Difference between revisions of "Team:NTNU Trondheim/Experiments"

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<li> Dillute the overnight cultures for biofilm assay. </li>  
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<p> Dillute the overnight cultures for biofilm assay. </p>  
 
<li> Do Crystal Violet assay after 3 h, 5 h and 8 h of incubation of a 96-well plate. </li>
 
<li> Do Crystal Violet assay after 3 h, 5 h and 8 h of incubation of a 96-well plate. </li>
 
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Revision as of 16:09, 3 September 2018

In our lab journal we have thoroughly documented what we have done in the laboratory, what our goals each session has been, how we have worked and results from each session. If you want a closer look on our methods/receipies, take a look at our protocol.




  • Week
    25

    Week 25

    • Jun
      20

      Our first day in the laboratory!


      Goal: Incubate the Escherichia coli (E. coli) samples which carry pgRNA and pdCas9 on agar plates separately with antibiotics.

      1. LA- medium (1.5%) was made.
      2. Two different agar plates were made – one containing ampicillin (AMP) and one containing chloramphenicol (CM).

      Incubation of bacteria
      Two Escherichia coli (E. coli) samples which carry pgRNA and pdCas9 plasmids respectively, were ordered from Addgene. E.coli carrying the pgRNA plasmid were plated on agar plates with AMP, while E.coli carrying the pdCas9 plasmid were plated on agar plates with CM. Incubation for 24 h at 37℃.

      Results:
      There was growth and single colonies on all the plates.
    • Jun
      21

      Inoculation of E.coli

      Goal: Inoculate a colony from each agar plate in LB- medium with antibiotics.

      Procedure
      One colony of pgRNA- and pdCas9- bacteria were picked and inoculated in LB-media (25 mL) with 25 µL of the associated antibiotic (AMP or CM). The cell cultures were incubated at 37℃ in a shaking incubator at 204 rpm.
    • Jun
      22

      Isolation and verification of pgRNA and pdCas9

      Goal: Isolate and verify that the E.coli carry pgRNA and pdCas9.

      Plasmid isolation
      pgRNA and pdCas9 were isolated from the incubated bacteria prepared yesterday (June 21) by following the miniprep protocol.
      Determine plasmid concentration and purity
      The concentration of plasmids and the purity of each sample were determined by using Nanodrop.

      Restriction digest and plasmid verification
      The presence of pgRNA and pdCas9 in the cell cultures were verified after following the restriction digest protocol and separation of the DNA fragments by gel electrophoresis.
  • Week
    26

    Week 26

    • Jun
      25

      Transformation of pgRNA and pdCas9 into competent cells

      Goal: Transform pgRNA and pdCas9 into competent cells (E.coli K-12 DH5α).

      Procedure:
      The extracted pgRNA and pdCas9 plasmid DNA were transformed into E.coli K-12 DH5α cells.
    • Jun
      26

      Transfer successful transformed cells to LB-medium and preparation of standard curves for the InterLab study

      Goal:
      Inoculate successful transformed competent cells to LB-medium, and prepare the standard curves for InterLab study.

      Procedure:
      Inoculate colony from agar plates to LB-medium:
      A colony from each agar plate was selected and inoculated to LB- medium with antibiotics. Competent cells with pgRNA were transferred to LB- medium with AMP, while competent cells with pdCas9 were inoculated into LB- medium with CM. The cell cultures were incubated at 37℃ with shaker.

      Preparation of 1xPBS solution for the InterLab-study.
      Preparation of agar plates with CM, LA- and LB-medium.
      Preparation of standard curves for InterLab study:

      Calibration 1: OD 600 reference point – LUDOX protocol
      A conversion factor to transform absorbance data from plate reader into OD600 was obtained in spectrometer by measuring four replicates of LUDOX CL-X and ddH2O. The data were imported into Excel sheet.

      Calibration 2: Particle standard curve – Microsphere protocol
      The solution of silica beads provided in the kit from iGEM HQ, was vortexed before dilution with water (96 μL Silica beads in 904 μL dd H2O). A serial dilution of microspheres was obtained by adding 100 μL dd H2O into 4x11 wells (E2, F2, G2, H2…E12, F12, G12, H12) on a 96 well plate. Microsphere stock solution (200 μL) was pipetted into E1 and 100 μL of the solution was transferred to E2 and mixed well before transferring 100 μL to E3. The procedure repeated until 100 μL solution was pipetted into E11, and 100 μL was transferred to liquid waste. The dilution series was repeated for row F, G and H. The absorbance of the samples was measured by a plate reader with shaker at 24℃. The data were exported to Excel sheet.

      Results:
      Sett inn bilde her.
    • Jun
      27

      Isolation of pgRNA and pdCas9 from compentent cells (DH5α) and preparation of stock solution for calibration 3 for InterLab study

      Goal:
      Isolate plasmids from competent cells (DH5α), and prepare the fluorecein stock solution for InterLab study.

      Procedure:
      Dilution of cell cultures:
      Each cell cultures (successful transformed competent cells from day 5 – 26.06.2018) (2.5 mL) were transferred into fresh LB-medium (22.5 mL) with CM (22.5 μL). The new cultures were incubated for about 2 hours at 37℃ with shaking .

