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 it into DH5α strain.
  • Growth was found on the negative control plates so re-transformed DNA
  • Inoculated transformed cells containing constitutive promoters in 10 mL of LB broth with Carbenicillin 50.

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 first batch of DH5α chemical competent cells using TSS (Transformation and Storage Solution) method.
  • Restriction enzyme digest of constitutive promoters and control(pUC19) for validation with EcoRI and PstI (1 μL each).
figure 1 figure 1

Pilot Experiment

  • Still awaiting the arrival of the BMG Labtech FLUOstar Omega plate reader.

Inducible Gene Switch

  • Prepared more DH5α chemical competent cells for transformation
  • Prepared Chloramphenicol antibiotic stock for future use.
  • 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 plasmids containing required DNA(RBS, amilCP (BBa_K1033930, blue chromoprotein) and amilGFP(BBa_K1033931, yellow chromoprotein)) from iGEM Distribution kit and transformed it 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 in 10 mL of LB broth with Carbenicillin 50.

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 with EcorI-HF and SpeI-HF for ligation of CP2 into BBa_J61002 plasmid(backbone for CP1 and CP3) for rfp quantification as CP2 was originally in pSB1A2 vector.
  • Gel extraction of CP2 (insert) and CP1 (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



    Ran samples on 1% agarose gel with 2-log ladder. Restriction digest was successful only for CP1. CP1 was successfully extracted from the gel.
    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 (Cm resistance). Re-streaked them onto Cm plates, incubate overnight at 37°C 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 (1 ɥL DNA).
    • figure 10


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

  • Ran CP2 PCR product on 4% agarose gel with 2-log DNA ladder and low molecular weight (MW) ladder to check if amplified region was correct
    • figure 12
    Results:CP2 PCR was successful but ran out of sample stock. Inoculated CP2 PCR product in 10 mL LB broth with Carbenicillin 50

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

  • Important dates:
    12,th August- IDT genes arrived -- alcR and alcA2
    • figure 13


  • Restriction digest of alcA 1 and amilCP to construct alcA1 + amilCP. alcA1 was digested with EcoRI + SpeI while amilCP was digested with EcoRI and XbaI. Control, pUC19 was digested with EcoRI and XbaI Expected band sizes after digest with respective enzymes (highlighted band sizes to be gel extracted):

    Insert: alcA1 (22.4 ng/ɥL) - 866 + 2047
    Vector: amilCP (69.3 ng/ɥL) - 15 + 2742
    Control: pUC19 (48.6 ng/ɥL) - 27 + 2659
    • figure 14

    figure 15

    Restriction digest did not work



  • Inoculated CP1, CP2, CP3, alcA1, amilCP, amilGFP, spisPink in 10 mL LB broth with appropriate antibiotic
    • figure 16


  • Restriction digest for plasmid verification by using different enzymes with standard 10X Cutsmart buffer
    • figure 17

    Expected band sizes:

    figure 18

    figure 19
  • Miniprep inoculated cells and measured the concentrations
    • figure 20
  • Ethanol precipitation for CP2
    • figure 21
  • PCR purification of CP1
    • figure 22
  • Restriction enzyme digest to verify alcA1 using EcoRI and EcoRV
    • figure 23

    figure 24

    figure 25

  • Inoculated CP2_pSB1C3, CP1 and CP3 for rfp quantification
  • Miniprep inoculated samples
    • figure 26

  • RFP quantification for CP1, CP2_pSB1C3, CP3 - Results were not comparable due to: different backbones (origin of replication) and different incubation time for cultures
  • Newly synthesised genes from IDT arrived -- alcR and alcA2

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

  • Restriction enzyme digest of CP1 and chromoproteins for chromoprotein quantification. Ran on gel for gel extraction.
    • figure 27

    figure 28

    figure 29

    Highlighted band sizes were extracted.



  • Inoculated pUC19 in 10 mL LB broth with Carbenicillin 50.
  • Re-suspended genes from IDT in 100 ɥL of sterile milli-Q for 1 hour. Transformed it into DH5α for each gene using chemical transformation protocol. We plated 20 ɥL and 200 ɥL of each sample respectively onto Carbenicilin plates. pUC19 was transformed as a control.

    Results of re-suspension of IDT genes:
    • figure 29.1

    Highlighted band sizes were extracted.



