Lab Journal

• Week

  25

  Getting started

  • Jun

    20

    Our first day in the laboratory!

    Goal: Incubate the Escherichia coli (E. coli) samples which carry pgRNA and pdCas9 on agar plates with antibiotics.
    
    Preparation of LA- medium
    LA- medium (1.5%) were made after the following recipe, and autoclaved for 20min at 121°C.

    • 1L Distilled water
    • 5g Yeast extract
    • 10g Tryptone
    • 5g NaCl
    • 15g Agar

    
    Agar plates with antibiotics
    Two different agar plates were made – one type contains ampicillin (AMP) while the other type contains chloramphenicol (CM), and were prepared after the following recipe:
    
    LA- medium with AMP:

    • 0.5L LA-medium
    • 0.5mL AMP (50mg/mL)

    LA-medium with CM:

    • 0.5L LA-medium
    • 0.5mL CM (35mg/mL)

    
    The LA-media with antibiotic were poured on petri dishes.
    
    Incubation of bacteria
    Two Escherichia coli (E. coli) samples which carry pgRNA and pdCas9 plasmids respectively, were ordered from Addgene. E.coli which carry the pgRNA plasmid were plated on agar plates with AMP, while E.coli which carry the pdCas9 plasmid were plated on two agar plates with CM, and incubated at 37℃.
    
    Results:
    The E.coli with pgRNA and pdCas9 formed colonies on the agar plates with AMP and CM
  • Jun

    21

    Inoculation of E.coli

    Goal: Inoculate a colony from each agar plate which were prepared from the previous day, in LB- medium with antibiotics.
    
    Procedure
    One colony of pgRNA- and pdCas9- bacteria grown overnight on agar plates were picked and inoculated in LB-media (25mL) with AMP (25μL) and CM (25μL), respectively. The cell cultures were incubated at 37℃ in a shaking incubator at 204rpm.
    
  • 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 protocol of ZR Plasmid Miniprep Kit:
    

    1. 1mL of each cell culture (which either carry pgRNA or pdCas9) were transferred to eppendorf tube and centrifugated for 20s at 16000g
    2. The supernatant was discarded, while the pellet was resuspended in buffer
    3. Lysing buffer was added to the sample and carefully mixed in 2min
    4. Neutralizing buffer was added to the sample, and mixed until the colour of the solution became yellow
    5. The solution was centrifugated for 2min at 16000g
    6. The supernatant was collected and transferred to column in a collection tube, and centrifugated for 30s at 16000g
    7. The supernatant in the collection tube was discarded
    8. Endo-wash buffer was added to the column and centrifugated for 1min at 16000g
    9. Plasmid- wash buffer was added to the column and centrifugated for 1 min at 16000g
    10. The solution in the collection tube was discarded, while the column with plasmids was transferred to a new collection tube
    11. The plasmids in the column were eluted by adding 30μL distilled water, and centrifugated for 30s in 16000g

    
    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 whinto competent cells (E.coli K-12 DH5α).
    
    Procedure:
    pgRNA and pdCas9 which were isolated from day 3 (22. June. 2018) were transformed into E.coli K-12 DH5α cells after the following transformation protocol:

    1. Thaw competent cells on ice for 10min.
    2. Use a pipette and transfer 2μL of mini-prepped plasmids into DH5α samples. Pipette up and down while stirring the pipette tip gently for 5-10s.
    3. Incubate the cells on ice for 20min.
    4. Heat shock the cells for 45s at 42℃. Immediately put the samples on ice for 2min.
    5. Add 900μL LB medium to the cells. Incubate at 37℃, 200RPM for 1h.
    6. Plate out 100μL of the transformation medium into agar plates with selection agent (in our case, we chose chloramphenicol).
    7. Incubate the cells at 37℃.

    
    Procedure:
    Sett inn bilder.
  • Jun

    26

    Transfer successful transformed cells to LB-medium and preparation of standard curves for 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 were 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.
    
