Team:USTC/InterLab
This year we are very honored to participate in the Fifth International InterLab Measurement Study. We chose to follow the Plate Read and CFU Protocol in the measurement. Firstly we made three sets of unit calibration measurements: an OD600 reference point, a particle standard curve, and a fluorescein standard curve. Then we finished the cell measurement and measured both fluorescence and absorbance in hour0 and hour6. We also made the Colony Forming Units (CFU) measurement and calculated the CFU per 1 mL. Finally we uploaded our data on time and it had been accepted.
1. Introduction
A key component to all engineering disciplines is reliable and repeatable measurement which is the same holds true for synthetic biology. The goal of the iGEM InterLab Study is to identify and correct the sources of systematic variability in synthetic biology measurements, so that eventually, measurements that are taken in different labs will be no more variable than measurements taken within the same lab. In the previous studies, iGEM InterLab Study showed that by measuring GFP expression in absolute fluorescence units calibrated against a known concentration of fluorescent molecule, the variability in measurements between labs can greatly be reduced. However, when taking bulk measurements of a population of cells (such as with a plate reader), there is still a large source of variability in these measurements: the number of cells in the sample.
Because the fluorescence value measured by a plate reader is an aggregate measurement of an entire population of cells, which need to divide the total fluorescence by the number of cells in order to determine the mean expression level of GFP per cell. Usually we do this by measuring the absorbance of light at 600nm, from which we compute the “optical density (OD)” of the sample as an approximation of the number of cells. OD measurements are subject to high variability between labs, however, and it is unclear how good of an approximation an OD measurement actually is. If we used a more direct method to determine the cell count in each sample, then potentially we could remove another source of variability in our measurements.
This year, the iGEM Measurement Committee want our help in answering the following question: Can we reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of OD?
In order to compute the cell count in our samples, we used two orthogonal approaches:
Absorbance measurements use the way that a sample of cells in liquid scatter light in order to approximate the concentration of cells in the sample. In this year’s Measurement Kit, we provide you with a sample containing silica beads that are roughly the same size and shape as a typical E. coli cell, so that it should scatter light in a similar way. Because we know the concentration of the beads, we can convert each lab’s absorbance measurements into a universal, standard “equivalent concentration of beads” measurement.
A simple way to determine the number of cells in a sample of liquid media is to pour some out on a plate and see how many colonies grow on the plate. Since each colony begins as a single cell (for cells that do not stick together), we can determine how many live cells were in the volume of media that we plated out and obtain a cell concentration for our sample as a whole. We will have you determine the number of CFUs in positive and negative control samples in order to compute a conversion factor from absorbance to CFU.
By using these two approaches, we will be able to determine how much they agree with each other, and whether using one (or both) can help to reduce lab-to-lab variability in measurements.
Our team choose to follow the Plate Read and CFU Protocol. Following is what we do this year.
2. Plate Read and CFU Protocol
Before beginning our experiments, the following information is about our plate reader.
| Instrument brand and model |
BMG LABTECH CLARIOstar |
|
|---|---|---|
| Our instrument can measure both absorbance and fluorescence |
||
| Our instrument has pathlength correction, and it can be disabled |
||
| Our instrument has variable temperature settings, and this can be set
tore |
||
| Information about filter |
the bandpass width |
530 nm / 30 nm bandpass, 25-30nm width |
| excitation |
485 nm |
|
| emission |
520-530 nm |
|
| Our instrument uses top optics |
||
Then we will need all of the following supplies and reagents to complete this entire protocol. We have checked that we have all of these supplies and reagents.