      Measurement of the cell density:
      The cell density of the new cultures was measured at OD 600, after calibration with water (blank sample).

      Plasmid isolation and determination of concentration:
      The plasmids were isolated from the competent cell after the protocol of ZR plasmid miniprep kit (see lab journal from day 3 - 22. June. 2018).
      The concentration of the plasmids was determined by using Nanodrop.

      Restriction digest:
      The plasmids from each culture (5 μL) were digested by mixing with 10xbuffer (2 μL), distilled water (12.5 μL) and restriction enzyme BspHI (0.5 μL). The solution was centrifuged down before the samples were incubated over night at 37℃.

      Results:
    • Jun
      28

      Plasmid verification and storing the cell cultures

      Goal:
      Verify that the successfully transformed DH5α cells carry pgRNA or pdCas9, and store the cell cultures for future use.

      Procedure:
      Plasmid verification:
      The digested plasmid samples from yesterday’s incubation (lab 6 – 27. June.2018) were separated by gel electrophoresis.

      Bacteria glycerol stocks:
      The cell cultures made from 27. June were stored in a glycerol stock (500 µL cell culture and 500 µL 50% glycerol) in the freezer at -80℃.
      Results:
      Plasmid verification:
      L, pR1, pR2, pC1 and pC2 are the DNA-fragments from ladder solution, the two samples of pgRNA and pdCas9, respectively. From Figure 1 the two fragments with 1.5 kb and 1.0 kb were found from the fragments of pgRNA after digestion by BspHI. The fragment with 100 bp from the digested pgRNA is not visible on the gel, probably due to masking of the dye colour at the bottom of the gel. Two bands from the digested pdCas9 with 2.7 kb and 4 kb were also detected. By comparing the results og gel elecrophoresis obtained from today's experiment with the results from day 3 (22. June. 2018), one can clearly see that the bands with the expected length from successful transformed competent cells, are much clearer. Hence, this also indicate the samples from the successful transformed DH5α cells are purer, which is also true by comparing the results from Nanodrop (see day 6 - 27. June. 2018, and day 3 - 22. June. 2018).
    • Jun
      29

      Insertion of anti-luxS via PCR and fluorescence standard curve for InterLab study

      Goal:
      Insert anti-luxS in pgRNA plasmid, and generate fluorescence standard curve for the InterLab study.
      Procedure:
      Insertion of anti-luxS gene and amplification of pgRNA with PCR.

      InterLab – Calibration 3
      A fluorescence standard curve was generated from the data obtained after measuring the fluorescence of the serial dilution of fluorescein.

      The fluorescence was measured with the following settings:

      Temperature: 24℃

      Gain (manual): 56

      Wave length [nm] Band width [nm]
      Excitation 494 4
      Emission 525 20

      Results:
      InterLab – Calibration 3:
      Figure 1 shows the florescence standard curve in the concentration interval [0, 2.5 μM], with a R2 value of 0,9956. The model of the fluorescence standard curve is:
  • Week
    27

    Week 27

    • Jul
      2

      Separation, isolation and transformation of linear DNA (pgRNA with anti-luxS)

      Goal:
      1. Separate the PCR product from circular DNA
      2. Isolate the linear DNA (pgRNA with anti-luxS from the gel
      3. Transform the linear DNA into competent cells
      4. InterLab study - transformation of plasmids to competent cells

      Procedure:
      Separation of linear DNA from circular DNA:
      The leaner DNA obtained from PCR (from day 8 – 29. June. 2018), was separated from the circular DNA (original plasmids) after running the sample (25 μL) mixed with loading dye (5 μl) on gel electrophoresis (gelGreen) simultaneously with a ladder (1 μL gene ruler + 1 μL loading dye + 4 μL dH2O) for 50 min at 90 V. The sample and the ladder were prepared by the gel electrophoresis protocol.

      Gel digestion and DNA isolation:
      The agarose was digested and the DNA was isolated from the sample following the gel purification protocol.
      Determine the DNA concentration and purity:
      The concentration and the purity of the isolated DNA was determined by using Nanodrop.

      Transformation of PCR product to competent cells: The PCR product (5 μL) was transformed to competent cells after following the transformation protocol.
      InterLab – transformation of plasmids to competent cells:
      The plasmids listed on the protocol, was transformed into competent cells (DH5α) after the following protocol:
      1. Thaw the competent cells on ice (10 min), and pre-chill the eppendorf tubes.
      2. Resuspend the DNA in the wells (provided by iGEM HQ) with dH2O (10 μL).
      3. Add competent cells (50 μL) to the eppendorf tubes.
      4. Add resuspended DNA (1 μL) to the Eppendorf tubes.
      5. Incubate on ice for 25 min
      6. Heat shock the cells for 45 s at 42℃ in water bath
      7. Incubate on ice for 5 min.
      8. Add LB-medium (950μL) to the tubes
      9. Incubate for 1h at 37℃ with shacker
      10. Pipette each sample (100 μL) to agar plate with CM
      11. Centrifuge the samples for 3 min at 6800g, discard supernatant (800 μL) and resuspend the pellet with the remaining supernatant
      12. Pipette each sample (100 μL) to agar plate with CM
      13. Incubate overnight at 37℃.

