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Revision as of 12:14, 2 October 2018

iGem Groningen 2018

menu

Notebook

  • Week 25

    • Monday June 18th

      Who:

      Aim

    • Tuesday June 19th

      Who:

      Aim

    • Wednesday June 20th

      Who:

      Aim

    • Thursday June 21st

      Who:

      Aim

    • Friday June 22nd

      Who:

      Aim

    • Saturday June 23rd

      Who:

      Aim

    • Sunday June 24th

      Who:

      Aim

  • Week 26

    • Monday June 25th

      Who:

      Aim

    • Tuesday June 26th

      Who:

      Aim

    • Wednesday June 27th

      Who:

      Aim

    • Thursday June 28th

      Who:

      Aim

    • Friday June 29th
      thing we did
    • Saturday June 30th

      Who:

      Aim

    • Sunday July 1st

      Who:

      Aim

  • Week 27

    • Monday July 2nd

      Who:

      Aim

    • Tuesday July 3rd

      Who:

      Aim

    • Wednesday July 4th

      Who:

      Aim

    • Thursday July 5th

      Who:

      Aim

    • Friday July 6th

      Who: Rianne & Phillip

      Aim Competent cell test

      To test the competence of our cells, we performed a competent cell test according to the IGEM Competent Cell test kit protocol.

      We dissolved the dried-down purified plasmid DNA from BBa_J04450 into 50 µl of water to obtain 100 pg/µl and 10 pg/µl concentrations. In short, 1 µl of each DNA concentration was added to 50 µl of competent cells in duplicates. We obtained plenty of colonies and the efficiency of transformation was determined sufficient. This batch of competent E. coli DH5ɑ cells was used for all of the following E. coli transformations.

    • Saturday July 7th

      Who:

      Aim

    • Sunday July 8th

      Who:

      Aim

  • Week 28

    • Monday July 9th

      Who:

      Aim

    • Tuesday July 10th

      Who: Rianne & Phillip

      Aim Transformation of competent E. coli DH5ɑ with the devices for the iGEM Interlab study

      The next devices were provided to us by iGEM in the plates in the distribution kit. The following devices were resuspended into 10 µl sterilized deionized water.

      DeviceLocation on plate 7
      Negative control2D
      Positive control2B
      Device 12F
      Device 22H
      Device 32J
      Device 42L
      Device 52N
      Device 62P

      1 µl of this solution was transformed into 50 µl of competent DH5ɑ cells using the following protocol:

      1. LB plates with the appropriate antibiotics were prepared as described here (link to our protocol page LB agar plates)
      2. Eppendorf tubes (1.5 ml) were pre-chilled on ice and competent cells were thawed on ice
      3. 50 µl of competent cells was added to the pre-chilled eppendorf tubes, together with the DNA
      4. 30 min incubation on ice
      5. Heat shock for 45 sec in a 42℃ water bath
      6. Incubation on ice for 5 min
      7. 950 µl of LB broth was added to the cells
      8. Incubation for 1 hour, 37℃, 200 rpm
      9. Plating of the cells on the LB-agar plates. 100 µl one one plate, then the culture is centrifuged and the supernatant is partially discarded. The cell pellet is resuspended in approximately 100 µl and plated on a separate plate.
      10. Overnight incubation at 37℃ (agar side up)
    • Wednesday July 11th

      Who: Rianne

      Aim Growth of the colonies used in the iGEM Interlab study

      We obtained between 10-100 colonies per transformation. Two colonies were picked with a sterile pipette tip and transferred into 5 ml of LB with chloramphenicol. The cultures were grown in the appropriate broth overnight at 37℃ in a shaking incubator at 200 rpm.

      Who: Rianne

      Aim To purify the plasmids from Wen. et al out of E. coli for transformation into yeast

      • 1882: PYD1 - CipA1 - EGII
      • 1883: PYD1 - CipA3 - EGII
      • CB: PRS425 - CBHII - BGLI

      These three plasmids that we received from Wen. et al were kindly transformed by Vakhil Takhaveev into E. coli and plated onto LB agar plates. In order to purify the plasmids out of these cells, I grew three colonies of each strain into 10 ml of LB with the appropriate antibiotics. The next day, the plasmid was extracted from the full-grown cultures using a plasmid extraction kit. The following concentrations (ng/µl) were obtained:

      Results

      18821883CB
      1278,15572,80443,80
      2664,1098,0361,70
      3321,25652,0439,95

      The plasmids were stored at -20℃ until further use.