  • Gel extraction of digested CP1 and amilCP, amilGFP and spisPink
    • figure 30

    figure 31

    Repeated digest as the DNA concentration was not satisfactory



  • -Inoculated CP1, amilCP, amilGFP and spisPink in 10 mL LB broth with its respective antibiotic to obtain more DNA
  • Transformation of genes from IDT worked. Picked single colonies and inoculated them 10 mL LB broth with the respective antibiotic.
  • Performed miniprep for the overnight cultures. Results:
    • figure 32

    Digested products were run on a 1% gel at 120V for 35 minutes. amilGFP and spisPink digest did not work. CP1 and amilCP worked so proceeded to perform gel extraction to obtain the necessary band.



    figure 33

    *all the cut bands were loaded into 1 tube



    figure 34


  • Redid the digest for amilGFP and spispink and control plasmid(given by Marc) using XbaI and PstI-HF. Ran on 1% gel and it worked so proceeded to gel extract. Ligation with CP1.

    Concentrations of samples used:
    amilGFP - 124.4 ng/ɥL
    spispink - 136.5 ng/ɥL
    control plasmid - 500 ng/ɥL
    figure 35

    *the three bands for each sample that was cut was evenly distributed into 2 tubes



    figure 36


  • Chemical transformation of control plasmid and pUC19 (control vector) into DH5α competent cells to get more DNA. Plated onto Carbenicillin plates
  • Inoculated alcA2, alcR and pUC19. Miniprep results:
    • figure 37


  • Made glycerol stocks of alcA2 and alcR using 400 ɥL of 50% sterile glycerol + 600 ɥL inoculated culture and stored in -80°C for future use.
  • Ligated CP1 with chromoproteins and transformed into DH5α. Transformants were plated onto Carbenicillin plates and left to grow overnight in the 37°C incubator.
  • Inoculated pTE055.term to make glycerol stocks.
  • Plasmid verification of alcR and alcA 2
    • figure 38

    figure 39


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

  • Restriction digest & gel extraction for ligationSet A = CP1/2/3 (vector, SpeI/PstI-HF) + alcR (insert, XbaI/PstI-HF/ZraI)

    Expected band size (bp):
    • CP1 = 887 + 2096
    • CP2 = 18 + 2096
    • CP3 = 887 + 2096
    • alcR = 565 + 2204 + 2639


    Set B = alcA1/2 (insert, EcoRI-HF/SpeI) + ChP (vector, EcoRI-HF/XbaI)
    Expected band size (bp):
    • alcA1 = 886 + 2047
    • alcA2 = 785 + 2772
    • amilCP = 15 + 2742
    • amilGFP = 15 + 2772
    • spispink = 15 + 2752


    Set C =
    (a) RFP (from CP1, insert, SpeI/PstI-HF) + CP2 (pSB1C3, vector, SpeI/PstI-HF)
    (b) CP1 (vector, EcoRI-HF/PstI-HF) + ligated set C (a) (EcoRI-HF/PstI-HF)
    Final construct = CP2+RFP in BBa backbone

    Set D = Positive controls - pTE, puc19 vectors
    Expected band size (bp):
    pTE
    • SpeI/PstI-HF = 89 + 1197 + 3761
    • XbaI/PstI-HF = 16 + 2622 + 2409
    • EcoRI-HF/SpeI = 89 + 249 + 4709
    • EcoRI-HF/XbaI = 932 + 2638 + 1477


    Prepared 3 vials of 25 ɥL for each sample
    Set A = CP1/2/3 (vector, SpeI/PstI-HF) + alcR (insert, XbaI/PstI-HF/ZraI)
    DNA concentration:
    • CP1 = (a) 83.6ng/ɥL, (b) 323.6ng/ɥL
    • CP2 = (a) 103.9ng/ɥL, (b) 32.3ng/ɥL, (c) 22.3ng/ɥL
    • CP3 = 195.4ng/ɥL
    • alcR = 390.3ng/ɥL


