Team:NTNU Trondheim/Experiments
Lab Journal
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Week25
Getting started
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Jun20
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.
LA- medium (1.5%) were made, and autoclaved for 20min at 121°C.
Two different agar plates were made – one type contains ampicillin (AMP) while the other type contains chloramphenicol (CM).
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 -
Jun21
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.
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Jun22
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:
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.
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Jun25
Transformation of pgRNA and pdCas9 into competent cells
Goal: Transform pgRNA and pdCas9 into 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.
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Jun26
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:
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Jun27
Isolation of pgRNA and pdCas9 from compentent 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: -
Jun28
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:- Add cell culture (500μL) to glycerol stock (500μL, 50%) in cryo tubes.
- 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). -
Jun29
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)
- Forward anti-luxS primer (6μL) and reverse anti-luxS primer (6μL) were added to a PCR tube
- pgRNA (2) (0.5μL, 44.2ng/μL) was added to the PCR tube
- Takara pre-mix (2x6.5μL) was added to the solution
- 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:
Week27Week 27
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Jul2
Separation, isolation and transformation of linear DNA (pgRNA with anti-luxS)
Goal:
- Separate the PCR product from circular DNA
- Isolate the linear DNA (pgRNA with anti-luxS from the gel
- Transform the linear DNA into competent cells
- 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:- Add 3 volumes of ADB buffer to each volume of agarose excised from gel.
- Incubate the sample for 5-10min at 50℃.
- 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).
- Add DNA wash buffer (200μL). Centrifuge for 30s at 16000g (the DNA in the column was washed twice by repeating this step)
- Discard the flow though
- 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).
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:
- Thaw the competent cells on ice (10min), and pre-chill the eppendorf tubes.
- Resuspend the DNA in the wells (provided by iGEM HQ) with d H2O (10μL).
- Add competent cells (50μL) to the eppendorf tubes.
- Add resuspended DNA (1μL) to the Eppendorf tubes.
- Incubate on ice for 25min
- Heat shock the cells for 45s at 42℃ in water bath
- Incubate on ice for 5min.
- Add LB-medium (950μL) to the tubes
- Incubate for 1h at 37℃ with shacker
- Pipette each sample (100μL) to agar plate with CM
- Centrifuge the samples for 3min at 6800g, discard supernatant (800μL) and resuspend the pellet with the remaining supernatant
- Pipette each sample (100μL) to agar plate with CM
- 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. -
Jul3
Inoculation of successful transformed competent cells and InterLab study
Goal:
- Inoculate colonies of successful transformed competent cells
- 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: -
Jul4
Verification of successful inserted anti-luxS in pgRNA
Goal:
- InterLab - transformation of plasmids into competent cells
- 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.
Plasmid isolation: Table 2: Concentration and the relative purity of isolated pgRNA(2) from the two cell cultures.Culture OD600 pR2 (1) 0.954 pR2 (2) 0.998 Concentration (ng/μL) 260nm/280nm 260nm/230nm pR2(1) 37.2 2.06 4.29 pR2(2) 123.3 1.77 1.01 -
Jul5
Double transformation and InterLab study - Inoculation
Goal:
- Transform both pdCas9 and pgRNA with anti-luxS into competent cells
- 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:
- Thaw competent cells on ice.
- Add pdCas9(2) (5μL) and pgRNA(2) with anti-luxS (5μL) to the same tube containing competent cells.
- Incubate the cells on ice for 20min.
- Heat shock the cells for 45s at 42℃.
- Incubate the cells on ice for 2min.
- Add LB- medium (900μL).
- Incubate the cells for 1.5h (max. 2h) at 37℃ with shaker.
- Plate the cells (100μL) on agar with AMP and CM (make minimum 5 plates)
- Centrifuge the cell culture for 3min at 6800g.
- Discard the supernatant (200μL)
- Resuspend the pellet with the remining supernatant in the tube
- Plate the cells (100μL) on agar with AMP and CM.
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:
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Jul6
Inoculation of successful double transformed cells, interLab study - Cell measurement
Goal:
- Inoculate successful double transformed cells (probably carrying pdCas9 and pgRNA with anti-luxS)
- InterLab study - measure absorbance and the fluorescence of each cell cultures
Procedure:
Inoculation of successful double transformed cells
- 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).
- 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:
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Jul7
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
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:
Restriction digest and gel electrophoresis:Sample Concentration [ng/μL] 260nm/280nm 260nm/230nm 1 219.9 2.01 1.82 2 35.3 2.03 3.86
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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Jul9
Monday
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Jul10
Tuesday
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Jul11
Wednesday
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Jul12
InterLab Study: Revive bacteria glycerol stock
Goal : InterLab Study: Revive bacteria glycerol stock
Procedure: The frozen bacteria glycerol stock made from day 13- 06. July. 2018, were thawed on ice. Each culture were inoculated in LB medium with CM (5mL), and incubated over night at 37C. -
Jul13
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:
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)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 -
Jul14
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
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Jul16
Transformation of the RFP-biobrick (test)
Goal:
1. Transform the RFP-biobrick (BBa_J04450) into competent DH5α-cells
Procedure :
1. Thaw competent cells on ice for approximately 10 minutes
2. Take 10 µL distilled water and resuspend the biobrick in the plate (position 23O, plate 7)
3. Add 2µL of the biobrick-solution to the tube containing competent cells. Pipette up and down while stirring carefully with the tip.