      Results:
      Separation of linear DNA from circular DNA:
      Figure 1 shows the image of the gel where the linearized DNA was separated from the circular DNA. The linearized DNA is approximately 2 kb. The linearized DNA fragment was then cut out from the gel and stored in eppendorf tube.

      Note! Due to UV-light exposure, the DNA may mutate. In this case, this may affect the later steps of our project. The worse scenario, mutation of anti-luxS. Hence our CSRPRi system may not recognize luxS in the bacteria. Biofilm production will not be inhibited…

      Determine the DNA concentration and purity:

      Table 1 shows the results of the linear pgRNA (2) from PCR (probably with anti-luxS gene).

      Table 1: The concentration and the relative purity of the pgRNA (2) from PCR.
      Concentration [ng/μL] 260 nm/280 nm 260 nm/230 nm
      52.8 1.90 0.27

      From the 260/230 ratio, the sample has a relatively a small amount of nucleic acids. Probably, it is contaminated by the agarose from the gel electrophoresis. On the other hand, the 260/280 ratio is ok, indicating that the sample contains a relatively larger amount of RNA than DNA. InterLab – transformation of plasmids to competent cells:
      No visible colonies were detected on agar plates, except the positive and negative control cells.
    • Jul
      3

      Inoculation of successful transformed competent cells and InterLab study

      Goal:
      1. Inoculate colonies of successful transformed competent cells
      2. InterLab study - transformation of plasmids to competent cells

      Procedure:
      InterLab – transformation of plasmids to competent cells:
      No visible cell colonies were formed on the agar plates that were incubated on day 9 - 02. Junly. 2018. Only the positive and negative control cells formed colonies.

      Therefore, the remaining plasmids (6 μL) were transformed to the competent cells and plated on agar based on the same protocol from day 2. Jul. 2018. During the incubation at 37℃ with shaker, the incubation time was extended to 2 h. The cell cultures were centrifuged, and 700 μL supernatant was discarded, and the pellets were resuspended with the remaining supernatant. The cell cultures were then plated on agar plated with CM, and incubated overnight at 37℃.

      Inoculation of successful transformed competent cells:
      Colonies of successful transformed competent cells with pgRNA (probably containing anti-luxS) were inoculated from the agar plates (from day 9 – 02. July. 2018) to LB-medium with AMP. The cell cultures were incubated with shaker overnight at 37℃.

      Results:
    • Jul
      4

      Verification of successful inserted anti-luxS in pgRNA

      Goal:
      1. InterLab - transformation of plasmids into competent cells
      2. Verification of succsesful insertion of anti-luxS in pgRNA(2)

      Procedure:
      InterLab – Transformation of plasmids to competent cells
      There has been a mistake of numbering the plates with plasmids provided by iGEM HQ. Therefore, we did not get any cell colonies from the previous two transformations…

      The plasmids (1.5 μL) from the right wells were transformed to competent cells by following the transformation protocol from day – 02. July. 2018, after resuspension of the plasmids in the wells.

      Cell density:
      The cell cultures with pgRNA(2) (probably with anti-luxS), inoculated from day 10 – 04. July. 2018, were diluted 10 times by following the same dilution procedure from day 6 – 27. June. 2018. The cells were incubated with shaker for 1h before measuring at OD 600.

      Plasmid isolation:
      The plasmids from the two cell cultures were isolated after the same mini-prep protocol from day 3 – 22. June. 2018. The concentration and the purity of the isolated plasmids were measured by Nanodrop.

      Restriction digest and gel elecrophoresis:
      Insertion of anti-luxS gene was verified after the protocol of restriction digest and gel electrophoresis (see the protocols from day 3 – 22. June. 2018). The restriction enzyme PstI was chosen, and the expected length of the plasmid fragments are 490 bp and 2094 bp.

      Note! PstI has a cut site at the anti-luxS gene in the plasmid.

      Bacteria glycerol stock:
      Two samples of the diluted cell cultures (made at this morning), were stored in 50% glycerol solution at -80℃ (the same green box with the other successful transformed cells with pgRNA (without anti-luxS) or cells with pdCas9 plasmids).

      Results:
      InterLab - Transformation:
      Colonies were formed on agar plates with CM.

      Cell density:

      Table 1: Absorbance of the cell cultures with pgRNA(2) after 1 h incubation at 37℃ with shaker.
      Culture OD 600
      pR2 (1) 0.954
      pR2 (2) 0.998
      Plasmid isolation: Table 2: Concentration and the relative purity of isolated pgRNA(2) from the two cell cultures.
      Concentration (ng/μL) 260 nm/280 nm 260 nm/230 nm
      pR2(1) 37.2 2.06 4.29
      pR2(2) 123.3 1.77 1.01
    • Jul
      5

      Double transformation and InterLab study - Inoculation

      Goal:
      1. Transform both pdCas9 and pgRNA with anti-luxS into competent cells
      2. InterLab study - inoculate colonies in LB-medium

      Procedure:
      Double transformation of pdCas9 and pgRNA with anti-luxS:
      Both pdCas9(2) and pgRNA(2) with anti-luxS (isolated from day 11 – 05. July. 2018) were simultaneously transformed into competent cells (DH5α). The amount of added pdCas9(2) (5 μL) and pgRNA(2) with anti-luxS was 5 µL per/plasmid, and it was added to the same tube containing competent cells. The cells were plated on agar with both AMP and CM. A couple of steps were added at the end, and the purpose was to increase the cell concentration plated on agar, and probably increase the possibility of colony formation. The cell culture was therefore centrifuged for 3 minutes at 6800g, and the supernatant discarded. The pellet was then resuspended and cells were plated.

