Team:CMUQ/Part Collection



CMUQ





CMUQ

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  • Week 1
    • Tuesday, 8 May

      Preparation

        Dina, Sondoss, Sakina
      • Prepare LB-amp and LB-Chl plates for autoclave tomorrow
      • Prepare LB media for autoclave tomorrow


        • Haya
      • Took pictures of lab work

    • Wednesday 9 May

      Preparation

        Dina, Sondoss
      • Autoclave LB media and agar plates (LB-amp) not LB-Chl
        • After Autoclave

      • Continue primer design for pRSET em-GFP plasmid with Dsp expression cassette and restriction sites

    • Thursday 10 May

      Preparation

        Dina, Sondoss
      • The prSET 211 plasmid- containing bacteria was streaked on LB-Amp plates (duplicates) and incubated at 37˚C in the teaching lab.
      • The LB-Chl plates were poured. The plates are placed in the iGEM box in 2033 along with LB-Amp plates.
      • Design primers to amplify the Dsp and GFP segment on the pRSET plasmid, with cut sites that allow insertion in g-block in pSB1C3 plasmid
      • Team members registered on the iGEM roster
      • iGEM calendar sync to google drive to tackle deadlines

  • Week 2
    • Sunday 13 May

      Preparation

        Dina, Sondoss
      • Start overnight culture for the prSET 211 plasmid
      • Streak the pSB1C3 plasmid onto LB-Chl plates and incubate overnight at 37˚C
      • Prepared 5mL of 25mg/mL Chl stock solution.
      • Prepared 30 mL of 100mg/mL Amp stock solution.
        • coacervation

        coacervation

        coacervation

    • Monday 14 May

      Preparation

        Dina, Sondoss
      • Prepare LB-Chl media
      • Start overnight culture for the pSB1C3 plasmid in LB-Chl medi, picked from two duplicate plates
        • coacervation

        coacervation

      • Inoculated pSB1C3 plasmid.
      • Plasmid extraction of the prSET 211 plasmid.
      • Run agarose gel electrophoresis to check for the plasmid DNA extracted (pRSET 211), include a 1Kb ladder
        • coacervation

    • Tuesday 15 May

      Preparation

        Dina, Sondoss
      • Troubleshoot the agarose gel electrophoresis image for prSET 211 plasmid.
      • Plasmid extraction of the pSB1C3 plasmid.
      • Overnight culture of pRSET 211 clones.
      • Run agarose gel electrophoresis to check size for pSB1C3 plasmid DNA extracted (expected size 2.07 kb), include a 1Kb ladder
        • coacervation

    • Wednesday 16 May

      Preparation

        Dina, Sondoss
      • Plasmid extraction (mini prep) of the prSET 211.
      • Nanodrop the samples to know the DNA mass in the prSET 211 samples.
      • One prSET 211 sample= 156.4 ng/uL
      • Another prSET 211 sample= 186.5 ng/uL

      Restriction Enzyme DNA 10x NEBuffer Final Volume
      10 units = 1 µL 1 µg 5 µl 50 uL

      Results

    • Thursday 17 May

      Preparation

        Dina, Sondoss
      • Prepare SOC media to use next week
      • Perform restriction digestion using HindIII enzyme and NEBuffer 2.1.
      • Run agarose gel electrophoresis for the uncut pRSET plasmid and cut plasmid using HindIII restriction enzyme. (expected size= 4.734 kb compared to 3.6 kb intact original prSET without Dsp insert).

        • electrophoresis
  • Week 3
    • Sunday 20 May

      Preparation

        Dina, Sondoss
      • Start Day 1 of SDS-PAGE protocol (overnight culture of prSET 211 in DH5a) to test for expressions.
      • Prepared the SDS-PAGE gel and stored in the fridge in the teaching lab.
      • Autoclaved the SOC media.

    • Monday 21 May

      Preparation

        Dina, Sondoss
      • Perform IPTG induction.

      # Time Minutes OD Reading
      1 0 40
      2 15 41
      3 30 49
      4 45 70
      5 60 89
      6 75 100
      Results
    • Tuesday 22 May

      Preparation

        Dina, Sondoss
      • Test for GFP fluorscence using fluorescence plate reader.