    Per vial
    figure 40

    figure 41


    Set B = alcA1/2 (insert, EcoRI-HF/SpeI) + ChP (vector, EcoRI-HF/XbaI)
    DNA concentration:
    • alcA 1 = 84.9ng/ɥL
    • alcA 2 = 743.3ng/ɥL
    • amilCP = 181.1ng/ɥL
    • amilGFP = 53.2ng/ɥL
    • spispink = 230.8ng/ɥL
    figure 42

    figure 43


    Set C (b) = CP1 (vector, EcoRI-HF/PstI-HF) + ligated set C (a) (EcoRI-HF/PstI-HF)
    figure 44


    Set D = Positive controls - pTE, puc19 vectors
    Prepared a 10ɥL reaction rather than a 25ɥL
    DNA concentration:
    • pTE = 124.6ng/ɥL
    • SpeI/PstI-HF
    • XbaI/PstI-HF
    • EcoRI-HF/SpeI
    • EcoRI-HF/XbaI
    • puc19 = 417.5ng/ɥL
    • EcoRI-HF/ZraI
    • PstI-HF/ZraI

    figure 45

    figure 46

    figure 47


  • Inoculated CP1, CP2, BL21 cells
  • Restriction enzyme digest for alcA1, alcA2, alcR, ChP
    Concentrations:
    alcA 1 - 84.9 ng/ɥL
    alcA 2 - 743.3 ng/ɥL
    pTE - 124.6 ng/ɥL
    Expected band size (bp):
    • alcA1 = 886 + 2047
    • alcA2 = 785 + 2772
    • amilCP = 15 + 2742
    • amilGFP = 15 + 2772
    • spispink = 15 + 2752
    • alcR = 565 + 2204 + 2639

    figure 48


    Concentrations:
    • amilCP - 181.1 ng/ɥL
    • amilGFP - 53.2 ng/ɥL
    • spispink - 230.8 ng/ɥL
    • pTE - 124.6 ng/ɥL

    figure 49


    Concentrations:
    • alcR - 390.3 ng/ɥL
    • pUC19 - 250.0 ng/ɥL
    figure 50

    figure 51

    Result: Bands were distorted severely, most likely due to not properly dissolved agarose powder.



    figure 52

    Result: Only one band was seen instead of two. May have missed out adding any of the reagents/problems with the DNA. Should change a new aliquot of positive control DNA from this point onwards.



  • Gel extraction of alcA1, alcA2, alcR and digested products
    figure 53

    *CP2 to be PCR purified,/p>



  • PCR purification of amilCP, amilGFP, spispink
    figure 54


    • Re-did overnight ligation of CP1 with chromoproteins (ChP)
    figure 55

    Concentrations:
    • amilCP - 20.2 ng/ɥL
    • amilGFP - 10.4 ng/ɥL
    • spispink - 10.7 ng/ɥL/li>

    1:7 ligation DNA mass:
    • amilCP - 59.53 ng
    • amilGFP - 62.03 ng
    • spispink - 60.36 ng/li>


  • Ligation of alcA 2 with amilCP
    figure 56


  • Re-did alcA1/alcA2/alcR digest with pTE1055.term controls and gel extracted appropriate sizes.
    figure 57

    figure 58

    *all samples were digested at 37ç for a Total of 2 hours. For alcR & pTE1055, X/P was added first for 1 hour before adding ZraI and left to digest for another hour.
    *All samples were run on a 1% gel.



    alcA 1: 886 + 2047
    alcA 2: 785 + 2772
    alcR : 565 + 2204 + 2639
    figure 59

    Result: The correct band sizes were extracted for alcR and gel extraction was performed as per protocol. For alcA2, the wrong band size was extracted so digest would be repeated. alcA1 did not work.



    figure 60

    After soaking in ethidium bromide, alcR bands were clearly seen and extracted
    figure 61
  • Transformed overnight ligated samples (CP 1 + chromoproteins (Carbenicillin), alcA2 + amilCP (CmR) ) into DH5alpha along with a pUC19 (Carbenicillin) positive control using protocol. They were plated into the appropriate Ab plates and SOC medium was used as a negative control.
  • Ligation of CP1 + alcR and CP2 + alcR using Roche ligation kit and transformed into DH5α. Plated onto Carbenicillin plates, left in 37°C incubator overnight.
    Insert: CP1, CP2
    Vector: alcR (482.1ng/ɥL)
    figure 62


  • Repeated alcA 1 & alc A 2 digest using same measurements as before and used pTE1055 control. alcA 1 did not work again. alcA 2 worked and the correct band size (785) was extracted. Gel extraction protocol was used to obtain the DNA.
    figure 63


