Team:Manchester/Notebook

Manchester iGEM 2016

Notebook

  • * Inducible Gene Switch - All activities have been performed by Sathya, Hui Wen and Prakrithi.
  • * Cell Free Mechanism - All activities have been performed by Guada, Mala and Svetlana.
  • * Pilot Experiment - All activities have been performed by Guada, Mala and Svetlana.

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 (GOx) solution 0.0025 g/mL, horseradish peroxidase (HRP) solution 250 μL/mL, 2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS) 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 Labtech FLUOstar Omega plate reader.

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: GOx, HRP and ABTS (table 1). PBS was used as a solvent.
    • Final reagent concentration, (μg/ml)
    GOx 60
    HRP 60
    ABTS 100
    Table 1.Master mix concentrations.



  • 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 concentrations.



  • 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 μl of master mix was added into each well.
  • Results
    1. The rate of reaction did not level off and poor colour intensity was observed.
  • 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.
  • Results
    1. 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 (i.e. absorbance measured every 2 sec for 5 min). However this time we doubled the concentration of each reagent that made up the master mix (table 3).
    • Final reagent concentration, (μg/ml)
    GOx 120
    HRP 120
    ABTS 200
    Table 3. Master mix



  • Result
    1. Increasing the concentration of master mix reagents did not yield intense colour changes






  • 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)
    GOx 625
    HRP 62.5
    ABTS 625
    Table 4. Master mix


  • Result
    1. 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 (i.e. absorbance measured every 2 sec for 5 min).
    • Final glucose concentration, (μg/ml)
    0.50
    1.00
    .25
    1.50
    1.75
    2.00
    Table 5

  • Result
    1. At the end of our experiment we noticed very intense colour change as shown in figure 1.




    • figure 2
    figure 2.1
    Figure 1: 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) (table 6).
    • Final ABTS concentration, (μg/ml)
    1
    5
    10
    15
    20
    Table 6
  • Measured absorbance of water at 420 nm
  • Measured absorbance of each of the reagents at 420 nm at the following concentrations (table 7).
    • Concentration, (μg/ml)
    HRP 0.25
    HRP 2.50
    H2O2 5.08
    Table 7


  • Set up the reaction with 3 reagents: ABTS, H2O2, HRP. Measured absorbance every 25 sec for 900 sec (table 8)
    • ABTS concentration (μg/ml) H2O2 concentration (μg/ml) HRP concentration (μg/ml)
    1.25 1.25 0.0625
    1.875 1.875 0.0625
    2.5 2.5 0.0625
    3.125 3.125 0.0625
    3.750 3.750 0.0625
    4.375 4.375 0.0625
    5.000 5.000 0.0625
    5.625 5.625 0.0625
    6.25 6.25 0.0625
    6.875 6.875 0.0625
    7.5 7.5 0.0625
    8.125 8.125 0.0625
    8.750 8.750 0.0625
    9.375 9.375 0.0625
    10 10 0.0625
    10.625 10.625 0.0625
    11.25 11.25 0.0625
    11.875 11.875 0.0625
    12.5 12.5 0.0625
    13.125 13.125 0.0625
    Table 8.


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.

Cell-Free Mechanism

  • Cloned Pet28b Vector from Nick Weise.
  • The 20 μL plate (pET28b) showed single colonies.
  • Control Plate (Puc19) there was no growth.
  • Grew overnight cultures and then mini-prepped the Pet28b Vector.
  • Results of Mini-Prep:
    Sample Concentration (ng/μl) 280/280 260/230
    pet28b-sample1 90.6 1.89 1.74
    pet28b-sample2 95.3 1.79 1.41
  • Set up Restriction enzyme digests for validation with NdeI and SalI.
  • Both samples of Pet28b were not cut by restriction digest.
  • Performed more mini-preps.
  • We set up a double digest using EcoRI and ClaI.
  • Results:
    figure 1
    Expected-
    EcoRI - 5368bp
    ClaI - 5368 bp
    1. Double digest (EcoRI + ClaI) – 3924bp and 1444, suggests that double digest worked at Pet28b is characterised.

Inducible Gene Switch

Cell-Free Mechanism

  • pucIDT_AOx arrived!
  • Transformed the IDT_AOx Vector into DH5α Strain.
  • Performed mini-prep of IDT_AOx Vector.
  • Results:
    Samples Sample 1 AOx Sample 2 AOx Sample 3 AOx Sample 4 AOx
    Concentration (ng/μl) 497.1 750.9 857.5 671.5
    260/280 1.85 1.86 1.87 1.87
    260/230 2.22 2.27 2.33 2.30
  • Performed restriction digest of pucIDT_AOX with NdeI and SalI.
  • Results:
    figure 2
  • Didn’t work as 3 bands are shown for the double digest of NdeI and SalI, suggesting star activity.
  • Ordered new SalI and discussed restriction digest complications with Supervisors.
  • Inoculated more Pet28b into LB Broth.
  • Mini-prepped Pet28b Vector.
  • Results:
    Samples Sample 1 pet28b Sample 2 pet28b Sample 3 pet28b Sample 4 pet28b
    Concentration (ng/μl) 132.4 131.5 91.0 76.9
    260/280 1.96 1.95 1.90 1.81
    260/230 0.18 1.77 2.07 1.37

Inducible Gene Switch

Cell-Free Mechanism

  • Characterised pucIDT_AOX with double digest using NdeI and SalI (using 2uL in the digest reaction mixture). We also validated this with single digests of EcoRI and PstI.
  • Results:
    figure 3
    Digestion with SalI+ NdeI gave 2 bands. In this gel you can only see 1 band of 2781 bp size because the other band (2015 bp) was cut. This picture was taken after cutting the gel to minimise exposure of UV light. Negative control in NEB 3.1 Buffer worked. Single digests with EcoRI and PstI also worked- band of 4796bp. Double digest with EcoRI+PstI also worked. Negative control in NEB 2.1 buffer also worked.