    Preperation of 1xPBS splution:
    1xPBS solution for the interLab study was made after the following recipe:

    • 1L Distilled water
    • 8g NaCl
    • 0.2g KCl
    • 1.42g Na2HPO4
    • 0.24g KH2PO4

    
    Prepareation of agar plates, LA- and LB-medium:
    LA- and LB- medium was made after the protocols from day 1 (20. June. 2018).
    Agar plates with CM were prepared after the protocol from day 1(20. June. 2018).
    
    Preparation of standard curves for interLab study:
    Calibration 1: OD600 reference point – LUDOX protocol
    A conversion factor to transform absorbance data from plate reader into OD600 was obtained in spectrometer by measuring 4 replicates of LUDOX CL-X and dd H2O. The data were imported into Excel sheet.
    Calibration 2: Particle standard curve – Microsphere protocol
    Solution of silica beads provided in kit from iGEM HQ, was vortexed before dilution with water (96μL Silica beads in 904μL dd H2O). A serial dilution of microspheres was obtained by adding 100μL dd H2O into 4x11 wells (E2, F2, G2, H2…E12, F12, G12, H12) on a 96 well plate. Microsphere stock solution (200μL) was pipetted into E1 and 100μL of the solution was transferred to E2 and mixed well before transferring 100μL to E3. The procedure repeated until 100μL solution was pipetted into E11, and 100μL 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 copentent cells (DH5alpha) and preparation of stock solution for calibration 3 for interLab study

    Goal:
    Isolate plasmids from competent cells (DH5α), and prepare the fluorecin stock solution for interLab study.
    
    Procedure:
    Dilution of cell cultures:
    Each cell cultures (successful transformed competent cells from day 5 – 26.06.2018) (2.5mL) were transferred into fresh LB-medium (22.5mL) with CM (22.5μL). The new cultures were incubated for about 2 hours at 37℃ with shaker.
    
    Measurement of the cell density:
    The cell density of the new cultures was measured at OD600, 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 (5μL) were digested by mixing with 10xbuffer (2μL), distilled water (12.5μL) and restriction enzyme BspHI (0.5μL). 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 (see the protocols from lab 3 – 22. June. 2018).
    
    Bacteria glycerol stocks:
    The cell cultures made from 27. June were stored in glycerol stock after following protocol:

    1. Add cell culture (500μL) to glycerol stock (500μL, 50%) in cryo tubes.
    2. Freeze the samples at -80℃.

    
    The glycerol stock was prepared after following recipe:

    • 25mL Glycerol (99.56%)
    • 25mL Distilled water

    
    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 1.5kb and 1.0kb were found from the fragments of pgRNA after digestion by BspHI. The fragment with 100bp 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 2,7kb and 4kb 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 ant-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:
    Reverse anti-luxS primer (26.2nmol) and forward anti-luxS primer (30nmol) was suspended with 261μL and 299μL water, respectively, to obtain 100μM solutions. Each primer (10μL) were diluted with water (990μL) to obtain a final concentration of 1μM.
    
    The 20bp of anti-luxS gene was inserted into pgRNA (2) via PCR after the following protocol:
    

    • 12.5μL Takara high five pre-mix (do not contain primers or templates)
    • 7.5pmol Primer (forward (FW) and reverse (RV) anti-luxS primers)
    • >100ng Template (we use >50ng)

    
    

    1. Forward anti-luxS primer (6μL) and reverse anti-luxS primer (6μL) were added to a PCR tube
    2. pgRNA (2) (0.5μL, 44.2ng/μL) was added to the PCR tube
    3. Takara pre-mix (2x6.5μL) was added to the solution
    4. The sample was amplified by PCR, and stored in freezer

    
    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, 2.5μM], 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 (25μL) mixed with loading dye (5μl) on gel electrophoresis (gelGreen) simultaneously with a ladder (1μL gene ruler + 1μL loading dye + 4μL dH2O) for 50min at 90V. The sample and the ladder were prepared after the same protocol from day 3 – 22. June. 2018.
    