  • CP1 + amilCP ligation product restriction enzyme digest to verify the plasmid. pUC19 was used as a positive control.
    figure 64


  • Sent alcA 1 for sequencing due to repeated failure in restriction enzyme digest. sequencing results of alcA 1indicate that alcA1 did not contain XbaI / SpeI site.
    figure 65


  • Inoculated cultures and miniprep results
    figure 66


  • Validation of miniprep ligated product- alcA 2 + amilCP by restriction enzyme digest and sent for sequencing using VF2 primer

    Expected band sizes:
    pTE1055
    • PstI-HF/ZraI: 2867 + 2180
    • XhoI/PstI-HF: 2820 + 2040 + 187

    alcA 2 + amilCP
    • PstI-HF/ZraI: 1921 + 1475 + 131
    • XhoI/PstI-HF: 1622 + 1013 + 892

    figure 67

    figure 68

    figure 69

  • RE digest of all chromoproteins, constitutive promoters, alcA1, alcA2, alcR and pTE 1055(control)
    figure 70


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

  • Important dates:
    29th August-First BioBrick ready
  • Restriction enzyme digest
    figure 71

    *ZraI was added to both samples after 1 hour of incubation

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    figure 72

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  • PCR purification - CP2, amilCP, amilGFP, spispink
    figure 73


  • alcA2 + amilCP sequencing results with VF2 primer → alcA2 + amilCP ligation was successful (FIRST BIOBRICK READY!)
    figure 74


  • Received alcA 1 forward and reverse primers from IDT. Added appropriate amounts of milli-Q as instructed by IDT to make a stock concentration of 100 ɥM. Did a dilution to make aliquots of 10 ɥM of primers to use for PCR.
    figure 75


  • Q5 PCR to insert XbaI/SpeI in alcA1. Also used an a negative control (empty vector from previous day’s ligation) → failed as too much dNTP was used resulting in Mg2+ depletion

    alcA1 - 349.6 ng/ɥL
    figure 76


    PCR reaction conditions:
    figure 77

    figure 78


    Restriction Enzyme digest → Gel extraction → overnight ligation of:
    Insert CP3 (S/P) + Vector amilCP (X/P)
    Insert CP3 (S/P) + Vector amilGFP (X/P)
    Insert CP3 (S/P) + Vector spispink (X/P)
    Insert CP3 (S/P) + Vector alcR (X/P/Z)

    Controls:
    pTE1055 (S/P) & pTE1055 (X/P/Z)
    Concentrations:
    • CP3 = 526.9 ng/µL
    • amilCP = 146.2 ng/µL
    • amilGFP = 273.5 ng/µL
    • spispink = 235.3 ng/µL
    • alcR = 855. 0 ng/µL
    • pTE1055.term = 1074.4 ng/µL

    figure 79

    *for alcR, ZraI was added after 1 hour. Total digest time = 2 hours in a 37°C water bath.



  • Overnight ligation in 4°C cold room
    figure 80


  • noculated cultures of ligated products from 30th Aug using colonies from plate in Carbenicillin + LB. Miniprep results:
    figure 81


  • CloneAmp PCR to insert XbaI/SpeI in alcA1 as Q5 PCR has been failing.

    alcA1 - 366.0 ng/ɥL
    figure 82


  • Ran CloneAmp products (alcA1 + XbaI/SpeI) on a 1 % gel → PCR was successful so proceeded to PCR purify the product
    figure 83

    figure 84

  • Restriction digest of miniprep ligation product in to verify plasmid
    figure 85

    figure 86


  • Restriction digest of PCR purified alcA1+ XbaI/SpeI with EcoRI-HF and SpeI-HF → Gel extract (866bp band) for ligation with spispink (E/X)
    figure 87


    Gel extraction was not successful. Repeat the PCR
    figure 88

    * bottom half of the gel is supposed to be verification of ligated products but there was something wrong with the gel and hence nothing could be seen. Re-do digest on Monday to verify the ligated product.



  • Heat inactivation of overnight ligation product at 65C for 10 minutes. They were then transformed into DH5α competent cells and plated on Carbenicillin and left on bench over the weekend.
    • Transformed CP1 + alcR and CP2 + alcR into BL21 competent cells. Plated onto Carbenicillin and left on bench over the weekend.