        • electrophoresis

        electrophoresis

        electrophoresis

        Wavelengths: 520nm (Green)/580-640 at high speed
        Sample Time/Minutes Supernant Pellet
        0 0.00195 0.00123
        15 0.00193 0.00128
        30 0.00192 0.00132
        45 0.00194 0.00162
        60 0.00194 0.00144
        75 0.00195 0.00172
        384 0.00204 0.00265

        Results

    • Wednesday 23 May

      Preparation

        Dina, Sondoss
      • Run the SDS-PAGE gel to test expression of protein from pRSET 211 in DH5a.
      • Preform double-digest restiction enzyme reaction. One restriction enzyme that can be used in the digestion is HindIII.
      • Assign roles: people have to do research to see application of detecting Calcium
      • Hardware: heating block and fluorescence device linked to an app

  • Week 4
    • Tuesday 29 May

      Preparation

        Dina, Sondoss
      • MathWorks Account setup for iGEM

  • Week 5
    • Monday 11 June

      Interlab

        Sakina, Sondoss
      • Prepared approx 20mL 25mg/mL stock solution of Chlororamphenicol.

    • Tuesday 12 June

      Interlab

        Sakina, Sondoss
      • Pour-plated 1L of LB-Chl plates with Chl concentration of 25μg/mL.

    • Saturday 16 June

      Preparation

        Dina
      • Found CRISPR-Cas12a sequence to order:

        • electrophoresis
  • Week 6
    • Sunday 17 June

      Preparation

        Joanna
      • Searched for some of the most common genetic diseases in the region (Middle East) to consider testing for with some custom designed guide-RNA

    • Monday 18 June

      Preparation

        Dina
      • Finalize project description and share with Dr. Vincent, Cheryl and Joana to review.

    • Tuesday 19 June

      Preparation

        Dina
      • Searched for doctors and organizations to reach out in Syria, to inquire about their need of field-ready diagnosis technique Finished editing the project description; looking forward for feedback.

    • Wednesday 20 June

      Preparation

        Dina
      • CRISPR-Cas12a team submissions due.

  • Week 7
    • Sunday 24 June

      Interlab

        Sakina, Sondoss
      • Add deionized water and other solutions to the weighted flasks of 1L SOC media and 250mL LB broth and autoclave. The prepared solutions would be 1L of SOC media and LB broth. The remaining step would be to add filtered glucose solution and MgCl2 to cooled down SOC media.

    • Monday 25 June

      Interlab

        Sakina,Sondoss
      • Start and finish the 2 calibration protocols and procedures.: Calibration #1 and calibration #2.
      • Meet up with Dina the procedure to ensure we are on track.
      • Started and finished transformations for the 9 samples (1 negative control- from the kit, 1 positive control-from the kit, 1 control DNA- from the component cell test kit, 6 test samples).
      • For each sample, including controls, has 2 plates. One plate contains transformed cell sample, the other plate contains a more concentrated cell sample (10X more concentrated).

        • coacervation

        # Plate Column 1 2 3 4 5 6 7 8 9 10 11 12
        1 A 0.74 0.417 0.239 0.149 0.103 0.076 0.059 0.05 0.049 0.044 0.042 0.04
        2 B 0.709 0.336 0.219 0.187 0.094 0.077 0.063 0.053 0.05 0.052 0.043 0.04
        3 C 0.65 0.434 0.248 0.165 0.114 0.078 0.065 0.054 0.052 0.044 0.042 0.039
        4 D 0.662 0.414 0.259 0.162 0.118 0.075 0.06 0.052 0.051 0.048 0.042 0.04

        Results



        # LUDOX CL-X ddH20
        replicate 1 0.074 0.047
        replicate 2 0.075 0.046
        replicate 3 0.076 0.047
        replicate 4 0.075 0.048

        Results

    • Tuesday 26 June

      Interlab

        Sakina,Sondoss
      • The transformed cells (DH5α by Dr. MB lab) did not grow. The positive and negative control did not have any cells grown. The cell DNA control was re-done and spread on LB plates rather than LB-Chl plates. This was one mistake done: incubate cell control DNA in LB-Chl plates. The protocol done today was to add 50μL of competent cells only and proceed to steps 12, 14-16 (no heat shock, ect).

    • Wednesday 27 June

      Interlab

        Sondoss
      • Cell Control was successful, transformed cells from Monday still didn't show any colonies.
        • coacervation
      • Perform Competent cell test on the DH5α provided by Dr. MB lab using iGEM test kit.

    • Thursday 28 June

      Interlab

        Sakina,Sondoss
      • Preform calibration protocol #3.
      • Preform component cells test kit on ultra-competent cells provided by Professor Vincent
      • The competency test for the competent cells provided by Dr. MB did not work as there were no colonies observed.