    Gel digestion and DNA isolation:
    The agarose was digested and the DNA was isolated from the sample after the following protocol:

    1. Add 3 volumes of ADB buffer to each volume of agarose excised from gel.
    2. Incubate the sample for 5-10min at 50℃.
    3. Transfer the melted sample to column with collection tube. Centrifuge for 60s at 16000g. Discard the flow-through (repeat this step until all solution of the sample has been transferred to the column).
    4. Add DNA wash buffer (200μL). Centrifuge for 30s at 16000g (the DNA in the column was washed twice by repeating this step)
    5. Discard the flow though
    6. Add d H­2O (20μL) directly to the column matrix. Centrifuge for 60s at 16000g. (The collected flow- through was transferred back to the column matrix, and centrifuged and collected in Eppendorf tube).

    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 (5μL) was transformed to competent cells after following the transformation protocol from day 4 – 25. June. 2018).
    
    InterLab – transformation of plasmids to competent cells:
    The plasmids listed on the protocol, was transformed to competent cells (DH5α) after the following protocol:
    

    1. Thaw the competent cells on ice (10min), and pre-chill the eppendorf tubes.
    2. Resuspend the DNA in the wells (provided by iGEM HQ) with d H2O (10μL).
    3. Add competent cells (50μL) to the eppendorf tubes.
    4. Add resuspended DNA (1μL) to the Eppendorf tubes.
    5. Incubate on ice for 25min
    6. Heat shock the cells for 45s at 42℃ in water bath
    7. Incubate on ice for 5min.
    8. Add LB-medium (950μL) to the tubes
    9. Incubate for 1h at 37℃ with shacker
    10. Pipette each sample (100μL) to agar plate with CM
    11. Centrifuge the samples for 3min at 6800g, discard supernatant (800μL) and resuspend the pellet with the remaining supernatant
    12. Pipette each sample (100μL) 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 2kb. 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]	260nm/280nm	260nm/230nm
    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 (6μL) were transformed to the competent cells and plated on agar based on the same protocol from day 4 – 24. June. 2018. During the incubation at 37℃ with shaker, the incubation time was extended to 2h. The cell cultures were centrifuged, and 700μL 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 (1.5μL) from the right wells were transformed to competent cells after 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 the OD600.
    
    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 490bp and 2094bp.
    
    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 1h incubation at 37℃ with shaker.

    Culture	OD600
    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)	260nm/280nm	260nm/230nm
    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) after the following protocol:
    

    1. Thaw competent cells on ice.
    2. Add pdCas9(2) (5μL) and pgRNA(2) with anti-luxS (5μL) to the same tube containing competent cells.
    3. Incubate the cells on ice for 20min.
    4. Heat shock the cells for 45s at 42℃.
    5. Incubate the cells on ice for 2min.
    6. Add LB- medium (900μL).
    7. Incubate the cells for 1.5h (max. 2h) at 37℃ with shaker.
    8. Plate the cells (100μL) on agar with AMP and CM (make minimum 5 plates)
    9. Centrifuge the cell culture for 3min at 6800g.
    10. Discard the supernatant (200μL)
    11. Resuspend the pellet with the remining supernatant in the tube
    12. Plate the cells (100μL) on agar with AMP and CM.

    Steps 9 – 12 are optional. The purpose of the last four steps are to increase the cell concentration plated on agar, and probably increase the possibility of colony formation.
    
    InterLab – Inoculation of colonies
    A colony from each agar plate was inoculated in LB-medium (5mL) with CM (5μL). 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 (20mL) with AMP (25μL) and CM (25μL).
    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 OD600 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, after the following protocol:
    
    The other protocol:

    • 7.1μL Plasmids
    • 0.5μL PstI
    • 0.5μL BamHI-HF
    • 2μL NEBuffer 1.1
    • 10.4μL ddH2O

    The samples were incubated for 1.5h 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 after the protocol from day 3 – 22. June. 2018.
    
    

    Results:

    Plasmid verification:
    Plasmid isolation:
    

    Sample	Concentration [ng/μL]	260nm/280nm	260nm/230nm
    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.
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