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

  • Q5 PCR to insert XbaI/SpeI to alcA1 →Run on 1% gel → PCR purify → restriction enzyme digest with EcoRI/SpeI → gel extraction → ligation with spispink (E/X) overnight

    alcA1 - 366.0 ng/µL, amplified fragment side = 886 bp
    figure 89

    figure 90

    figure 91


  • PCR purification of alcA1 using protocol
    figure 92


  • Restriction enzyme digest of PCR purified alcA1 with EcoRI-HF and SpeI. Digested at 37°C in PCR machine for 1 hour.

    Band expected: alcA1 = 886bp, pTE1055.term = 4709 + 89 + 249
    figure 93

    figure 94

    Digest failed so repeated it again. Repated digest worked, so proceeded to gel extract the fragment.


    figure 95
  • Gel extraction of alcA1 digested with E/S using protocol.
    figure 96


  •  overnight ligation of alcA1 (E/S) with spispink (E/X) in 4°C (cold room)

    Insert: alcA1 (E/S) - 886 bp, 92.4 ng/ɥL
    Vector: spispink (E/X) - 2752 bp, 37.8 ng/ɥL
    figure 97


  • Restriction digest to verify ligated products - CP1 + alCR, CP2 + alcR, CP2 + rfp
    figure 98

    figure 99

    figure 100

    Results: No bands were seen except for positive controls (pTE1055). Re-made overnight cultures to miniprep and validate again.



  • New ligation of CP + alcR and CP + Chromoproteins. Ligation was done using Roche kit for 30 mins on ice. This time transformed into Marc’s competent cells (to eliminate the possibility of contaminated competent cells) and plated onto Carbenicillin plates, left overnight.

    Ratio - 1:1
    figure 101


    Ratio - 1:3
    figure 102

    figure 103

    figure 104


  • Colony PCR (with VF2 and VR primers)

    Mastermix:
    figure 105

    figure 106 figure 106.1
    Made overnight cultures of the correct samples
  • Glycerol stock and miniprep overnight cultures - alcA1 + spispink
    figure 107
  • Digest alcA1 + spispink with EcoRV-HF and SphI-HF to verify plasmid. alcR used as control
    figure 108

    figure 109
    Sent alcA1 + XbaI/SpeI that was done using PCR for sequencing
  • RE digest for CP, alcR and ChP for ligation

    Samples:
    Eg: Vector + insert
    • CP1 (S/P) 2096bp + alcR (X/P/Z) 2639bp
    • CP2 (S/P) 2096bp + alcR (X/P/Z) 2639bp
    • CP3 (S/P) 2096bp + alcR (X/P/Z) 2639bp
    • CP1 (S/P) 2096bp + amilCP (X/P) 713bp
    • CP2 (S/P) 2096bp + amilGFP (X/P) 743bp
    • CP3 (S/P) 2096bp + spispink (X/P) 723bp
    figure 110


  • Ligation
    Conc. of alcR used = 31.4 ng/ɥL. Concentrations of the other DNA used are in G) below.

    Ratio 1:1
    figure 111


    Ratio 1:3
    figure 112


    Ratio 1:5
    figure 113


    Controls
    figure 114

  • Gel extraction- Nanodrop concentration
    figure 115

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

  • Colony PCR

    Mastermix:
    figure 116

    figure 117

    figure 118

    figure 119


  • Miniprep CP1 + alcR (1:3)
    figure 120


  • RE digest to confirm CP1 + alcR (1:3) ligation was successful

    Did not perform as the DNA concentration was very low

  • Inoculated ligated products. Miniprep results:
    figure 121
  • Ligation of CP1 and ChP

    1:3
    figure 122

    1:5
    figure 123

    Controls
    figure 123

    Transformed ligated products

  • RE digest for verification
    figure 125

    figure 126


  • RE digest for ligation
    figure 127

    figure 128

    figure 129

    Something was wrong with the gel. Will re-run gel the next day. However, we loaded all of the pTE sample so there will be none for the re-run.