  • Performed Gel Extraction and cut band of 2015bp of pucIDT_AOX.
  • Performed double digest of Pet28b using NdeI and SalI:
    figure 4
    This photo shows a band at around 5kp and linearised Pet28b should be 5310.


  • Performed a PCR purification of Pet28b.
  • Nano-drop results of cut AOX and PCR purified Pet28b:
    AOx (2015 bp) Pet28b (5310 bp)
    Concentration (ng/μl) 11.1 9.9
    260/280 1.66 1.93
    260/230 0.16 1.78
  • Performed ligation of digested and purified AOX and Pet28b.
  • Transformed ligated Pet28b_AOX using Puc19 as a negative control for each ratio that was plated.
  • Created O/N cultures of 4 colonies.
  • Prepared glycerol stocks of Pet28b_AOX and performed mini-prep.
  • Performed restriction digest (with NdeI and SalI) to screen recombinant clones:
    figure 5
  • Out of the 4 samples we chose to mini-prep and run a digest on, none worked. They all show bands between 5-6kb this suggests the insert didn’t ligate with the Pet28b.

Inducible Gene Switch

Cell-Free Mechanism

  • We discussed last week’s apparent failure of the ligation and he suggested we colony PCR many samples and then digest these to check if the ligation worked.
  • We performed a colony PCR of many colonies from a variety ratios we had plated
  • Results:
    figure 6
  • Picture on the left shows expected amplified PCR AOX band (2319bp). Right picture shows colony PCR of samples 1 to 15.
  • Sample 3 in lane 4 shows a band of around 2300bp. This suggests AOX is present in the sample and thus possibly ligated. The other bands of 350 bp indicates a vector that has re-ligated to itself.
  • Made O/N of sample 3
  • Mini-prepped sample 3 (Pet28b_aox)
  • Pet28b_aox was digested with XbaI and BamHI and this didn’t work.

Inducible Gene Switch

Cell-Free Mechanism

  • More colonies were screened through colony PCR
  • Results:
    figure 7
  • This gel shows sample 7 in lane 9 and sample 15 in lane 17 have a band of around 2kb and IDT_AOX should be 2044.
  • These samples were taken to make O/N cultures
  • Samples 7 and 15 (of Pet28b_aox) were mini-prepped
  • Double digests (using NdeI and SalI) were performed on the two samples.
  • Result from Sample 7:
    figure 8

    In lane 4 there is band a 5000kb (indicating Pet28b) and a band at 2000kb (indicating AOx). This means that the ligation for sample 7 has worked. Lanes 3 and 5 are negative controls. Lane 2 is the same sample where the 5000kb band (indicating Pet28b) can be seen, however the 2000kb is too faint.



  • Result from Sample 15:
    figure 8

    In lanes 2 and 3 bands of 5000bp (indicating Pet28b) and 2000bp (indicating AOx) can be seen. This suggests that the ligation of Pet28b and AOx was successful.



  • Transformed, sample 7 and 15 into DH5α strain and left over the weekend on the bench. Puc19 was used as a control.

Inducible Gene Switch

Cell-Free Mechanism

  • Sent sample 15 for sequencing
  • Growth on plates was seen for both sample 7 and 15. No growth on control plate indicating transformation worked.
  • O/N cultures were made of above samples.
  • Transformed sample 7 and 15 (of Pet28b_aox) into DH5α Strain and BL21 strain.
  • Growth on plates from both of the strains. puC19 control worked too.
  • The BL21 cells were then IPTG induced. Pet28b (-ve control) and pBbESK_RFP (+ve control) were also induced.
  • Simultaneous to the above work, we started work on our submission vector for iGEM.
  • A double digest of PUCIDT_AOx, using EcoRI and PstI was performed.
  • Results:
    figure

    There is a band at 3000bp and a missing band at 2000bp. This is because the AOx was cut out and then the photo was taken.



  • A gel extraction of this AOX was done.
  • Ligation, using Roche Kit, of linearized (and supplied) pSB1C3 and gel extracted AOx was performed.
  • The ligation of pSB1C3 with AOX (pSB1C3_AOx) was transformed and left overnight.
  • A colony PCR of four random samples was then performed.
  • Results:
    figure

    Lanes 2 and 5 both show a band at 2000kb suggesting it is the AOx from pSB1C3. This indicates the ligation worked.



  • Performed restriction double digest to characterise the pSB1C3_AOX
  • Results:
    figure


  • Stored pellets from IPTG induced cells in -20 degrees freezer

Inducible Gene Switch

Cell-Free Mechanism

  • Prepared IPTG induced BL21strain with Pet28b_aox for SDS Page.
  • Sent the pSB1C3_aox for sequencing.
  • Performed SDS-PAGE of IPTG and non-IPTG induced cells.
  • Results:
    figure
  • Performed Western Blot of SDS-Page results.
  • Results:
    figure 12

    This shows the protein Alcohol dehydrogenase as well as truncated protein. However, this is in the insoluble fraction

Inducible Gene Switch

Cell-Free Mechanism

  • Repeated induction of cells with IPTG, SDS-AGE and western Blot from last week, to see if the protein will fall in to the soluble fraction.
  • Western Blot still shows correct protein in the insoluble fraction
  • Started to help the inducible gene switch mechanism

Inducible Gene Switch

Cell-Free Mechanism

  • Typed up lab work for this mechanism
  • Prepared PSB1C3_aox to be sent off to iGEM HQ
  • Helped the inducible gene switch experimental work

Inducible Gene Switch

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