      InterLab – Inoculation of colonies
      A colony from each agar plate was inoculated in LB-medium (5 mL) with CM (5 μL). The cell cultures were incubated overnight at 37℃ with shaker.

      Results:
    • Jul
      6

      Inoculation of successful double transformed cells, interLab study - Cell measurement

      Goal:
      1. Inoculate successful double transformed cells (probably carrying pdCas9 and pgRNA with anti-luxS)
      2. InterLab study - measure absorbance and the fluorescence of each cell cultures

      Procedure:
      Inoculation of successful double transformed cells
      1. A colony from each agar plates with AMP and CM (made from day 13 – 05. July 2018) was picked and inoculated in LB medium (20 mL) with AMP (25 μL) and CM (25 μL).
      2. The cell cultures were incubated at 37℃ with shaker.

      InterLab – Cell measurement
      We were not able to measure the cell cultures today, since the TECAN device was under reparation.
      Each cell cultures were stored with 50% glycerol (see day 7 – 28. June. 2018) at -80℃.

      Results:
    • Jul
      7

      Verification of pdCas9 and pgRNA with anti-luxS after double tranformation

      Goal:

      Verify that both pdCas9 and pgRNA with anti-luxS are tranformed into DH5α cells

      Procedure:

      Dilution of cell culture:
      Each cell cultures, inoculated and incubated from day 13 – 07. July. 2018, were diluted (1mL) with LB medium with antibiotics until the OD 600 reached 1.9.

      Plasmid verification:
      Plasmid isolation:
      The plasmids were isolated from each cell cultures after following the mini-prep plasmid protocol from day 3 – 22. June. 2018. The concentration and the purity of the samples were determined by Nanodrop.

      Restriction digest and gel electrophoresis:
      The plasmids in each sample were digested with restriction enzymes BamHI-HF and PstI with the following ingredients and amounts:
      • 7.1 μL Plasmids
      • 0.5 μL PstI
      • 0.5 μL BamHI-HF
      • 2 μL NEBuffer 1.1
      • 10.4 μL ddH2O
      The samples were incubated for 1.5 h at 37℃.
      Note! NEBbuffer 1.1 was chosen instead of Cut smart since the activity of PstI in Cut smart was 50%.
      The activity of the enzymes in NEBbuffer 1.1:
      BamHI-HF: 100%
      PstI:75%
      After the plasmids have been degraded by restriction enzymes, the fragments were separated by gel electrophoresis.

      Results:

      Plasmid verification:
      Plasmid isolation:
      Sample Concentration [ng/μL] 260 nm/280 nm 260 nm/230 nm
      1 219.9 2.01 1.82
      2 35.3 2.03 3.86
      Restriction digest and gel electrophoresis:
      The bands on the gel, corresponded with the expected fragments (6705bp, 2094bp, 465bp, 25bp) from digestion of the plasmids by PstI and BamHI-HF. Therefore, we concluded that both of pdCas9 and pgRNA with anti-luxS have successfully been transformed into DH5α cells.
  • Week
    28

    Week 28

    • Jul
      9

      No lab

    • Jul
      10

      No lab

    • Jul
      11

      No lab

    • Jul
      12

      InterLab Study: Revive bacteria glycerol stock

      Goal : InterLab Study: Revive bacteria glycerol stock

      Procedure: The frozen bacteria glycerol stock made from 6th of July were thawed on ice. Each culture were inoculated in LB medium with CM (5 mL), and incubated over night at 37°C.
    • Jul
      13

      InterLab Study: CFU/mL/OD calculations

      Goal:
      Count colony forming units on plates spread the day before.

      Procedure:
      Counted CFU on all 36 plates incubated 13. July.

      Results:
      The colonies on agar plates, from yesterday’s incubation, were counted and registered:
      CFU Dilution 3 Dilution 4 Dilution 5
      1.1 195 6 1
      1.2 185 2 0
      1.3 203 15 3
      2.1 222 19 2
      2.2 38 22 1
      2.4? 52 15 1
      3.1 113 56 3
      3.2 112 12 2
      3.3 127 25 2
      4.1 79 4 0
      4.2 75 10 1
      4.3 107 3 1
      where 1 and 2 are positive controls, 3 and 4 are negative controls. (= 2.1 means the second dilution of the second positive control culture)
    • Jul
      14

      InterLab – CFU/mL/OD calculation

      Goal:
      Counting colonies on each agar plate, and calculate CFU/mL in starting samples with an OD600 = 0.1.

      Procedure:
      The colonies on the agar plates, prepared from yesterday’s incubation, were counted and registered.

