Team:SSHS-Shenzhen/InterLab
Monday July 23, 2018 11:30
7/23: > Calibration 1: OD600 Reference point
Monday, July 23, 2018 9:00
1) Add 100 μl LUDOX into wells A1, B1, C1, D1
2) Add 100 μl of dd H2O into wells A2, B2, C2, D2
3) Measure absorbance at 600 nm of all samples in the measurement mode we plan to use for cell measurements
4) Record the data in both the table below and our notebook
5) Import data into Excel sheet provided
Monday, July 23, 2018 9:25(pm)
Specific data from our experiment
| LUDOX CL-X | H2O | |
| Replicate 1 | 0.058 | 0.038 |
| Replicate 2 | 0.059 | 0.037 |
| Replicate 3 | 0.053 | 0.037 |
| Replicate 4 | 0.055 | 0.036 |
| Arith. Mean | 0.056 | 0.037 |
| Corrected Abs600 | 0.019 | |
| Reference OD600 | 0.063 | |
| OD600/Abs600 | 3.238 |
Note: Carefully pipet liquids into the wells of 96 well plates in order to prevent the case of liquid sticking on the wall of wells and caused inaccurate data.
7/23:> Calibration 2: Particle Standard Curve
Monday, July 23, 2018 10:30
1) Obtain the tube labeled “Silica Beads” from the InterLab test kit and vortex 4 vigorously for 30 seconds.
2) Immediately pipet 96 μL microspheres into a 1.5 mL eppendorf tube
3) Add 904 μL of ddH2O to the microspheres
Monday, July 23, 2018 10:40
1）Add 100 μl of ddH2O into wells A2, B2, C2, D2....A12, B12, C12, D12
2）Vortex the tube containing the stock solution of microspheres vigorously for 10 seconds
3）Immediately add 200 μl of microspheres stock solution into A1
4）Transfer 100 μl of microsphere stock solution from A1 into A2.
5）Mix A2 by pipetting up and down 3x and transfer 100 μl into A3…
6）Mix A3 by pipetting up and down 3x and transfer 100 μl into A4...
7）Mix A4 by pipetting up and down 3x and transfer 100 μl into A5...
8）Mix A5 by pipetting up and down 3x and transfer 100 μl into A6...
9）Mix A6 by pipetting up and down 3x and transfer 100 μl into A7...
10）Mix A7 by pipetting up and down 3x and transfer 100 μl into A8...
11）Mix A8 by pipetting up and down 3x and transfer 100 μl into A9...
12）Mix A9 by pipetting up and down 3x and transfer 100 μl into A10...
13）Mix A10 by pipetting up and down 3x and transfer 100 μl into A11...
14）Mix A11 by pipetting up and down 3x and transfer 100 μl into liquid waste
Monday, July 23, 2018 9:00(pm)
Specific data from our experiment
Note: we need to be aware of Re-mixing each of the row immediately before putting the plate into the plate reader.
7/23:>Calibration 3: Fluorescence standard curve
Monday, July 23, 2018 2:00
1) Spinning down fluorescein kit tube.
2) Prepare 10x fluorescein stock solution (100 μM) by resuspending fluorescein in 1 mL of 1xPBS.
3) Dilute the 10x fluorescein stock solution with 1xPBS to make a 1x fluorescein solution with concentration 10 μM: 100 μL of 10x fluorescein stock into 900 μL 1x PBS
Monday, July 23, 2018 2:20
1) Add 100 μl of PBS into wells A2, B2, C2, D2....A12, B12, C12, D12
2) Add 200 μl of fluorescein 1x stock solution into A1, B1, C1, D1
3) Transfer 100 μl of fluorescein stock solution from A1 into A2.
4) Mix A2 by pipetting up and down 3x and transfer 100 μl into A3…
5) Mix A3 by pipetting up and down 3x and transfer 100 μl into A4...
6) Mix A4 by pipetting up and down 3x and transfer 100 μl into A5...
7) Mix A5 by pipetting up and down 3x and transfer 100 μl into A6...
8) Mix A6 by pipetting up and down 3x and transfer 100 μl into A7...
9) Mix A7 by pipetting up and down 3x and transfer 100 μl into A8...
10) Mix A8 by pipetting up and down 3x and transfer 100 μl into A9...
11) Mix A9 by pipetting up and down 3x and transfer 100 μl into A10...
12) Mix A10 by pipetting up and down 3x and transfer 100 μl into A11...
13) Mix A11 by pipetting up and down 3x and transfer 100 μl into liquid waste
Monday, July 23, 2018 3:00
1) Repeat dilution series for rows B, C, D
2) Measure fluorescence of all samples in instrument
3) Record the data in our notebook
4) Import data into Excel sheet
Specific data from our experiment
Note: In the process of calibration, only four 96 well plates are not enough for us because we sometimes made mistakes, such us drip into the wrong well. As a result, whenever we failed to drip, we had to clean up our fruits and dry the plates to ensure they can be used again.
Also, we found that silica suspension was easy to form precipitate, which causes a series of failure of our experiment. Thus, we shock it all the time to make sure it won’t precipitate to hinder.
Cell measurement
7/20:>Preparations
Friday July 18, 2018 9:00
Transform Escherichia coli DH5α with these following plasmids (all in pSB1C3):
| Device | Part Number | Plate | Location |
| Negative control | BBa_R0040 | Kit Plate 7 | Well 2D |
| Positive control | BBa_I20270 | Kit Plate 7 | Well 2B |
| Test Device 1 | BBa_J364000 | Kit Plate 7 | Well 2F |
| Test Device 2 | BBa_J364001 | Kit Plate 7 | Well 2H |
| Test Device 3 | BBa_J364002 | Kit Plate 7 | Well 2J |
| Test Device 4 | BBa_J364007 | Kit Plate 7 | Well 2L |
| Test Device 5 | BBa_J364008 | Kit Plate 7 | Well 2N |
| Test Device 6 | BBa_J364009 | Kit Plate 7 | Well 2P |
1. Resuspend DNA in the above wells in the Distribution Kit with 10µl dH20. Pipet up and down several times, let sit for a few minutes.