  • Made DH5α competent cells
  • Ran gel for digested products
    figure 130

    figure 131

    Both fragments of CP3 were gel extracted.
    Smaller fragment: 887 bp = rfp


    figure 132

    Bigger fragment: 2096 bp = CP3 vector Since the CP2 S/P/A control shows that the digest has worked, CP2 was PCR purified to lose the 18bp fragment and retain the CP2 vector.



  • PCR purification of CP2 (pSB1C3) S/P
    figure 133
  • Gel extraction of CP3 fragments (vector and rfp)
    figure 134
  • Before performing the CP2 + rfp ligation, we ran the PCR purified vector and gel extracted rfp on a gel just to confirm that the DNA is present.
    figure 135

    The correct band sizes are seen. Proceeded to perform CP2 + rfp ligation.

  • alcR restriction enzyme digest with XbaI/PstI-HF. No ZraI could be found, so we ran a 0.6% gel for a really long time hoping to be able to get the separation of the 2 bands.
    figure 136

    figure 137

    figure 138

    Separation of bands was not seen. Repeated digest with PvuI obtained from another lab.

  • CP2 (vector) + rfp (insert) ligation
    figure 139

    The ligation was left in the PCR machine at 24°C for 2 hours. It was then heat inactivated at 65C for 10 minutes and then transformed into DH5alpha. They were plated onto Carbenicillin plates.

  • Tested our new DH5alpha competent cells and BL21 cells by transforming them with pUC19 and plated onto Carbenicillin. Left overnight at 37°C.
  • Ran gel for alcR digest from 14th Sept (F). We did not have SybrSafe, so left the gel in Ethidium Bromide for half an hour before visualising gel.
    figure 140

    Proceeded to gel extract the appropriate alcR fragment.

  • Gel extraction of alcR
    figure 141
  • Restriction digest of alcA1, alcA2 and alcR to put into pSB1C3 submission vector.
    figure 142

    figure 143

    Samples were left in PCR machine at 37°C for 1.5 hours. ZraI was added after 45 minutes.

  • Gel extraction of fragments from digest
    figure 144

    figure 145


  • Submission vector ligation using fragments from D and pSB1C3 vector that has been digested with EcoRI-HF and PstI-HF by Mechanism 1 team.

    Concentration of pSB1C3 = 11.1 ng/ɥL
    figure 146

    Samples were left on the bench for 1 hour before transforming into Dh5α and plated onto Cm plates. Left in 37°C incubator.

  • Restriction digest of old alcA2 + amilCP product for verification
    figure 147

    figure 148
    Samples were left in the PCR machine at 37°C for 1.5 hours. It was then run on a 1% gel.
    figure 149
  • Ligation : CP3 + chromoproteins & CP + alcR
    figure 150

    figure 151

    Controls
    figure 152

    figure 153

    figure 154


  • Transformed ligated product onto appropriate plates
  • Made 5ml overnight culture for rfp quantification for CP1 and CP3 in the Bba_J61002 vector that contains rfp. Grown in 5ml LB + CmR (50 ɥg/ml). Also grew pBbB2K (kanamycin, ATC inducible) Kiesling vector as a control with rfp.
  • alcA1+spispink restriction enzyme digest to verify the plasmid
    figure 155

    figure 156

    figure 157

    alcA1 + spispink is not the correct product.



  • spispink restriction digest to obtain pSB1C3 vector.
    figure 158

    figure 159

    figure 160

    The larger fragment was extracted and proceeded to purify as per protocol.



  • Colony PCR
    Mastermix:
    figure 161

    figure 162

    figure 163


  • RFP quantification for CP1 and CP3 using the overnight culture.A kiesling vector - pBb2K (Kan) was used as a control that can be induced by aTC. 50nM of aTC was added to the induced wells during mid-way log growth. RFP was measured for 24 hours.

    Stock concentration of aTC = 10mM
    Concentration needed to add to 200ɥL cells in plate = 50nM

    A 1:1000 dilution of the stock concentration with 100% ethanol was made and a final concentration of 1mM was obtained. 1ɥL of aTC was added to each well. Important to note that aTC is light sensitive and hence the tube has to be wrapped in aluminium foil. OD 600 and RFP were measured every 30 mins for 24 hours. The cells were shaken at 500 rpm and at 37 °C.