        • coacervation
        Plate Column 1 2 3 4 5 6 7 8 9 10 11 12
        A 154.317 133.758 102.334 69.224 42.072 23.948 13.309 7.172 4.031 2.463 1.642 0.865
        B 151.654 135.298 102.291 67.249 42.085 23.603 12.919 7.626 3.357 2.313 1.552 0.898
        C 151.325 135.298 102.478 70.074 41.569 23.440 12.802 6.931 4.013 2.423 1.622 0.888
        D 151.970 134.203 101.371 68.129 41.813 23.292 12.848 7.024 3.885 2.364 1.617 0.895

        Results

  • Week 8
    • Sunday 1 July

      Interlab

        Sakina, Sondoss
      • The DNA samples (from kit plate 7, including positive and negative control) were transformed.
        • The competency test for the One shot- ultra component cells provided by Dr Vincent worked successfully.

        coacervation

        coacervation
    • Monday 2 July

      Interlab

        Sakina, Sondoss
      • Inoculated the transfomed samples (test samples 1-6, positive and negative controls).


        • coacervation

        coacervation
      • Below are picture of the transformed samples. The cell control was used as the negative control (as only cells were spread and these cells had no Chl resistance). The difference between the positive and negative control was the fluorescence and color of the colonies. The hypothesis is that the negative control would not emit fluorescence while the positive control would. This can only be confirmed after taking fluorescence readings.

        • Name of Sample Number of Colonies
        Cell control 0
        Negative control 160
        Positive control 504
        Test device #1 264
        Test device #2 320
        Test device #3 740
        Test device #4 352
        Test device #5 808
        Test device #6 84

        Results



        coacervation

        coacervation

        coacervation

        coacervation
    • Wednesday 4 July

        Interlab

        Sakina,Sondoss
      • Preformed Cell measurement protocol: Day 3 protocol. The cell samples were sampled and preformed an assay.
      • Measured the absorbance and fluorescence of the inoculated samples at t=0 and t=6 (incubated in the shaking incubator at 220 rpm, 37°C for 6 hours). The results were recorded.

        • Absorbance of 1:10 diluted inoculated samples (step2)
        A Positive Control Negative Control Test Sample 1 Test Sample 2 Test Sample 3 Test Sample 4 Test Sample 5 Test Sample 6 Test Sample LB+Chloramphenicol
        Colony 1 0.1 0.106 0.107 0.087 0.092 0.116 0.102 0.1 0.038
        Colony 2 0.107 0.109 0.108 0.097 0.11 0.114 0.138 0.142 0.038

        Results



        Absorbance of inoculated samples at t=0
        A Negative Control Positive Control Test Sample 1 Test Sample 2 Test Sample 3 Test Sample 4 Test Sample 5 Test Sample 6 Test Sample LB+Chloramphenicol
        Colony 1 0.084 0.061 0.068 0.066 0.068 0.07 0.07 0.067 0.054
        Colony 1 0.085 0.062 0.065 0.065 0.071 0.075 0.066 0.066 0.054
        Colony 1 0.082 0.06 0.065 0.065 0.067 0.069 0.068 0.068 0.051
        Colony 1 0.084 0.064 0.063 0.063 0.068 0.071 0.066 0.066 0.052
        Colony 2 0.063 0.075 0.061 0.061 0.064 0.059 0.066 0.07 0.051
        Colony 2 0.063 0.073 0.058 0.058 0.065 0.057 0.065 0.074 0.053
        Colony 2 0.067 0.071 0.057 0.057 0.066 0.061 0.067 0.073 0.05
        Colony 2 0.063 0.073 0.055 0.055 0.059 0.057 0.063 0.073 0.046
        y>

        Results



        Fluorescence of inoculated samples at t=0
        Sample Negative control Positive control Test device 1 Test device 2 Test device 3 Test device 4 Test device 5 Test device 6 Lb + Chlorophenicol
        A 1.034 1.740 2.391 1.806 1.057 2.356 2.110 1.503 1.015
        B 1.030 1.830 2.414 1.838 1.016 2.407 2.131 1.458 1.008
        C 1.030 1.852 2.453 1.865 1.063 2.378 2.140 1.492 1.011
        D 1.035 1.825 2.390 1.831 1.058 2.366 1.995 1.522 1.010
        E 1.047 1.765 3.387 1.646 1.062 2.681 1.148 1.702 1.056
        F 1.049 1.744 3.352 1.660 1.031 2.675 1.124 1.681 1.024
        G 1.008 1.766 3.339 1.671 1.039 2.673 1.099 1.691 1.023
        H 1.032 1.785 3.293 1.653 1.041 2.631 1.122 1.668 1.061