    • Thursday July 12th

      Who: Rianne

      Aim Production of glycerol stocks for the strains used in the iGEM Interlab study

      250 µl of culture was mixed with 250 µl of 50% glycerol and stored at -80℃. Simultaneously, a small amount of the glycerol stock was transferred into 5 ml of fresh medium supplemented with chloramphenicol and grown overnight at 37℃, 200 rpm.

    • Friday July 13th

      Who: Rianne & Phillip

      Aim First cell measurement for the iGEM Interlab study

      1. 500 µl of the overnight culture was transferred into 4,5 ml of LB with chloramphenicol
      2. OD600 measurement of 1:10 dilutions
        3. Colony 1Colony 2
        Negative control0.1910.192
        Positive control0.1980.204
        Device 10.1520.149
        Device 20.1890.208
        Device 30.190.191
        Device 40.150.171
        Device 50.0940.109
        Device 60.2040.195
      3. Dilution of these 1:10 cultures to target OD600 0.2 in a final volume of 12 ml

        4. 10 ml of culture was already pipetted into the 50 ml falcon tubes for all cultures, except for cultures of device 5, for which 9 ml was used. To these tubes, the following volumes (µl) were added:

        Colony 1Cell cultureLBColony 2Cell cultureLB
        Negative control1257743Negative control1249751
        Positive control1212788Positive control1176824
        Device 11579421Device 11611389
        Device 21269731Device 21154846
        Device 31263737Device 31257743
        Device 41599401Device 41404596
        Device 52553447Device 52202798
        Device 61176824Device 61231769
      4. 1000 µl of the 0 hour time point culture was pipetted into an eppendorf tube and kept on ice until further use in a box with ice at 4℃.
      5. Six hours of incubation at 37℃, 200 rpm
      6. 1000 µl of the 6 hour time point culture was pipetted into an eppendorf tube and kept on ice until further use in a box with ice at 4℃.
      7. 100 µl of these cell cultures were transferred into wells of a 96-microtiter plate.

      However, while analyzing our results, we realized that we did not set the ‘gain’ setting for fluorescence measurement in the plate reader to a fixed number across the different measurements. Therefore, we need to repeat this measurement.

      Aim Correlate OD600 measurements in our plate reader to colony forming units (CFU)

      We first diluted the overnight cultures of the negative and positive control 1:8 and measured OD600 in our plate reader. We then further diluted these cultures to target OD600 0.1, confirmed by our plate reader and plated onto plates in several dilutions.

      Aim Calibration experiments for the Interlab study

      LUDOX, silica beads and fluorescein experiments as described on our interlab page were also performed. As described above, the fluorescein calibration needs to be repeated.

      The exact protocol can be found here and the results of these experiments can be found on our Interlab page.

    • Saturday July 14th

      Who: Rianne & Phillip

      Aim Counting colony forming units

      We checked the colonies and counted them, to calculate how many cells were present in our culture of which the plate reader displayed the OD600 to be 0.1. The following numbers of colonies were found, where + and - stand for positive and negative control, respectively, and 1 and 2 stand for the two colonies that were picked at day 11-7-18. (4) and (5) stand for the dilutions plated as performed on 13-7-18.

      +1(4)+1(5)+2(4)+2(5)-1(4)-1(5)-2(4)-2(5)
      1181111922319191709
      215011208141121720219
      316619184222131120516
    • Sunday July 15th

      Who:

      Aim Growth of yeast strain YGEN 0013 and the interlab strains for second measurement

  • Week 29

    • Monday July 16th

      Who: Rianne

      Aim Growth of yeast strain YGEN 0013 and the interlab strains for second measurement

      YGEN 0013 colonies were picked and grown in Verduyn medium supplemented with the appropriate vitamins and trace elements, with addition of uracil, tryptophan and leucin. The strain was grown in 5 ml overnight at 30℃, 200 rpm.

      To repeat the Interlab fluorescence measurements, the glycerol stocks of the interlab strains 12-7-18 were taken out of the -80℃, and grown in 5 ml of LB and the appropriate antibiotic overnight at 37℃, 220 rpm.

