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 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 plasmids containing required DNA(RBS, amilCP(blue chromoprotein) and amilGFP(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 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 (Cm resistance). Re-streaked them onto Cm plates, incubate O/N 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 (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

  • Important dates:
    12,sup>th August- IDT genes arrived -- alcR, alcA2, AOX
    • 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 (22.4ng/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 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
  • IDT genes arrived -- alcR, alcA2, AOX

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
  • Re-suspended genes from IDT in 100ɥL of sterile milli-Q for 1 hour. Transformed them into DH5α for each gene using chemical transformation protocol. We plated 20ɥL and 200ɥL 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. spispink
    • figure 30

    figure 31

    Repeated digest as the DNA concentration was not satisfactory



  • -Inoculated CP1, amilCP, amilGFP and spispink to obtain more DNA
  • Transformation of genes from IDT worked. Picked single colonies and inoculated them.
  • Miniprep 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 as per protocol 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 PTE 1055.term(control) 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 - 124.4ng/ɥL
    spispink- 136.5 ng/ɥL
    pTE1055.term - 500ng/ɥL
    figure 35

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



    figure 36


  • Chemical transformation of PTE1055.term and pUC19 (control vector) into DH5α competent cells to get more DNA. Plated onto Carbenicillin plates
  • Inoculated alcA 2, 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

    "


    figure 72

    >br />
  • 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

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

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

Cell-Free Mechanism

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

Inducible Gene Switch

Sponsors

Follow us on