overnight cultures
  • Prepared fresh batch of DH5α chemical competent cells
  • Restriction enzyme digest of constitutive promoters and control(pUC19) for validation with EcoRI and PstI
figure 1 figure 1

Pilot Experiment

  • Still awaiting the arrival of the BMG spectrophotometer

Inducible Gene Switch

  • Prepared DH5α chemical competent cells
  • Prepared Chloramphenicol antibiotic stock
  • Restreaked alcA 1 (BBa_K678001) and spispink(BBa_K1033923 pink chromoprotein)

Pilot Experiment

  • We began by preparing master mix containing three reagents glucose oxidase, HRP and ABTS (table 1)
    • Final reagent (μg/ml)
    Glucose oxidase 60
    HRP 60
    ABTS 100
    Table 1. Master mix



  • Six different glucose concentrations were then made as depicted in table 2
    • Final glucose concentration (μg/ml)
    0.50
    1.00
    .25
    1.50
    1.75
    2.00
    Table 2. Glucose concentration



  • Protocol
    1. 50 μl of glucose solution was added into each well. Samples were run in triplicates
    2. Tube containing master mix was placed into spectrophotometer.
    3. Spectrophotometer was then used to measure absorbance of our green coloured product-oxidised ABTS at 420 nm. Absorbance values were taken every 2 sec for 3 min once 150 ul of master mix was added into each well.
  • Results
    1. The rate of reaction did not level off and poor colour intensity was observed.
    2. We repeated the same experiment we did earlier this week. However instead of measuring absorbance for 3 min absorbance was measured every 2 sec for a 5 min period.
    3. Results - Reaction rate did level off after measuring absorbance for 5 min. However, colour intensity was still very poor.

Inducible Gene Switch

  • Resuspended DNA(RBS, amilCP(blue chromoprotein) and amilGFP(yellow chromoprotein)) from iGEM Distribution kit and transformed them into DH5α strain.
  • Inoculated alcA 1 and spispink for miniprep
  • Important event:
    18th July - Resurrected DNA from iGEM DNA distribution kit

Pilot Experiment

  • To increase the brightness of the colour change we repeated the experiment we performed on 22/07/2016. However this time we doubled the concentration of each reagent that made up the master mix
    (table 3).
    • Final reagent concentration (μg/ml)
    Glucose oxidase 120
    HRP 20
    ABTS 200
    Table 3. Master mix

Inducible Gene Switch

Pilot Experiment

Inducible Gene Switch

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