2. Label 1.5ml tubes with part name. Fill lab ice bucket with ice, and pre-chill 1.5ml tubes in a floating foam tube rack.
3. Thaw competent cells on ice
4. Pipette 50µl of competent cells into 1.5ml tube.
5. Pipette 1µl of resuspended DNA into 1.5ml tube. Keep all tubes on ice.
6. Pipette 1µl of control DNA into 2ml tube.
7. Close 1.5ml tubes, incubate on ice for 30 mins.
8. Heat shock tubes at 42°C for 45 sec.
9. Incubate on ice for 5min.
10. Pipette 950µl SOC media to each transformation.
11. Incubate at 37°C for 1 hours, shaking at 200-300rpm
12. Pipette 100µL of each transformation onto petri plates.
13. Spin down cells at 6800g for 3mins and discard 800µL of the supernatant. Resuspend the cells in the remaining 100µL, and pipette each transformation onto petri plates.
14. Incubate transformations overnight at 37°C.
Examples of the colonies
BBa_R0040
BBa_I20270
BBa_J364001
7/19:
The plates were taken out from the incubator and stored at 4oC (since we did not have time to do the next step until 7/22)
7/22:> Preparations
Sunday July 22, 2018 9:00
Pick 2 colonies from each of the transformation plates and inoculate in 5-10 mL LB medium + Chloramphenicol. Grow the cells overnight (16-18 hours) at 37°C and 220 rpm.
7/23:> Cell growth, sampling, and assay
Monday, July 23, 2018 3:30
1) make a 1:10 dilution of each overnight culture in LB+Chloramphenicol
2) measure Abs600 of these 1:10 diluted cultures
3) record the data in our notebook
4) dilute the cultures further to a target Abs600 of 0.02 in a final volume of 12 ml LB medium + Chloramphenicol in 50 mL falcon tube
5) take 500 µL samples of the diluted cultures at 0 hours into 1.5 ml eppendorf tubes, prior to incubation.
6) place the samples on ice.
7) Incubate the remainder of the cultures at 37°C and 220 rpm for 6 hours
Monday July 23, 2018 10:00(pm)
1) take 500 µL samples of the cultures at 6 hours of incubation into 1.5 ml eppendorf tubes
2) place samples on ice
3) measure the samples
4) Record data in the notebook
5) Import data into Excel sheet
Colony Forming Units per 0.1 OD600 E. coli cultures
Monday July 23, 2018 7:00(pm)
1) Measure the OD600 of cell cultures
2) Dilute overnight culture to OD600 = 0.1 in 1mL of LB + Cam media
3) Check the OD600
Monday July 23, 2018 8:10(pm)
Making the dilution Series
1) prepare three 2.0 mL tubes with 1900 μL of LB + Cam media for Dilutions 1, 2, and 3
2) prepare two 1.5 mL tubes with 900 μL of LB + Cam media for Dilutions 4 and 5
3) label each tube for each Starting Sample.
4) pipet 100 μL of Starting Culture into Dilution 1. Discard tip.
5) repeat Step 4 for each dilution through to Dilution 5
6) aseptically spead plate 100 μL on LB + Cam plates for Dilutions 3, 4, and 5.
7) Incubate at 37°C overnight and count colonies after 20 hours of growth
Tuesday July 24, 2018 10:00
Count the colonies on each plate with fewer than 300 colonies.
Multiple the colony count by the Final Dilution Factor on each plate
Examples of the colonies
BBa_R0040 Culture 1 Dilution 4 replicate 1
BBa_I20270 Culture 1 Dilution 4 replicate 1
BBa_R0040 Culture 1 Dilution 5 replicate 1
BBa_I20270 Culture 1 Dilution 5 replicate 1
| Sample ID | Colonies for dilution 5 | Colonies for dilution 4 | CFU/ml |
| BBa_I20270 Culture 1 Dilution replicate 1 | 18 | 78 | 6.24x10^7 |
| BBa_I20270 Culture 1 Dilution replicate 2 | 30 | 62 | 4.96x10^7 |
| BBa_I20270 Culture 1 Dilution replicate 3 | 12 | 56 | 4.48x10^7 |
| BBa_I20270 Culture 2 Dilution replicate 1 | 36 | 169 | 1.35x10^7 |
| BBa_I20270 Culture 2 Dilution replicate 2 | 8 | 176 | 1.41x10^7 |
| BBa_I20270 Culture 2 Dilution replicate 3 | 21 | 135 | 1.08x10^7 |
| BBa_R0040 Culture 1 Dilution replicate 1 | 66 | 209 | 1.67x10^7 |
| BBa_R0040 Culture 1 Dilution replicate 1 | 15 | 176 | 1.41x10^7 |
| BBa_R0040 Culture 1 Dilution replicate 1 | 23 | 212 | 1.70x10^7 |
| BBa_R0040 Culture 1 Dilution replicate 1 | 20 | 230 | 1.84x10^7 |
| BBa_R0040 Culture 1 Dilution replicate 1 | 18 | 217 | 1.74x10^7 |
| BBa_R0040 Culture 1 Dilution replicate 1 | 14 | 108 | 108 |
Table. Data for Colony Forming Units per 0.1 OD600 E. coli cultures