        Results



        Absorbance of inoculated samples at t=6
        A Negative Control Positive Control Test Sample 1 Test Sample 2 Test Sample 3 Test Sample 4 Test Sample 5 Test Sample 6 Test Sample LB-Chloramphenicol
        Colony 1 0.243 0.345 0.482 0.407 0.326 0.469 0.341 0.0331 0.056
        Colony 1 0.261 0.397 0.429 0.429 0.393 0.530 0.370 0.327 0.055
        Colony 1 0.420 0.419/td> 0.480 0.348 0.286 0.579 0.328 0.269 0.049
        Colony 1 0.292 0.499 0.532 0.347 0.367 0.570 0.309 0.365 0.051
        Colony 2 0.473 0.295 0.345 0.304 0.331 0.320 0.378 0.393 0.050
        Colony 2 0.439 0.387 0.399 0.269 0.307 0.389 0.343 0.425 0.048
        Colony 2 0.425 0.324 0.411 0.313 0.323 0.378 0.368 0.411 0.051
        Colony 2 0.406 0.313 0.327 0.347 0.324 0.374 0.306 0.390 0.047

        Results



        Fluoresence of inoculated samples at t=6
        Sample Negative Control Positive Control Test Sample 1 Test Sample 2 Test Sample 3 Test Sample 4 Test Sample 5 Test Sample 6 Test Sample LB+Chloramphenicol
        A 1.002 6.659 2.755 7.491 1.187 3.952 3.288 4.363 1.000
        B 1.068 6.890 2.710 7.707 1.183 4.125 3.416 4.440 1.033
        C 1.031 7.115 2.795 7.7664 1.169 4.286 3.394 4.406 1.000
        D 0.983 6.936 2.739 7.253 1.157 4.333 3.461 4.455 1.089
        E 1.034 5.742 3.424 8.345 1.196 4.822 1.143 4.592 1.045
        F 0.969 5.920 3.289 8.480 1.204 4.726 1.072 4.264 0.990
        G 1.043 5.706 3.293 8.339 1.197 5.102 1.105 4.567 1.003
        H 1.013 6.074 3.237 8.192 1.192 4.991 1.119 4.527 0.975

        Results

    • Thursday 5 July

      Interlab

        Sakina,Sondoss
      • Preformed Cell measurement protocol: Day 3 CFU to OD600 (steps 1 and 2).
      • Absorbance of Positive and Negative controls (steps 1 and 2 of Cell measurement protocol: Day 3: CFU to OD600:
      • The 1.1-4.3 samples are triplets of the samples that were prepared to have final volume of 1mL (remaining volume was LB-Chl) and OD=0.1 (#1 is positive control sample #1, #2 is positive control sample #2, #3 is negative control sample #1 and #4 is negative control sample #2).
        • A #1 #2 1.1 1.2 1.3 2.1 2.2 2.3
        10 units = 1 µL 1 µg 5 µl 50 uL 50 uL 50 uL 50 uL 50 uL 50 uL
        10 units = 1 µL 1 µg 5 µl 50 uL 50 uL 50 uL 50 uL 50 uL 50 uL
        10 units = 1 µL 1 µg 5 µl 50 uL 50 uL 50 uL 50 uL 50 uL 50 uL

        Results

  • Week 9
    • Sunday 8 July

      Interlab

        Sakina, Sondoss
      • Spread-plated the positive and negative controls triplets of the protocol Cell measurement: Day 3: CFU to OD600 protocol.

    • Monday 9 July

      Preparation

        Sakina, Sondoss
      • Counted colonies from the spread-plated samples yesterday.
        • Sample 1
        Name of Sample 1 Triple Number of Colonies
        1.1 Dilution 10^-3 1
        1.1 Dilution 10^-4 0
        1.1 Dilution 10^-5 0
        1.2 Dilution 10^-3 9
        1.2 Dilution 10^-4 0
        1.2 Dilution 10^-5 0
        1.3 Dilution 10^-3 8
        1.3 Dilution 10^-4 0
        1.3 Dilution 10^-5 0
        Sample 1
        Name of Sample 2 Triple Number of Colonies
        2.1 Dilution 10^-3 24
        2.1 Dilution 10^-4 4
        2.1 Dilution 10^-5 0
        2.2 Dilution 10^-3 25
        2.2 Dilution 10^-4 3
        2.2 Dilution 10^-5 0
        2.3 Dilution 10^-3 18
        2.3 Dilution 10^-4 3
        2.3 Dilution 10^-5 0
        Sample 1
        Name of Sample 3 Triple Number of Colonies
        3.1 Dilution 10^-3 2400
        3.1 Dilution 10^-4 640
        3.1 Dilution 10^-5 69
        3.2 Dilution 10^-3 139
        3.2 Dilution 10^-4 0
        3.2 Dilution 10^-5 1
        3.3 Dilution 10^-3 96
        3.3 Dilution 10^-4 10
        3.3 Dilution 10^-5 1
        Sample 1
        Name of Sample 4 Triple Number of Colonies
        4.1 Dilution 10^-3 163
        4.1 Dilution 10^-4 5
        4.1 Dilution 10^-5 1
        4.2 Dilution 10^-3 82
        4.2 Dilution 10^-4 5
        4.2 Dilution 10^-5 0
        4.3 Dilution 10^-3 55
        4.3 Dilution 10^-4 6
        4.3 Dilution 10^-5 2

        Results



      • The following picture is an example of how the plates looked like when counting. The samples shown were 3.3 and 4.1. The last 2 samples were dilution 3 for each triplet and the top samples were dilution 5 for each triplet.