    • Tuesday July 17th

      Who: Rianne & Phillip

      Aim Second Interlab measurement

      The same protocol as described on 13-8-18 and in the Interlab study was used. The starting OD600 of the 1:10 diluted overnight cultures were:

      Colony 1Colony 2
      Negative control0.190.182
      Positive control0.1670.168
      Device 10.2050.181
      Device 20.170.175
      Device 30.1850.19
      Device 40.1680.184
      Device 50.1780.185
      Device 60.1950.174

      Dilution of these 1:10 cultures to target OD600 0.2 in a final volume of 12 ml: 10 ml of culture was already pipetted into the 50 ml falcon tubes for all cultures. To these tubes, the following volumes (µl) were added:

      Colony 1Cell cultureLBColony 2Cell cultureLB
      Negative control1263737Negative control1319681
      Positive control1437563Positive control1429571
      Device 11171829Device 11326674
      Device 21412588Device 21371629
      Device 31297703Device 31263737
      Device 41429571Device 41304696
      Device 51348652Device 51297703
      Device 61231769Device 61379621

      The cell measurements and the fluorescein measurements were repeated, but now the gain settings were fixed between the measurements. You can find the results on our Interlab page.

    • Wednesday July 18th

      Who: Rianne & Ingeborg

      Aim Purification of PhipZ

      E. coli containing PhipZ (with both an ampicillin and zeocin marker) was grown in LB containing ampicillin and the plasmid was extracted using a plasmid extraction kit. Four parallel cultures were used to purify PhipZ and the following concentrations were obtained (ng/µl): 381.7, 319.9, 419.2 and 389.05. This will be sufficient for the rest of our project. The plasmids were stored at -20℃.

    • Thursday July 19th

      Who:

      Aim

    • Friday July 20th

      Who:

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    • Saturday July 21st

      Who:

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    • Sunday July 22nd

      Who:

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  • Week 30

    • Monday July 23rd

      Who: Rianne

      Aim PCR on EGII and CBHI gene fragments

      Primers for the gene fragments were diluted in MQ to 100 µM (a working stock of 50 µM was also prepared). Synthesized gene fragments were diluted in MQ to 5 ng/µl. For both EGII and CBHI, three PCR reactions were performed:

      ABC
      PCR buffer (5X)101010
      Primer A (50 µM)111
      Primer B (50 µM)111
      Polymerase111
      Template (5ng/µl)111
      Q buffer10
      Mg2+2
      MQ362634

      Primer melting temperatures:

      EGII:

      • FW = 51,3
      • Rev = 49,2

      CBHI:

      • Fw = 51,3
      • Rev = 48,6

      PCR programme run in a thermocycler:

      1. 95℃ - 5 min
      2. 94℃ - 15 sec
      3. 44℃ - 1 min
      4. 72℃ - 1 min
      5. 72℃ - 10 min
      6. 4℃ on hold

      Steps 2-4 were repeated 35 times

      Who: Rianne

      Aim Count colonies & pick colonies from yeast transformation

      • Negative control
      • -Leu, -Trp: 0 colonies
      • -Trp: at least 10 colonies
      • -Leu: 0 colonies

      1882+CB1883+CB18821883CB
      10%12615521
      90%3232manymanymany

      Note: -trp plates look different than others. They are white-ish and the colonies are small.

      Two colonies of each plate were used to inoculate 5 ml of Verduyn medium with the appropriate uracil, leucin or tryptophan supplementation. The cultures were grown overnight at 30℃ and 200 rpm

    • Tuesday July 24th

      Who: Rianne

      Aim Agarose gel of 23-07-18 PCR products

      3 µl of the ladder was loaded, 12 µl of the PCR products were mixed with 3 µl of sample buffer. NC = negative control (no template added). L = ladder.

      • Upper lane: L - EGII(A)-NC(A)-EGII(B)-NC(B)-EGII(C)-EGII(C)-NC(C)-CBHII(A)-NC(A)
      • Lower lane: L- CBHII(B)-NC(B)-CBHII(C)-NC(C)-ctrl EGII-ctrl CBHII
    • Wednesday July 25th

      Who: Rianne

      Aim Sending plasmids pNZ8048 and pPMK4 to Leiden for collaboration

      A small volume of purified plasmid pNZ8048 (obtained from Alisa Garaeva) and PMK4 (obtained from Buu Minh Tran) were transferred to screw-capped microtubes, and, accompanied by a dried drop on Whatman filter paper, sent to Leiden by mail.