      Results:

      Table 1: The average values of counted colonies from two cultures of positive and negative controls with different final dilution factor (dilution 1 = 8x104, dilution 2 = 8x105, dilution 3 = 8x106).
      Positive control Negative control
      Culture 1, dilution 1 194.3 117.3
      Culture 1, dilution 2 7.7 31.0
      Culture 1, dilution 3 1.3 2.3
      Culture 2, dilution 1 104.0 87.0
      Culture 2, dilution 2 18.7 5.7
      Culture 2, dilution 3 1.3 0.7
  • Week
    29

    Week 29

    • Jul
      16

      Transformation of the RFP-biobrick (test)

      Goal:
      Transform the RFP-biobrick (BBa_J04450) into competent DH5α-cells.

      Procedure :
      Before doing the transformation the biobrick was resuspended with 10 µL dH2O in the plate (position 23O, plate 7). The cells with and without the biobrick (negative control) were both incubated and plated after the transformation. The negative control was plated on LA-petri dishes while the transformed cells was plated on LA-petri dishes with CM.
      Results:
      There was only one colony on each plate
    • Jul
      17

      Colony picking biobrick (test) and transformation

      Goal :
      1. Get colonies of the transformed DH5α (biobrick) 2. Get colonies on the negative control plate

      Procedure:
      1. Due to the lack of colonies on the negative control plates, new plates was streaked.
      2. New LA-plates without antibiotics were made.
      3. Colonypicking the two colonies from the plates with the transformed bacteria in to liquid medium.
      4. The transformation of bacteria with the biobrick plasmid was redone on four plates because of the lack of growth on the plates (see 16.07.18).

      Results:
      There were more colonies on the four new plates, and they were light red as well (About 20 colonies). The new negative control plates had colonies as well.

    • Jul
      18

      RFP biobrick fluorescens measurement (test)

      Goal:
      1. To get a descriptive curve for the rise in fluorescens (transformed DH5α)
      2. Find out how often the measurements need to be done.

      Procedure:
      The second batch of plates from transformation and the negative control:
      1. Colonypicking and exchange to liquid medium

      Measurements of the first batch (Tecan):
      1. Measured the OD of the blank (LB and CM), and measured the OD of the two cultures.
      2. For the next measurements we diluted the cultures by 1:100 in separate tubes.
      3. Incubation of the tubes
      4. The first measurement was done in the Tecan, and repeated for every 30 minutes and then 1 hour (because of minor changes in values)
      5. The measurements was set overnight as well using the following script: FluorescenceLong. The data was set to 588 nm absorbance, 584 nm excitation, 607 nm emission, 37°C, 40 gain, 150 kinetic cycles, 300s shaking (orbital 3 mm amplitude).

      Results:
      The second batch from transformation has turned red. From the measurements of the test batch we have an idea of the increase in fluorescence intensity and the change in absorbance over time. This can be used to determine the timing of the second batch with the negative control as well.
    • Jul
      19

      Biobrick: Preparations, Fluoresence microscopy

      Goal:
      Prepare for measurements tomorrow.
      Learn about fluorescence microscopy

      Procedure:
      Preparations:
      Made LA-petri dishes with CM. Diluted cultures of the biobrick Bba_J04450 and negative control and incubated them at 37°C overnight.

      Demonstration and training in fluorescence microscopy:
      Astrid Bjørkøy showed us how to use the fluorescence microscope, and we tried it out by taking photos of cultures of the Bba_J04450 biobrick. She also told us how to count cells by adjusting a photo's threshold and calculate based on image size.

      Results:
      The biobrick Bba_J004450 was very bright. We used excitation 561 nm and emission ca 625-680 nm as the biobrick was too bright at emission 607 nm. Consider using another laser further away from excitation point (584 nm) for the real photos of the biobrick, and the improved biobrick.
    • Jul
      20

      Biobrick: Red Fluorescent Protein measurement (FAILED)

      Goal:
      Measure the expression of the Bba_J04450 biobrick in transformed dH5α cells. This is done by measuring fluorescence.

      Procedure:
      1. Incubated dH5α, J04450 transformed cells were dilluted 1:100 with LB
      2. 200 µL, 5 replicates of LB, LB with Chloramphenicol and 5 dH5α dillutions made from different batches were applied to a 96 well plate
      3. Tecan plate reader was used to measure absorbance at 588 nm and fluorescence at excitation 584 nm and emission 607 nm with the following parameters:
      • Temperature: 37,0°C, varying from 36,5°C to 37,5°C
      • Kinetic cycles: 750
      • Gain: 40
      • Z position: 18055 µM
      • Shaking: 300 s
      • Orbital shaking amplitude: 3 mm


      Results:
      The wells dried out, presumably due to us choosing not to use a lid on the plate, and the measurements were therefore aborted by the machine.
  • Week
    30

    Week 30

    • Jul
      23

      Make super competent cells of the TG1

      Goal:
      Make supercompetent cells of the E.coli - TG1 strain

      Procedure:

      DAY 1:

      - Made Psi media, transformation buffer 1 and transformation buffer 2.
      - Made an E.coli TG1 culture in a small flask with 10 mL Psi- medium. Inoculated culture at the end of the day and left the flask for incubating while shaking and at 37°C overnight.