        • coacervation

    • Wednesday 11 July

      Improvement

        Dina
      • Finalized the lab flow for improvement project

  • Week 10
    • Tuesday 17 July

      Improvement

        Dina
      • Sent new Improvement sequences to order

    • Thursday 19 July

      Improvement

        Dina, Joanna, Sondoss,Sakina
      • Meeting with Mexico TecCEM iGEM team 2018
      • Discussed Projects, and agreed on a collaboration of plasmid characterziation and samples exchange.

  • Week 11
    • Sunday 22 July

      Preparation

        Dina, Sondoss
      • Restriction digest and agarose gel of pSB1C3 (BBa_J04450) using the protocol:
      • Nano-drop the pSB1C3 (BBa_J04450) from previous mini-prep to know volume of DNA to add.
      • Single digest the pSB1C3 (BBa_J04450) with EcoR1 restriction enzyme.
      • Run agarose gel electrophoresis to pSB1C3 (BBa_J04450) to check size (estimated size = 3.1 kb).
        • RESULTS:
      • We used the pSB1C3 plasmid provided in kit plate 7 (BBa_J04450) which has a reporter protein (RFP) but we don't want the fluorescence as we are using our own FP, therefore we need to transform the one in the linearized plasmid kit and perform digest and gel.
        • coacervation

    • Monday 23 July

      Preparation

        Dina, Sondoss
      • PCR amplify the Dsp-T7 fragment using the protocol:

    • Tuesday 24 July

      Preparation

        Improvement project (Dina and Sondoss):
      • Run agarose gel electrophoresis on the PCR amplification of the Dsp-T7 fragment (expected size= 1.232 Kb).
        • RESULTS:
      • The PCR for Dsp-T7 fragment failed. Will check reagents and conditions and repeat.
        • coacervation

    • Wednesday 25 July

      Preparation

        Dina
      • Preform a PCR reaction on the Dsp-T7 fragment
      • changed annealing temp from 58˚C to 54˚C
      • changed annealing time from 15 sec to 30 sec
      • Run an agarose gel electrophoresis of Dsp-T7 fragment PCR (expected size= 1.232 Kb).

        • Sondoss
      • Transform DH5α cells with the pSB1C3 plasmid (the plasmid was obtained from the small iGEM grey box and it is the linearized plasmid with no fluorescent proteins).

    • Thursday 26 July

      Preparation

        Sakina,Sondoss
      • If PCR of Dsp-T7 was successful → PCR cleanup, Restriction digest with EcoRI and PstI
      • If PCR of Dsp-T7 was unsuccessful → troubleshoot and repeat PCR + gel
      • Check plates of the transformed DH5α cells with the linearized pSB1C3 plasmid and proceed with overnight culture if successful.

        • Results
      • Transformation of linearized pSB1C3 backbone (found in small iGEM grey box with no fluorescnet proteins) in DH5-α cells. The colonies showed red colour which means it has the fluorescence proteins
      • The PCR of Dsp-T7 after changing the conditions failed. In the 1st lane is the 1kb and the PCR product is supposed to show in the 3rd lane.
        • coacervation

        coacervation

        coacervation

        coacervation

  • Week 12
    • Sunday 29 July

      Preparation

        Dina,Sondoss
      • Inoculate the transformed colonies into duplicates (2 colonies inoculated in 5mL of LB-Chl media each).
      • Nanodrop the gBlocks DNA sample to know the DNA concentration.
      • Trouble-shoot the PCR and preform a PCR reaction on the Dsp-T7 fragment:
        1) Changed the annealing temp from 54 to 60˚C
        2)Change template DNA volume from 10 µL to three different volumes in separate PCR tubes: 1 µL, 3µL and 5µL
      • Run an agarose gel electrophporesis of the three Dsp-T7 PCR products (expected size= 1.232 Kb).
        • Results
      • Results: Third trial of Dsp-T7 PCR with less template and higher annealing temp (60˚C) didn't work. will perform a positive control with one of the parts in iGEM 2017 kit using the same primers and the same PCR conditions
        • coacervation

        coacervation

    • Monday 30 July

      Preparation

        Dina,Sondoss
      • Mini prep on cultures of linearized pSB1C3 plasmid in DH5α cells
      • Ran a positive control for PCR using 10µL of the part BBa_K1763000 (iGEM 2017 Kit plate 7, well 1A) and the same conditions as yesterday, used new reagents from the gold amplitaq kit and made fresh dilutions of primers and taq in nuclease-free water
        • Results
      • The PCR positive control using BBa_K1763000 and fresh reagents failed. Will check primer sequences
        • coacervation