      The plasmids arrived in Leiden soon!

      The Leiden Igem team with the eppendorf tubes containing the plasmids.

    • Thursday July 26th

      Who: Rianne & Bram

      Aim PCR of EGII

      Because two bands appeared in the previous PCR reaction, we want to change the reaction conditions to see if we can obtain a single product with the right length. Two conditions were tested. The temperature was increased to prevent non-specific binding and DMSO was added.

      AB
      PCR buffer (5X)1010
      Primer A (50 µM)11
      Primer B (50 µM)11
      Polymerase11
      Template (5ng/µl)22
      DMSO3
      MQ3532

      Primer melting temperatures:

      EGII

      • FW = 51,3
      • Rev = 49,2

      PCR programme run in a thermocycler:

      1. 95℃ - 5 min
      2. 94℃ - 15 sec
      3. 46℃ - 1 min
      4. 72℃ - 1 min
      5. 72℃ - 10 min
      6. 4℃ on hold

      Steps 2-4 were repeated 35 times

      Ladder - EGII A - EGII B

      Gel was kept in the fridge overnight before use, which is probably the reason why the bands are not as clear as desired

    • Friday July 27th

      Who:

      Aim

    • Saturday July 28th

      Who:

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    • Sunday July 29th

      Who:

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  • Week 31

    • Monday July 30th

      Who:

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    • Tuesday July 31st

      Who:

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    • Wednesday August 1st

      Who:

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    • Thursday August 2nd

      Who:

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    • Friday August 3rd

      Who:

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    • Saturday August 4th

      Who:

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    • Sunday August 5th

      Who:

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  • Week 32

    • Monday August 6th

      Who: Rianne

      Aim Restrictions of cellulosome PCR products or gene fragments

      Restriction reactions were performed in a 50 µl total reaction volume. (1µl of each restriction enzyme, 5 µl of 10X restriction buffer, 1 µg of DNA). For all reactions, NEB buffer 3.1 was used with enzymes BamH1 and Xho1.

      Concentration stock (ng/µl)For ~1 µg (µl)MQ
      CBHI225,5538
      EGII (PCR with DMSO, 26-7)1457,535,5
      EGII (no DMSO, 26-7)1507,535,5
      PhipZ300439

      Reactions were incubated at 37℃ for 1,5 hours.

      • PCR clean-up gave the following concentrations of restricted fragments:
      • -CBHI31,35
      • -EGII (DMSO)27,05
      • -EGII 28,35
      • -PhipZ32,55

      The restricted clean fragments were stored at -20℃ until further use. The genes Scaffold part 1 and Scaffold part 2 were diluted to 5 ng/µl by addition of 200 µl MQ to the delivered dried genes. One third (~300 ng in 65,5 µl) was used for a 75 µl restriction reaction with the following additions:

      Enzyme 1 (1 µl)Enzyme 2 (1 µl)Buffer (7,5 µl)
      Scaffold part 1BamH1Cla1NEB 3
      Scaffold part 2Xho1Cla1Cutsmart

      The mixtures were incubated for 2,5 hrs at 37℃ and then kept at 4℃ overnight. The genes BGL part 1 and BGL part 2 were diluted to 10 ng/µl by addition of 100 µl MQ to the delivered dried genes. One third (~300 ng in 30 µl) was used for a 50 µl restriction reaction with the following additions:

      Enzyme 1 (1 µl)Enzyme 2 (1 µl)Buffer (5 µl)
      BGL part 1BamH1Nru1NEB 3
      BGL part 2Xho1Nru1NEB 3

      The mixtures were filled up to 50 µl by addition of 13 µl MQ and incubated for 1,5 hrs at 37℃ and then kept at 4℃ overnight.

    • Tuesday August 7th

      Who: Rianne

      Aim Restriction cleanup and ligation

      PCR cleanup of the previous restriction reactions gave the following concentrations:

      • -Scaffold part 13,2 ng/µl
      • -Scaffold part 24,8 ng/µl
      • -BGL part 12,55 ng/µl
      • -BGL part 22,9 ng/µl

      For a vector:insert(:insert) equimolar ratio of around 1:3(:3), the following ligation mixtures were prepared:

      A, B: 100 ng plasmid. C, D: 50 ng plasmid reactions.