Cell-Free Mechanism

  • Prepared IPTG induced BL21 cells transformed 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

  • Miniprep results for ligated products
    figure 164


  • RE digest for verification of ligated products
    figure 165

    figure 166

    figure 167


  • Inoculated CP1, CP3, CP3 + amilCP, CP3 + spispink, Kiesling vector
  • overnight ligation into pSB1C3- alcA 1, alcR, alcA 2
    Vector: pSB1C3 - 31.6 ng/ɥL
    Insert(all cut with E/P): alcA 1 + X/S - 46.4 ng/ɥL, alcR - 42.2 ng/ɥL, alcA 2 - 29.5 ng/ɥL

    1:1
    figure 168
    1:3
    figure 169
    Controls
    figure 170


  • RE digest to re-verify CP3 + alcR
    figure 171

    figure 172

    figure 173

    Lane order: Ladder, A(P/B), B(P/B), C(P/B), pTE1055(P/B), A(E/P), B(E/P), C(E/P), amilCP(E/P), amilCP(E/P), Ladder



  • Protein expression overnight culture- 5 mL culture, CARB 100, 30℃ incubator. 2 replicates of CP3+alcR, CP3+rfp and BL21 were grown.
  • Chromoprotein comparison - the same amount of cells were added onto a plate and the time taken for a visible colour change was seen.
    figure 174


  • Made overnight culture for rfp quantification - CP1, CP3 and Kiesling vector pBbB2K
  • Submission vector ligation - alcA2, alcA1 and alcR into pSB1C3. Ligation was done in the PCR machine at 24°C for 2 hours. Heat inactivation at 65C for 10 minutes then transformed into DH5alpha and plated onto Carbenicillin. Plates were left at 37°C, overnight.

    1:1
    figure 175
    1:3
    figure 176
    Controls
    figure 177


  • Restriction digest to obtain pSB1C3 backbone using spispink. Digest was run on a PCR machine at 37°C for 1.5 hours and then viewed on a 1% gel . since the bands were correct, we proceeded to gel extract the larger fragment (pSB1C3 backbone).
    figure 178

    figure 179
  • Protein expression

    Made a 1:1000 dilution of the overnight cultures in a Total volume of 25ml (in a 250 mL Flask) = 24.75 mL LB + 0.25mL culture *sterile conditions, no antibiotic
    The culture was shaken at 37°C and the OD was measured at several timepoints. Some of the cultures needed dilutions to be made to enable OD 600 measurements to be taken.
    figure 180
    At each OD reading, 500ɥL of the culture was taken and placed into a 2mL tube. It was spun down at 12 000rpm, 4°C for 10 minutes. The supernatant was discarded and pellet was kept. 50 ɥL of 2X SDS-PAGE dye was used to resuspend the pellet and transferred into a PCR tube. The samples were boiled at 99°C in the PCR machine for 20 minutes. All the samples were kept in a 4°C fridge until ready to be loaded onto the SDS gel. SDS gel was run at 300V for 20 minutes.
    The remaining culture was transferred to a falcon tube and spun down at 10000 rpm , 4°C for 20 minutes. Supernatant was discarded and the pellet was stored in the -20°C for future use.
  • CP rfp quantification
    RFP quantification for CP1 and CP3 using the overnight culture.A kiesling vector - pBb2K (Kan) was used as a control that can be induced by aTC. 1ɥL of 1mM aTC was added to the induced wells during mid-way log growth. RFP was measured for 24 hours. OD 600 and RFP were measured every 30 mins for 24 hours. The cells were shaken at 500 rpm and at 37 C.
  • SDS-PAGE did not show presence of protein. Repeated SDS-PAGE but this time using stored pelleted cells. Cells were first separated into soluble and insoluble fractions.
    Pellets were first resuspended in 500ɥL Bugbuster (must be at room temperature) and transferred to a 2ml tube. They were then shaken on a rocker for 20mins before being spun down at 12 000 rpm at 4°C for 20 mins. Supernatant which contains the soluble fraction was transferred to a fresh 2ml tube.
    Pellets containing the insoluble fraction were resuspended in 200ɥL Buffer SB1. In a PCR tube, 0.3ɥL of the resuspended pellet + 0.5ɥL DTT + 5ɥL 2X dye + 4.2ɥL milli-Q were added. They were then boiled on the PCR machine at 98°C for 7 minutes. Supernatant containing the soluble fraction : In a PCR tube, 2.5ɥL of the supernatant + 0.5ɥL DTT + 5ɥL 2X dye + 2ɥL milli-Q were added. They were then boiled on the PCR machine at 98°C for 7 minutes. Both the pellet and supernatant fraction samples were then loaded onto a gel and run at 300V for 25-30minutes (4 gels were running at the same time. If only 2 gels, 20 minutes is enough). alcR size = 97.7kDa, hence a pre-made 10% gel was used.
  • Rfp quantification was repeated.
    RFP quantification for CP1 and CP3 using the overnight culture.A kiesling vector - pBb2K (Kan) was used as a control that can be induced by aTC. 1ɥL of 1mM aTC was added to the induced wells during mid-way log growth. RFP was measured for 24 hours. OD 600 and RFP were measured every 30 mins for 24 hours. The cells were shaken at 500 rpm and at 37 C.
    figure 181
  • colony PCR to check pSB1C3 + alcR/alcA1/alcA2 transformed and plated on 21st August.