    • Tuesday 31 July

      Preparation

        Sakina,Sondoss
      • Restriction digest with EcoRI for the extracted linearized pSB1C3 plasmids and gel electrophoresis
      • Ran a positive control for PCR using 5µL of the iGEM 2017 gBlocks (WT_ProU and WT_ProU_RFP) using the ampli Taq kit but followed protocol
      • Ran a PCR of Dsp T7 following the protocol and varied template: had 10µL and 20 µL

        • Results
      • The PCR positive control using iGEM 2017 gBlocks was successful!
      • The digest of linearized plasmid
        • coacervation

    • Wednesday 1 Aug

      Improvement Project

        Dina,Sondoss
      • Run agarose gel on PCR of Dsp T7 prepared using the ​Inaccessible Protocol protocol (but varied template amounts: 10 µL and 20 µL DNA)
      • Make another PCR reaction of Dsp T7 but increase the number of cycles from 30 to 35 and extension time from 1.5 min to 2 min.

        • Sakina, Sondoss
      • Repeated the calibration 3 (fluorescence calibration) and changed some parameters (the excitation and emission wavelengths were the same and the slit width and gain setting/voltage were changed). There were 4 sets of readings recorded with the same samples measured:
      • slit width=5 nm and low (voltage= 400V)

        • Slit width = 5nm, Low Voltage=400V
        Plate Column 1 2 3 4 5 6 7 8 9 10 11 12
        A 164.160 142.847 110.025 74.842 45.846 25.994 14.278 7.893 4.411 2.657 1.708 0.801
        B 169.160 141.862 106.457 74.435 46.544 26.961 15.125 8.281 4.630 2.810 1.816 0.793
        C 165.996 145.045 110.701 74.003 44.875 25.435 13.813 7.281 4.298 2.624 1.709 0.806
        D 167.309 144.083 109.446 73.933 46.028 26.427 14.680 7.902 4.370 2.671 1.774 0.822


        Slit Width = 5nm, Medium Voltage = 600V
        Plate Column 1 2 3 4 5 6 7 8 9 10 11 12
        A 1000 1000 1000 1000 1000 1000 726.547 402.176 225.262 134.521 87.634 40.300
        B 1000 1000 1000 1000 1000 1000 755.953 422.776 236.679 144.484 92.966 40.622
        C 1000 1000 1000 1000 1000 1000 691.225 371.729 214.275 130.302 86.462 39.826
        D 1000 1000 1000 1000 1000 1000 703.900 383.425 210.325 132.125 88.312 39.641


        Slit Width=25 nm and Low Voltage=400V
        Plate Column 1 2 3 4 5 6 7 8 9 10 11 12
        A 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 839.825
        B 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 839.314
        C 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 929.452 844.954
        D 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 972.109 840.484


        Slit Width =25 nm and Medium Voltage = 600 V
        Plate Column 1 2 3 4 5 6 7 8 9 10 11 12
        A 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000
        B 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000
        C 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000
        D 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000
        Result: The best setting was with slit width = 5nm and low voltage=400V
        PCR of Dsp T7 failed
        coacervation

    • Thursday 2 Aug

      Improvement Lab

        Dina,Sondoss
      • Run agarose gel electrophoresis for Dsp-T7 with increase number of cycles from 30 to 35 and extension time from 1.5 min to 2 min.
      • Run PCR on one of the fluorescent proteins.

        • Results
      • The PCR positive control using iGEM 2017 gBlocks was successful!
      • The digest of linearized plasmid
        • coacervation

  • Week 13
    • Sunday 5 Aug

      Skype Meeting with our iGEM Mentor

      • Advised us about outreach and human practices
      • Elaborated on the validation process and the proof of concept where we must ensure and try that everything is working"as expected" (eg: DNA well purified for use withe the right concentration) & Shed light on the fact we need an approval from the ministry of ethics if we are to collect saliva samples from patients.
      • Emphasized that sequences must be submitted in DNA form (make a corresponding sequence of the RNA and submit it)
      • Cheap Assays are preferred by iGEM .