      A (µl)B (µl)C (µl)D (µl)Control
      PhipZ3.13.11,541,543,1
      CBH3
      EGII3.12
      BGLI part 113.2
      BGLI part 216.1
      Scaffold part 111
      Scaffold part 27
      T4 ligase11221
      Ligase buffer113.52.51
      MQ1.91.7814.9
      Total volume1010352510

      The ligation mixtures were incubated for 2 hours at 37℃, following 20 minutes at 80℃ to heat-kill the enzymes.

      Who: Rianne

      Aim Transformation

      Ligated amountInto X competent cells (µl)
      Aall75
      Ball75
      C35200
      D25150
      Vall75

      Following the same protocol as previously used. Cells were plated on LB-ampicillin plates (100µl, 250µl and rest) and incubated at 37℃ overnight.

    • Wednesday August 8th

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      Aim

    • Thursday August 9th

      Who:

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    • Friday August 10th

      Who: Rianne

      Aim Grow the cellulosome-strains on cotton

      Common cotton pads were purchased from a local supermarket and 20 mg cotton was weighed and transferred into glass tubes. The tubes were subsequently autoclaved.

      Glass tubes with 2%, 0,2%, 0,02%, 0,002% and 0,0002% galactose were prepared. Both with and without the cotton. 10 ml of Verduyn medium (supplemented with the appropriate vitamins, trace elements and with uracil) was added to each tube. As a positive control, LB and E. coli bacteria were added to a glass tube containing cotton, to exclude non-growth because of chemicals in the cotton pads. Also, Verduyn medium containing 2% glucose was added to one of the glass tubes with cotton, to ensure viability of the yeast strains.

      Colonies of CB1883 and CB1882 were taken from the plate and inoculated into the tubes. The cultures were incubated at 30℃, 150 rpm.

      Results Growth in both positive controls, no growth in the remaining tubes.

    • Saturday August 11th

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    • Sunday August 12th

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  • Week 33

    • Monday August 13th

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    • Tuesday August 14th

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    • Wednesday August 15th

      Who:

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    • Thursday August 16th

      Who:

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    • Friday August 17th

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    • Saturday August 18th

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    • Sunday August 19th

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  • Week 34

    • Monday August 20th

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    • Tuesday August 21st

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    • Wednesday August 22nd

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    • Thursday August 23rd

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    • Friday August 24th

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    • Saturday August 25th

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    • Sunday August 26th

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  • Week 35

    • Monday August 27th

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    • Tuesday August 28th

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    • Wednesday August 29th

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    • Thursday August 30th

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    • Friday August 31st

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    • Saturday September 1st

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    • Sunday September 2nd

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  • Week 36

    • Monday September 3rd

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    • Tuesday September 4th

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    • Wednesday September 5th

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    • Thursday September 6th

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    • Friday September 7th

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    • Saturday September 8th

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    • Sunday September 9th

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  • Week 37

    • Monday September 10th

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    • Tuesday September 11th

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    • Wednesday September 12th

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    • Thursday September 13th

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    • Friday September 14th

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    • Saturday September 15th

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    • Sunday September 16th

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  • Week 38

    • Monday September 17th

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    • Tuesday September 18th

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    • Wednesday September 19th

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    • Thursday September 20th

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    • Friday September 21st

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    • Saturday September 22nd

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    • Sunday September 23rd

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  • Week 39

    • Monday September 24th

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    • Tuesday September 25th

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    • Wednesday September 26th

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    • Thursday September 27th

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    • Friday September 28th

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    • Saturday September 29th

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    • Sunday September 30th

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  • Week 40

    • Monday Oktober 1st

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    • Tuesday Oktober 2nd

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    • Wednesday Oktober 3rd

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    • Thursday Oktober 4th

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    • Friday Oktober 5th

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    • Saturday Oktober 6th

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    • Sunday Oktober 7th

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  • Week 41

    • Monday Oktober 8th

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    • Tuesday Oktober 9th

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    • Wednesday Oktober 10th

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    • Thursday Oktober 11th

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    • Friday Oktober 12th

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    • Saturday Oktober 13th

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    • Sunday Oktober 15th

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