4. Incubate the cells on ice for 20min.
5. Heat shock the cells for 45s at 42℃.
6. Incubate the cells on ice for 3 min.
7. Add LB- medium (900μL).
8. Incubate the cells for 1 h at 37℃ with shaker.
9. Plate the cells (100μL) on agar with CM (1 plate)
10. Centrifuge the cell culture for 3 min at 6800g.
11. Discard the supernatant (200μL)
12. Resuspend the pellet with the remining supernatant in the tube
13. Plate the cells (100μL) on agar with CM (1 plate).
14. Add LB-medium to the DH5α-cells that doesn´t contain CM resistance, and plate the negative control on agar (1 plate)
15. Repeat 10, 11 and 12 for the negative control and plate the cells on agar (1 plate)
Steps 10–12 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. The total amount of plates are 4
Results:
There was only 1 colony on each plate -
Jul17
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, crossing lines was made with the bacteria on new plates.
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 4 plates because of the lack of growth on the plates (see 16.07.18).
Results:
There were more colonies on the 4 new plates, and they were light red as well (About 20 colonies). The new negative control plates had colonies as well.
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Jul18
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.
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Jul19
Biobrick: Preparations, Fluoresence microscopy
Goal:
Prepare for measurements tomorrow.
Learn about fluorescence microscopy
Procedure:
Preparations:
Made agar-plates from one litre LA+CM solution in sterile cabinets. Diluted cultures of the biobrick 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 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 J004450 was very bright. We used excitation 561 nm and emission ca 625-680 as the biobrick was too bright at emission 607. Consider using another laser further away from excitation point (584nm) for the real photos of the biobrick, and the improved biobrick.
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Jul20
Biobrick: Red Fluorescent Protein measurement (FAILED)
Goal:
Measure the expression of the 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:
4. Temperature: 37,0°C, varying from 36,5 °C to 37,5 °C
5. Kinetic cycles: 750
6. Gain: 40
7. Z position: 18055 µM
8. Shaking: 300 s
9. 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.
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Jul23
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 10mL Psi- medium. Inoculated culture at the end of the day and left the flask for incubating while shaking and at 37degrees overnight.
Psi media:
- Tryptone 20 g
- Yeast extract: 5g
- MgCl2 : 5g
Adjust to pH=7.6 with KOH. Add water to 1L and autoclave.
TFB1:
Add the following to a 250mL flask:
- Potassium acetat : 0.588g
- Rubidium chloride: 2.42g
- Manganese chloride: 2.00g
- Glycerol: 30mL
Fill up with MiliQ to a volume of 180mL Adjust pH to 5.8 with a pH-meter by adding diluted acetic acid (0.2M) Fill up with MiliQ to a total volume of 200mL
TFB2:
Add the following to a 250mL flask:
- MOPS: 0.21g
- Rubidium chloride: 0.121g
- Calcium chloride: 1.1g
- Glycerol: 15mL
Fill up with MiliQ to a total volume of 90mL Adjust pH to 6.5 with a pH-meter by adding diluted NaOH (0.2M) Fill up with MiliQ to a total volume of 100mL
Results:
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Jul24
Make supercompetent cells of the TG1, day 2
DAY 2:
1. Transferred 1mL culture to a new shaking flask with 100mL Psi-medium. Grew in shaking incubator until OD(600) reached 0.4
2. Placed the culture on ice for 15 minutes, along with TFB1 and TFB2
3. Centrifuged the culture for 5 minutes at 4000 rpm at 4 degree Celcius. Poured of the supernatant.
4. Resuspended the pellet carefully in 40mL TFB1. Let sit on ice for 5 min.
5. Centrifuged for 5 minutes at 4000rpm at 4degree celcius. Poured of supernatant and resuspended pellet carefully in 3 mL TFB2.
6. Marked ca 20 2 mL cryotubes at aliquoted 100 mL of resuspended cells in each tube. Left all on ice until finished aliquoting.
7. Flash freeze for 5-10 secs in liquid nitrogen. Then stored at -80 degree celcius.
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Jul25
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 plasmid was isolated and extracted during the following miniprep protocol:
ZR plasmid miniprep kit:
1.1mL of each cell culture (which carry both pgRNA and 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.
Transformation into TG1-cells:
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 (CM and AMP).
7. Incubate the cells at 37℃.
Results: -
Jul26
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, see protocol.
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Jul27
Make supercompetent cells of TG1, second attempt
Goal:
1. Make supercompetent cells of TG1 by following an improved protocol.
Week31Week 31
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Jul30
Miniprep and transformation of TG1-cells
Goal:
1. Transformation of TG1-cells with DNA that includes pdCas9 and pgRNA with antiluxS-gene.
Procedure:
pgRNA with antiluxS and pdCas9 has already been transformed into E.coli DH5α. The plasmid was isolated and extracted during the following miniprep protocol:
ZR plasmid miniprep kit:
1.1mL of each cell culture (which carry both pgRNA and 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.
Transformation into TG1-cells:
1. Thaw competent cells on ice for 10min.
2. Use a pipette and transfer 5μL 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 (CM and AMP).
7. Incubate the cells at 37℃.
Results: -
Jul31
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