Difference between revisions of "Team:Newcastle/Proof"

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         <h3>Electrically Induced 'Light Bulb'</h3>
 
         <h3>Electrically Induced 'Light Bulb'</h3>
  
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        <p>In order to prove that our initial aims were correct; i.e. to induce GFP production by heat shocking the E. coli, we used a plate reader to measure cell growth and fluorescence over a 24 hour period.</p>
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<p> We aimed to engineer Escherichia coli that it increases fluoresce when an electrical current is passed through the growth medium, via the use of inducible promoters that respond to the heat-stress created by an electrical current.</p>
  
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        <p> Our 96 well plate was set up as seen below in Diagram 1.</p>
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<p> We designed two parts (BBa_K1895000 and BBa_K1895006) which respond to the heat-stress in two different ways:</p>
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<li><od>BBa_K1895000 contains a HtpG promoter which is induced by a sigma-factor (-32). This sigma factor is produced by cells when under different forms of stress. This part also contains a -32 coding region which should create a positive feedback loop and therefore increase fluorescence.</li>
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<li>BBa_K1895006 contains a DnaK promoter which is induced by dnaK, a product of other stress related responses within the cell.</li>
  
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        <p><b> INSERT DIAGRAM (see google drive) </p></b>
 
  
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        <p>All BioBricks were placed in the same pSB1C3 backbone and BL21 cells. The cells were grown up in liquid culture of LB broth with chloramphenicol overnight at 37°C. The following day, the cells were diluted down to an optical density of 0.05 at 600nm using LB broth with chloramphenicol. We then pipetted 100µl of the diluted cells into the corresponding wells, Diagram 1. The cells were laid out in this manner, with a border of LB broth, to allow for any inaccuracies that may occur because the plate reader heats the plate from the outside in.  </p>
 
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        <p>The plate reader was then set at either 30°C, 37°C and 42°C and measured for growth using OD600 and fluorescence of GFP using 485nm excitation wavelength and 520nm emission wavelength. The cells were left to grow for 24 hours and measured every five minutes by the plate reader. In between measurements, the plate reader was programmed to shake to ensure the cells didn’t clump together. </p>
 
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        <p>The results from the experiment can be seen below. </p>
 
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        <h4>Results</h4>
 
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        <p>INSERT GRAPHS</p>
 
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        <p>INSERT GRAPHS</p>
 
 
       </div>
 
       </div>
  

Revision as of 16:34, 16 October 2016

Electrically Induced 'Light Bulb'

We aimed to engineer Escherichia coli that it increases fluoresce when an electrical current is passed through the growth medium, via the use of inducible promoters that respond to the heat-stress created by an electrical current.

We designed two parts (BBa_K1895000 and BBa_K1895006) which respond to the heat-stress in two different ways:

  • BBa_K1895000 contains a HtpG promoter which is induced by a sigma-factor (-32). This sigma factor is produced by cells when under different forms of stress. This part also contains a -32 coding region which should create a positive feedback loop and therefore increase fluorescence.
  • BBa_K1895006 contains a DnaK promoter which is induced by dnaK, a product of other stress related responses within the cell.

    • Arabinose Controlled 'Variable Resistor'

    Our aim was to prove that the Arabinose Controlled ‘Variable Resistor’ grew in zinc (II) chloride? When arabinose is present, therefore showing that the SmtA is being produced and binding to the zinc (II).

    For this experiment, we used a plate reader and measured cell survival at various concentrations of zinc (II) chloride?, with and without arabinose present. The plate was set out as seen in Diagram 2.

    INSERT DIAGRAM OF THE PLATE LAYOUT

    The DH10(α)? cells with the pSB1C3 cells were grown up in liquid culture of LB broth with chloramphenicol overnight at 37°C. The cells were then diluted, using LB broth with chloramphenicol, to an optical density of 0.05 at 600nm.

    The zinc chloride was created using XXX of zinc diluted in XXXX? A serial dilution was then made of the zinc chloride in each of the corresponding wells. The cells were then placed in the correct wells.

    Arabinose (XXmM) was then added to each well correspondingly.

    The results from the experiment can be seen below.

    Results

    INSERT GRAPHS

    Battery

    To make a microbial fuel cell we followed the Reading University’s protocol

    We sourced the material such as the neoprene gaskets, carbon fibre electrode material, cation-exchange membrane, J-cloth from Professor Ian Head, Dr. Ed Milner and Paniz Izadi from the School of Civil Engineering and Geosciences.

    Results

    INSERT GRAPHS