      Results:
    • Jul
      24

      Make supercompetent cells of the TG1, day 2

      DAY 2:
      This was done from an overnight culture of TG1-cells, see protocol. Results:
      There was no growth on the plates with CM and AMP after the transformation (25. July. 2018), so the conclusion was that the cells were not supercompetent. The protocol was then updated and optimized for another trial.
    • Jul
      25

      Miniprep and transformation of TG1-cells

      Goal:
      1. Transformation of TG1-cells with DNA that includes pdCas9 and pgRNA with antiluxS-gene.
      2. Make LA-plates with both CM and AMP antibiotics

      Procedure:
      pgRNA with antiluxS and pdCas9 has already been transformed into E.coli DH5α. The plasmids were isolated and extracted by the miniprep protocol. Transformation of the TG1-cells with the extracted plasmids was done, and the bacteria were incubated.

      Results: No bacterial growth has been observed.
    • Jul
      26

      Preparations for biofilm measurements

      Goal:
      1. Make media for the biofilm measurements (LB and M63B1)
      2. Prepare TG1, TG2 buffers and Psi medium for supercompetent cells.

      Procedure:
      1. Made three versions of the two media with pH-values at 4.5, 7.2 and 9.5. The media was then autoclaved and sugar will be added before use of the media.
      2. Made TG1, TG2 buffers and Psi medium.

    • Jul
      27

      Make supercompetent cells of TG1, second attempt

      Goal:
      1. Make supercompetent cells of TG1 by following an improved protocol.
  • Week
    31

    Week 31

    • Jul
      30

      Transformation of TG1-cells and flow cytometry test

      Goal:
      1. Transformation of TG1-cells with DNA that includes pdCas9 and pgRNA with antiluxS-gene. 2. Learn and conduct a flow cytometry test of the E.Coli DH5a that contains the Bba_J04450-biobrick and a negative control. The goal was also to optimize a protocol for the biobrick measurements.

      Procedure:
      pgRNA with antiluxS and pdCas9 has already been transformed into E.coli DH5α. The plasmids were isolated and extracted by the miniprep protocol, and the cells transformed and incubated. For the transformation, 5 µL of the miniprepped plasmids were used instead of 2 µL.

      2.
      Results: Noticeable bacterial growth was observed on all plates, with no apparent contamination.
    • Jul
      31

      TG1 colonypicking


      Goal:
      1. Make cultures out of plates made on the 30th of July, prepare for test digest.
      2. Make DH5α cultures for later use in biobrick measurements.

      Procedure:
      Colonypicking into five beakers with 25 mL LB, 25uL Amp and 25 µL CM. Incubation at 37°C, 225 RPM overnight. New LB-media was made.
    • Aug
      1

      Miniprep, Nanodrop, Test digest

    • Aug
      2

      Verification of transformation of TG1 and preparation of media for biofilm measurements

      Goal:
      1. Confirm successful transformation of TG1 with pgRNA and PdCas9 with anti-luxS.
      2. Prepare media for biofilm measurements
      3. Inoculate TG1 in newly made media

      Procedure:
      Verification of the plasmids was done by miniprep, nanodrop, test digest and gel electrophoresis. Different amounts of glucose was added to the respective media.
    • Aug
      3

      Measure OD + change medium

      Goal:

  • Week
    32

    Week 32

    • Aug
      6

      E.Coli TG1 biofilm measurement

      The two E.Coli TG1 96-well plates made yesterday (06.08.2018) underwent the Crystal Violet Assay protocol (see Protocols), and the biofilm produced was measured by measuring absorbance at 590 nm with Tecan Infinite 200 Pro plate reader.
    • Aug
      7

      Incubate DH5α and TG1 in chosen media for biofilm measurements.

      Goal:

      - Incubate DH5α and TG1 (with pgRNA and pdCas) in selected media (LB and M63B1, pH 7.2, 0.4%/0.8% Glucose) - Make Tetracycline stock solution (5.0 mg/ml)

      Procedure:

      Incubated the frozen DH5α-stocks in LB (with CM and AMP) for a few hours. Inoculated into the chosen media in eppendorf tubes. Incubated TG1 into the chosen media in eppendorf tubes overnight. Made 5mg/mL Tetracyclin-solution (inducer). Note: light sensitive.

      Protocol:
      - Tetracyclin-HCl(powder): 54 mg
      - Ethanol: 7 mL
      - dH2O: 3 mL
      After being incubated for some time tetracyclin solution was added to the eppendorf tubes do induce the transcription of Cas9, and the culture tubes were further incubated.
    • Aug
      8

      Incubation of cell cultures for biofilm assay and preparation of M63B1 medium

      Goal :

      1. Incubate cell cultures on 96-well plates for biofilm assay
      2. Make M63B1 medium

      Procedure :

      Preparation for biofilm assay:
      1. Overnight cultures (with TET) was diluted until OD600 = 0.1
      2. Each diluted culture (100μL) was added on 96-well plate and incubated for 4 h at 37℃
      3. After 4 h incubation, the non-adhered cells were removed by washing the wells with physiological saline and the supernatant was discarded.
      4. New medium (100 μL) was added to each well and the plate was incubated for 24 h at 37℃.
      Note! The media that were added to the wells right before 24 h incubation did not contain TET

      M63B1 medium:
      M63B1 medium was made after following the protocol from 26. July. 2018

      Results:
    • Aug
      9

      Preparation of media with TET, and incubation of cell cultures on 96-well plate for biofilm assay

      Goal :

      1. Preparation of media with tetracycline (2 μM)
      2. Incubate cell cultures with and without tetracycline (TET) on a 96-well plate for the 24 h biofilm assay.
      3. Measure the absorbance of CV- assay with and without TET, made from yesterdays’ incubation.
      4. Prepare TG1 and dH5α cell cultures in growth medium (LB or M631B) with or without TET (2 μM).