        • Results
      • The PCR positive control using iGEM 2017 gBlocks was successful!
      • The digest of linearized plasmid

        • Interlab

        Sondoss
        • The DNA samples (from kit plate 7, including positive and negative control) were transformed. The total number of DNA samples transformed in DH5α were 8 samples. The plates were incubated overnight to grow colonies.

          • Dina
          • PCR the Dsp but add more MgCl2 compared to.
          • PCR the RFP resuspended in nuclease free water not TE buffer
            • Reagent Volume(µL)
            Nuclease-Free water 30.5
            25mM MgCl2 6
            10X PCR Buffer 5
            10mM dNTPs 1
            Prefix primer (10µM) 1
            Suffix primer (10µM) 1
            Dsp-T7 gBlock DNA/ RFP DNA (10ng/µL) 5
            Ampli Taq gold (2.5U/µL) 0.5
            Total reaction 50
            coacervation

    • Wednesday 8 Aug

      Improvement

      Dina, Sondoss, Aya
      • Ran Dsp and pSB1C3 digests (EcoRI and PstI) on 1% gel
      • Gel extraction
      • Nanodropped the digested DNA fragments extracted from the gel: pSB1C3: 15.2 ng/µL, Dsp: 1.3 ng/µL

        • Results
      • Successful digests for both the Dsp and pSB1C3, correct band sizes were observed. The bands extracted were the Dsp band and 2.0 kb band in the pSB1C3 backbone.
        • coacervation
        Gel extraction pictures and gels:
        The mass of microcentrifuge was tared and subtracted from the sample+microcentrifuge mass. The masses recorded were the masses of the gel extracted: Dsp band:
        • Mass of Dsp: 0.5229 grams
        • Mass (ng): 21 (as expected from the gel ladder bands)
        • Expected size (kb): 1.213
        • Observed size (kb): 1.2

          • Mass of pSB1C3 backbone: 0.2018 grams.
          • Mass (ng): 1 band was 250 ng and the other band was 375ng (as expected from the gel ladder bands)
          • Expected size (kb): 1.11 kb and 2.029 kb
          • Observed size (kb): 1.2 kb and 2.0 kb


            • The gel bands extracted were the Dsp band size 1.2 kb and the 2.0 kb pSB1C3 backbone using the Gel Extraction protocol.
          • Volume of QX1 added for pSB1C3 was: 600 μL as 3 folds more so mass=200 mg so 200*3=600
          • Volume of QX1 added for Dsp was: 1.5 mL as folds so mass=500 mg so 500*3=1500
            • coacervation
            Results

            A very faint band of size of ≈ 3 kb which is the band of interest was observed, along with other bands. This band is most likely the band of interest as it corresponds to expected ligated product of size 3.2 kb.
            coacervation

    • Thursday 9 Aug

      Improvement

      Dina, Sondoss, Aya
      • Ligation using master mix from NEB
      • Restriction digest with Sac1 and gel electrophoresis
        • < coacervation
    • Friday 10 Aug

      Improvement

      Dina, Sondoss, Aya
      • Transformed the ligated pSB1C3 backbone and Dsp then spread-plated on LB-Chl plate. The protocol used was Ultra-competent transformation Protocol.
        • The changes in the protocol were :
      • 5μL of ligation reaction was added to the 50μL One Shot cells vial (becasue we had very low
      • 150μL of SOC media was added to the transformation instead of 250μL. This was done in order to have total volume of 200μL to spread-plate.
      • Volume spread-plated was 200μL (the whole reaction) instead of 100μL.
        • coacervation

    • Saturday 11 Aug

      Improvement

      Sondoss
      • Inoculated 3 colonies in separate test-tubes with 3 mL LB-Chl media.
        • coacervation

  • Week 14
    • Sunday 12 Aug

      Improvement

      Dina, Sondoss
      • PCR of the remaining 3 fluorescent proteins (GFP, YFP, BFP) using the
      • Mini-prep the inoculated samples of the ligation reaction pSB1C3+Dsp. We preformed mini-prep on 2mL of each inoculated sample (total volume preformed mini-prep on=6 mL).
      • Ran agarose gel electrophoresis on the PCR products.

        • Results
        coacervation

        coacervation

        coacervation

        coacervation

    • Tuesday 17 Aug

      Improvement

      • Double Digested pSB1C3 and Dsp with EcoR1 and Pst1 using this protocol: Double Digestion using EcoR1 and Pst1
      • Run gel agarose electrophoresis (0.7%) of 1kb, negative control (water instead of digested sample), digested Dsp and digested pSB1C3
      • Run a gel agarose electrophoresis (0.7%) of

    • Tuesday 18 Aug

      Improvement

      Aya
      • Gel extraction of Dsp and pSBC13 EcoRI and PstI digested fragments.

