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Revision as of 13:58, 15 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 solution 0.005 g/mL, glucose oxidase solution for 0.0025 g/mL, horseradish peroxidase (HRP) solution 250 μL/mL, 2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid (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). PBS was used as a solvent.
    • Final reagent concentration, (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. PBS was used as a solvent.
    • 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

  • Important dates:
    18th July - Resurrected DNA from iGEM DNA distribution kit
  • 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

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



    Result

    Increasing the concentration of master mix reagent did not yield intense colour changes (figure 1)





    figure 1
    Figure 1: Colour change observed following absorbance of oxidised ABTS. Master mix containing: glucose oxidase, HRP and ABTS was added to 6 different concentrations of glucose.


  • Based on results from yesterday, further increases in in reagents concentration that make up the master mix were tested. We tested the same glucose concentration as on 22/07/2016. The same parameters for the absorbance reading were used (i.e. every 2 sec for 5 min). The only parameter that was changed this time was master mix reagent concentration (table 4)
    • Final reagent concentration (μg/ml)
    Glucose oxidase 625
    HRP 62.5
    ABTS 625
    Table 4. Master mix


    Result

    Strongly visible green colour was not observed following today’s experiment





  • Today it was decided to increase glucose concentration (table 5) following previous experimental conditions that did not result in appearance of intense green colour. Concentration of master mix reagents and absorbance time window was kept the same as on 22/07/2016. Samples were run in triplicates
    • Final glucose concentration (μg/ml)
    0.50
    1.00
    .25
    1.50
    1.75
    2.00
    Table 5. Glucose Concentration

    Result

    At the end of our experiment we noticed very intense colour change as shown in figure 2.





    figure 2
    Figure 2: Colour intensity following addition of master mix (ABTS, HRP and H2O2)


Inducible Gene Switch

  • Ordered genes from IDT: alcA2 and alcR
  • Inoculated CP1 and CP3

Pilot Experiment

  • Performed the following reaction where ABTS is oxidised in the presence of H2O2 (final concentration 250 μg/ml ) and HRP (final concentration 250 μg/ml
    • Concentration of ABTS (μg/ml)
    1
    5
    10
    15
    20
  • Just measured absorbance of water at 420 nm
    • Concentration (μg/ml)
    HRP 0.25
    HRP 2.50
    H2O2 5.08
    Measured absorbance of each of the reagents at 420 nm


  • Set up the reaction with all 4 reagents. Measured absorbance every 25 sec for 900 sec. Run samples in triplicates
    •   ABTS (μl) H2O2 (μl) HRP (μl) PBS (μl)
    Sample 1 10 5 5 180
    Sample 2 15 7.5 5 172.5
    Sample 3 20 10 5 165
    Sample 4 25 12.5 5 157.5
    Sample 5 30 15 5 150
    Sample 6; 35 17.5 5 142.5
    Volume of reagents to add per well

    Stocks
    H2O2 0.05g/ml
    ABTS 0.0025g/ml
    HRP 0.00025g/ml
    Concentration of stocks used

Inducible Gene Switch

  • Important dates
    2nd August- Received requested parts from iGEM HQ
    5th August- verified all parts from the DNA distribution kit
  • Restriction enzyme digest for ligation of CP2 into J61002 plasmid(backbone for CP1 and CP3) for rfp quantification as CP2 was originally in pSB1A2 vector which does not contain rfp.
  • Gel extraction of CP2 (insert) and (vector)

    Expected band size of
    CP2 = 2056 bp and 58 bp
    CP1 = 2925 bp and 58 bp
    * CP2- extract smaller fragment
    * CP1- extract larger fragment
    figure 3



    Ran samples on 1% agarose gel with 2-log ladder. Restriction digest was successful for both CP2 and CP1. However, only CP1 was successfully extracted from gel as CP2 was lost from the running gel due to small fragment size.
    figure 4

    figure 5
    Concentration of CP1 after gel extraction


  • Q5 polymerase PCR for CP2 using protocol
    Forward primer = V2 forward from kit
    Reverse primer = VR reverse from kit
    Tm = 70C
    figure 6
  • Received parts from iGEM HQ in the form of bacteria on agar. All came in pSB1C3 + CmR resistance. Re-streaked them onto CmR plates, incubate O/N at 37C to get single colonies next day.
    • figure 7


  • Overnight ligation of CP2 (insert) + CP1 (vector) with T4 ligase using protocol
    • figure 8


  • Transformation of ligated products in DH5α chemical competent cells
  • Colony PCR for CP1 and CP2 transformants
    • Expected colony PCR fragment size = 1142bp
    • Ran colony PCR products on 1% agarose gel to check if ligation was successful. No bands were seen indicating the ligation failed.
    • figure 9


  • Restriction digest to confirm all vectors plasmids that we need from BioBrick kit in 10 ɥl reaction (1ɥl DNA).
    • figure 10


  • Ran digested products on 1% agarose gel to check with a 2-log ladder
    • figure 11

  • Ran CP2 PCR product on 4% agarose gel with 2-log DNA ladder and low MW ladder to check if amplified region was correct
    • figure 12
    Results: CP2 PCR was successful but ran out of sample stock.

Pilot Experiment

Inducible Gene Switch

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