      Procedure :

      Preparation of media with TET (2 μM):
      1. Tetracycline (TET) (50 μL, 0.5 mg/mL) was added to a 25mL LB medium with 0.4% glucose, AMP and CM. The same amount of tetracycline was added to LB medium with 0.8% glucose, AMP and CM
      2. For the final M63B1 medium (0.5 L) with MgSO4*7H2O, AMP and CM and 0.4% or 0.8% glucose, TET (1mL, 0.5 mg/mL) was added.

      Preparation of 96-wells plate for 24h biofilm assay:
      1. Overnight cultures were diluted with or without TET (2 μM) containing media (LB or M63B1) until a target OD600 = 0.1.
      2. Each diluted culture (100 μL) were incubated on a 96-wells plate for 20 h at 37℃.

      Absorbance measurement of CV-assay:
      1. The supernatant was discarded from a 24h incubation of cell cultures on a 96-wells plate (prepared from 08. August. 2018) and rinsed with PS (100μL).
      2. The biofilm was fixed by adding methanol (100 μL, 99%) and waited for 15min. The supernatant was discarded, and the plate was airdried.
      3. CV-solution (100 μL, 0.1%) was added to each well and coloured the biofilm for 20 min. The excess CV was washed away with dH2O 4. Acetic acid (150 μL, 33%) was added to the each well.
      5. The absorbance was measured at Abs 590 nm

      Results:
    • Aug
      10

      Preparation of overnight cultures for biofilm assay, and storage of TG1 cells with pgRNA and pdCas9

      Goal :

      1. Prepare overnight cultures in LB (0.4% or 0.8% glucose, with AMP and CM) or M63B1 (0.4% or 0.8% glucose, with AMP and CM) with or without TET.
      2. Wash the 96-well plate (after 20 h adhesion - prepared from 09.08.2018) with physiological saline, add new media and incubate the plate.
      3. Make bacteria glycerol stocks of TG1 cells with pgRNA and pdCas9.

      Procedure :

      Preparation of overnight culture:

      TG1 and dH5α were incubated in eppendorf tubes with different media (LB or M63B1 with glucose (0.4% or 0.8%), AMP and CM) with or without TET (2 μM) for overnight at 37℃ with shaking.

      Preparation for biofilm assay:

      1. After 20 h adhesion (plate prepared from 09. August. 2018), the non-adhered cells were removed by washing the wells with physiological saline (PS) and the supernatant was discarded.
      2. New medium (100 μL) was added to each well and the plate was incubated for 24 h at 37℃.

      Bacteria glycerol stock preparation:

      TG1 cells with pgRNA and pdCas9 (500 μL) was stored with glycerol (500 μL, 50%) at -80℃.

      Results :
    • Aug
      11

      Preparation of overnight cultures for biofilm assay

      Goal :

      Prepare 96-well for cell adhesion and CV-assay

      Procedure :

      The OD of some of the overnight cultures was not optimal for continuing the procedure of CV assay. New cell cultures were made.

      96-well plates for 24 h, 48 h and 72 h incubation were made. However, since the cell cultures could not be diluted to OD = 0.1, the results of the CV assay will not be comparable.
    • Aug
      12

      Incubation of cell cultures for biofilm assay

      Goal :

      Preparation for cell adhesion on 96-well plates for 24 h, 48 h and 72 h incubation.

      Procedure :

      1. The overnight cultures were resuspended, before dilution until a target OD = 0.1.
      2. The diluted cell cultures were resuspended and added on 96-well plates and incubated for 4 h at 37℃.
      3. After 4 h incubation, the wells were washed with physiological saline (100 μL) and the supernatant was discarded.
      4. New media (100 μL) were added to the plates, and the plates were incubated at 37℃.

      Results :
  • Week
    33

    Week 33

    • Aug
      13

      Biofilm measurements after 24hrs

      Procedure:
      Followed the crystal violet assay:

      Changed medium in the plate for 48 and 72 hrs.