    Mastermix:
    figure 182

    figure 183
  • Co-transformation of CP3+alcR and alcA2+amilCP
    figure 184

    figure 185


  • Ran a 1% gel for colony PCR
    figure 186


  • Co-transformed colonies
    Picked 2 colonies from each plate, grow in 3mL LB with Carbenicillin and Chloramphenicol antibiotics to make cultures.
    Carb50 3ɥL
    CmR25 2.24ɥL
    Carb25+Cmr12.5 1.5ɥL Carb + 1.12ɥL CmR
    Incubate in 37°C shaker
    Stop the incubation when cultures reach approximately OD=0.1
    Make a 1:100 dilution for the culture with lowest OD
    Dilute other cultures accordingly to normalize all ODs (in reference to the lowest OD culture)
    Add different concentrations of ethanol to the cultures
    Measure absorbance of chromoproteins using a plate reader

  • RE digest for composite parts (CP3+alcR, CP3+amilCP, CP3+spispink) using EcoRI/PstI
    figure 187
    Result = All band sizes were correct exceptCP3+alcR. Gel extracted the correct fragments
    figure 188

    figure 189

Cell-Free Mechanism

  • Repeated induction of cells with IPTG, SDS-PAGE and western Blot from last week
  • Western Blot still shows correct protein in the insoluble fraction

Inducible Gene Switch

  • EcoRI/PStI digest for CP3 + alcR
    figure 190

    figure 191
  • Made cultures for co-transformation colonies with half antibiotic concentration until samples reached an OD of 0.6. Diluted the samples to normalise the OD before putting samples into the plate reader for comparison.
    figure 192


  • Re-did RE digest for verification of CP + alcR with SphI-HF and AhdI
    Expected band sizes:
    CP + alcR - 2472 + 1580 + 683
    pTE1055 - 1254 + 2073 + 172
    figure 193

    figure 194

    figure 195


  • Ran co-transformed colonies in plate reader. Normalise OD of samples in the plate reader until it reaches an OD of 0.6. Then, induce cells with ethanol.
    figure 196
  • Redid RE digest of CP3 +alcR with EcorI and PstI
    figure 197

    figure 198
    Gel extraction-not done as the band sizes for CP3 + alcR were not right. The control worked fine.

  • Miniprep overnight cultures of alcA 1, alcA 2, alcR in submission vector
    figure 199


  • Miniprep overnight cultures of alcA 1, alcA 2, alcR in submission vector
    figure 200


  • Miniprep overnight cultures of alcA 1, alcA 2, alcR in submission vector
    figure 201
    Note: Concentration/ratios were weird for miniprep products, proceed to RE digest for verification regardless

  • Absorbance measurement using plate reader (OD600, OD588, OD540)
    figure 202


  • RE digest for CP3+alcR_pSB1C3 with EcoRI/PstI/PvuI-HF
    figure 203


  • pSB1C3 verification using PstI/ZraI

    Expected band size:
    alcR - 2762+1921
    alcA1 - 1921+861+131
    alcA2 - 1921+780+131
    pTE - 2867+2180
    figure 204


Cell-Free Mechanism

  • Typed up lab work for this mechanism
  • Prepared PSB1C3_AOx to be sent off to iGEM HQ

Inducible Gene Switch

Cell-Free Mechanism

Inducible Gene Switch

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