    • Tuesday 19 Aug

      Improvement

      Joanna
      DNA Extraction: Using the protocol from Noth Carolina School of Medicine http://ncdnaday.org/modules/forensics/5%20minute%20DNA%20Extraction.pdf The following was done to extract DNA from saliva sample:
      • Add 1 mL of saliva to the the microtube
      • Add 2 drops of soap solution to the saliva micro- tube (cell membrane (fat) lysis)
      • Add 3 drops of contact lens multi-purpose solution (lysis enzymes/proteins)
      • Add a pinch of salt (aids in precipitation of DNA)
      • Addinvert microtube
      • Add 6 mL of isopropyl alcohol (precipitation)
      • Add 3 drops of contact lens multi-purpose solution (lysis enzymes/proteins)
      • Add 6 mL of isopropyl alcohol (precipitation)
      • Resuspend in Nuclease free water
      • Dissolve for the use of CRISPR by putting it in the 37 heating block.

        • Obeservations

        coacervation

  • Week 15
    • Tuesday 28 Aug

      Improvement

      Dina, Sondoss, Aya
      • Gel extraction pictures and gels:
      • The mass of microcentrifuge was tared and subtracted from the sample+microcentrifuge mass. The masses recorded were the masses of the gel extracted: Dsp band:
      • Mass of Dsp: ~0.479 grams
      • Mass (ng): same intensity as first band of 1.0 kb ladder (as expected from the gel ladder bands)
      • Expected size (kb): 1.213
      • Observed size (kb): 1.2

        • PSB1C3 backbone:
      • Mass of pSB1C3 backbone: ~0.2018 grams.
      • Mass (ng): band excised was half of the intensity of the 3.0 kb band (as expected from the gel ladder bands)
        • Expected size (kb): 1.11 kb and 2.029 kb Observed size (kb): 1.2 kb and 2.0 kb
      • The gel bands extracted were the Dsp band size 1.2 kb and the 2.0 kb pSB1C3 backbone using the Gel Extraction protocol
      • Volume of QX1 added for pSB1C3 was: 1076.1 μL as 3 folds more so mass=358.7 mg so 358.7*3=1076.1
      • Volume of QX1 added for Dsp was: ~1439 μL as 3 folds folds more so mass=~479 mg so 479*3=1439

  • Week 16
    • Thursday 4 Oct

      Crispr

      Dina
      • Calculations for next week experiments
    • Saturday 6 Oct

      Crispr

      Dina
      • Digested the pSB1C3 backbone (to submit CRISPR templates in) with EcoR1 and Spe1 according to https://nebcloner.neb.com/#!/protocol/re/sequential-heat/EcoRI,SpeI Ran the agarose gel electrophoresis along with Professor Vincent's samples.
      • Gel extracted the pSB1C3 backbone band 2.0 kb.

        • Mass of gel extracted= 0.242 g
        Size of band extracted= 48 ng
        Band size extracted= 2.0 kb
        Volume of QX1 buffer added= 726μL

        Volume of QIAEXII buffer added= 10μL
      • Nano-drop results= 3.9 ng/μL
      • Day 1 re-precipitation protocol was done using:
        • volume of 3M Na acetate added: 2μL
        volume of 96% ethanol added: 20μL
    • Sunday 7 Oct

      Crispr

      Dina
      • This is for submission of Template T (HBB ,mutant gene) in pSBC13 plasmid. The template digested was Template T.
      • Nanodrop Today: 3.0 ng/microliter but after heating at 37degees for 10 minutes --> 7.0 ng/microlter
      • Resuspended Template T (mutant HBB gene) in 25 microliter of nfw (20ng/microliter).
      • Digested Template with EcoRI and SpeI, added 0.2 microliter NaCl, total vol after digestion: 21 microliter
        • A B
        replicate 1 0.074
        replicate 2 0.075
        replicate 3 0.076
        replicate 4 0.075
        replicate 4 0.075

      • PCR cleanup: followed same protocol as given in manual (except added 4 vols of PB instead of 5 volumes), eluted with 20 microliter of EB, nanodrop: 6.8 ng/microliter Named tube: Temp T EcoRI SpeI PCR cleanup
      • Ligation of pSBC13 and Template T with Quick Ligation Kit (M2200S), followed same protocol.
        • A B
        replicate 1 0.074
        replicate 2 0.075
        replicate 3 0.076
        replicate 4 0.075
        replicate 4 0.075
      • Used 5 microliter of Ligation rxn to transform 50 microliter of DH5alpha, followed same protocol as on benchling but added 200 microliter SOC media instead