      Results:
      Results did not show any clear trend. Excel sheet attached.
      Wells with only medium, not cells, showed too high absorbance measurements, probably because of contamination of the mediums. Decided to autoclave all the mediums again. Will take measurements at 48 hrs, but not at 72 hrs.
    • Aug
      14

      Biofilm measurement after 48 hrs

      Goal:
      1. Measure biofilm after 48 hrs.
      2. Prepare RRVT-Biobrick and biobrick backbone
      3. Ligate and transform RRvT- biobrick and biobrick backbone into DH5alpha cells.
      Procedure:
      1. Performed the Crystal Violet Assay procedure for the plate designated for 48hrs and for the plate that initially was designated for 72 hrs measurement.
    • Aug
      15

      Biobrick ligation and transformation

      Goal:
      1. Ligate plasmid backbone and RRvT-plasmid and transform into cells.
      2. Due to the lack of colonies from yesterdays transformation the ligation of plasmid backbone and RRvT-plasmid and transformation into E.Coli Dh5α cells was redone.
      Procedure:
          Preparations of plasmid backbone and RRvT-biobrick:

          Made enzyme master mix for plasmid backbone by adding 5μL NEB buffer 2, 0.5 μL EcoRI-HF, 0.5 μL PstI, 0.5 μL BSA and 18.5 μL dH2O to an Eppendorf tube.

          Made enzyme master mix for RRvT-biobrick by adding 5μL NEB buffer 2, 0.5μL EcoRI-HF, 0.5 μL PstI and 19 μL dH2O to an Eppendorf tube. Incubated mix for 20 min at 50℃.

          Digested Plasmid backbone by adding 4 μL linearized backbone (25 ng/μL for 100 ng) and 4 μL of enzyme master mix for plasmid backbone.

          Digested RRvT-biobrick by adding 4 μL of the biobrick and 4 μL of the enzyme master mix for RRvT-biobrick. Digested both reactions at 37℃ for 90 min and did heat kill at 80℃ for 20 min.


          Ligation:

          Added 2 μL of digested plasmid backbone.

          Added 2 μL of digested RRvT-biobrick.

          Added 1 μL T4 DNA ligase buffer.

          Added 0.5 μL of T4 DNA ligase.

          Added water to 10 μL.

          Ligated at 16℃ for30min. Did heat kill at 80℃ for 20 min.


          Transformed E.Coli Dh5α cells with 1-2 μL of product following the protocol for transformation.

          Inoculated .. plates and let them incubate at 37℃ to the next day.

      Aug
      16

      Preparation for biobrick (RRvT) measurement and biofilm assay

      Goal:
      1. Inoculate successful transformed cells into LB medium with CM.
      2. Revive TG1 and DH5α cells in LB medium with AMP, CM and 0% glucose.
      Procedure:

      Preparation for biobrick (RRvT) measurement:

      Successful transformed cell colonies from yesterday's incubation were inoculated into LB - medium with CM and incubated at 37°C with shaking.

      Preparation for cell cultures for biofilm assay:

      TG1 and DH5α cells with pgRNA and pdCas9 were revived in LB medium with AMP, CM and 0% glucose. The cell cultures were incubated at 37°C with shaking.

    • Aug
      17

      Preparation for biofilm assay

      Goal:

      Dillute the overnight cultures for biofilm assay.

    • Do Crystal Violet assay after 3 h, 5 h and 8 h of incubation of a 96-well plate.

      • Procedure:

      The media for biofilm assay were made by adding the following ingredients:

    • LB 0.4% glucose, TET: 10 mL LB, 10µL AMP, 10µL CM, 0.2 mL 20% Glucose, 19.7µL 0.5mg/mL TET
    • LB 0.8% glucose, TET: 10 mL LB, 10µL AMP, 10µL CM, 0.4 mL 20% Glucose, 20µL 0.5mg/mL TET
    • LB 0.4% glucose: 10 mL LB, 10µL AMP, 10µL CM, 0.2 mL 20% Glucose
    • LB 0.8% glucose: 10 mL LB, 10µL AMP, 10µL CM, 0.4 mL 20% Glucose
    • M63B1 0.4% glucose, TET: 10 mL M63B1, 10µL AMP, 10µL CM, 10 µL 1M MgSO4*7H2O, 0.2 mL 20% Glucose, 19.7 µL TET
    • M63B1 0.8% glucose, TET: 10 mL M63B1, 10µL AMP, 10µL CM, 10 µL 1M MgSO4*7H2O, 0.4 mL 20% Glucose, 20 µL TET
    • M63B1 0.4% glucose: 10 mL M63B1, 10µL AMP, 10µL CM, 10 µL 1M MgSO4*7H2O, 0.2 mL 20% Glucose
    • M63B1 0.8% glucose: 10 mL M63B1, 10µL AMP, 10µL CM, 10 µL 1M MgSO4*7H2O, 0.4 mL 20% Glucose

      • Biofilm assay:

    • Pipette 3-4 mL overnight cultures to sterile tubes (total 16 tubes).
    • Centrifuge the cultures for 10 min at 2000 rpm at 9°C.
    • Remove the supernatant.
    • Add 3-4 mL media (LB/ M63B1, with 0.4%/ 0.8% glucose, with/ without TET) to 2 tubes each.
    • Resuspend/ vortex the cultures, and measure OD 600.
    • Dilute the cultures until target OD 600 = 0.1 and store the cells on ice until they are added to the wells.
    • Resuspend/ vortex the cultures, add 100 µL culture to each well on the plate.
    • Seal the top of the plate with parafilm, and incubate the plate at 37°C.
    • Do CV assay after 3 h, 5 h, 8 h, 24 h and 48 h incubation.
  • Week
    34

    Week 34

    • Aug
      20

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    • Aug
      21

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    • Aug
      22

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    • Aug
      23

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    • Aug
      24

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