❏ Measurement Kit (provided with the iGEM distribution shipment) containing:
❏ 1ml LUDOX CL-X
❏ 150 μL Silica Bead (microsphere suspension)
❏ Fluorescein (powder, in amber tube)
❏ iGEM Parts Distribution Kit Plates (you will obtain the test devices from the parts kit
plates)
❏ 1x PBS (phosphate buffered saline, pH 7.4 - 7.6)
❏ ddH2O (ultrapure filtered or double distilled water)
❏ Competent cells (Escherichia coli strain DH5α)
❏ LB (Luria Bertani) media
❏ Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH)
❏ 50 ml Falcon tube (or equivalent, preferably amber or covered in foil to block light)
❏ Incubator at 37°C
❏ 1.5 ml eppendorf tubes
❏ Ice bucket with ice
❏ Micropipettes (capable of pipetting a range of volumes between 1 μL and
1000 μL)
❏ Micropipette tips
❏ 96 well plates, black with clear flat bottom preferred, at least 3-4 plates (provided by
team)
2.1Calibration Protocols
Firstly we need to complete calibration protocols before cell measurements are taken. We need to make three sets of unit calibration measurements: an OD600 reference point, a particle standard curve, and a fluorescein standard curve.
2.1.1Calibration 1: OD600 Reference point
We used LUDOX CL-X (45% colloidal silica suspension) as a single point reference to obtain a conversion factor to transform our absorbance (Abs600) data from our plate reader into a comparable OD600 measurement as would cobe obtained in a spectrophotometer.
1ml LUDOX CL-X (provided in kit) ddH20 (provided by team) 96 well plate, black with clear flat bottom preferred (provided by team)
❏ Add 100 μl LUDOX into wells A1, B1, C1, D1
❏ Add 100 μl of dd H2O into wells A2, B2, C2, D2
❏ Measure absorbance at 600 nm of all samples in the measurement mode you plan to use
forcell
measurements
❏ Record the data in the table below or in your notebook
❏ Import data into Excel sheet provided (OD600 reference point tab)
The following table is our measurement data of OD600 Reference point.
Table1 OD600 reference point tab
| LUDOX CL-X | H2O | |
|---|---|---|
| Replicate 1 | 0.055 | 0.038 |
| Replicate 2 | 0.060 | 0.037 |
| Replicate 3 | 0.056 | 0.040 |
| Replicate 4 | 0.055 | 0.038 |
| Arith. Mean | 0.057 | 0.038 |
| Corrected Abs600 | 0.018 | |
| Reference OD600 | 0.063 | |
| OD600/Abs600 | 3.452 |
2.1.2Calibration 2: Particle Standard Curve
We prepared a dilution series of monodisperse silica microspheres and measure the Abs600 in our plate reader. The size and optical characteristics of these microspheres are similar to cells, and there is a known amount of particles per volume. This measurement will allow us to construct a standard curve of particle concentration which can be used to convert Abs600 measurements to an estimated number of cells.
300 μL Silica beads - Microsphere suspension (provided in kit, 4.7 x 10^8 microspheres) ddH20 (provided by team) 96 well plate, black with clear flat bottom preferred (provided by team)
Prepare the Microsphere Stock Solution:
❏ Obtain the tube labeled “Silica Beads” from the InterLab test kit and vortex vigorously
for 30
seconds. NOTE: Microspheres should NOT be stored at 0°C or below, as freezing affects the
properties
of the microspheres.
❏ Immediately pipet 96 μL microspheres into a 1.5 mL eppendorf tube
❏ Add 904 μL of ddH2O to the microspheres
❏ Vortex well. This is our Microsphere Stock Solution.
Prepare the serial dilution of Microspheres:
Accurate pipetting is essential. Serial dilutions will be performed across columns 1-11. COLUMN 12 MUST CONTAIN ddH2O ONLY. Initially you will setup the plate with the microsphere stock solution in column 1 and an equal volume of 1x ddH2O in columns 2 to 12. You will perform a serial dilution by consecutively transferring 100 μl from column to column with good mixing.
❏Add 100 μl of ddH2O into wells A2, B2, C2, D2....A12, B12, C12, D12
❏Vortex the tube containing the stock solution of microspheres vigorously for 10 seconds
❏Immediately add 200 μl of microspheres stock solution into A1
❏Transfer 100 μl of microsphere stock solution from A1 into A2.
❏Mix A2 by pipetting
up and down
3x and transfer 100 μl into A3…
❏Mix A3 by pipetting up and down 3x and transfer 100
μl into
A4...
❏Mix A4 by pipetting up and down 3x and transfer 100 μl into A5...
❏Mix
A5 by
pipetting up and down 3x and transfer 100 μl into A6...
❏Mix A6 by pipetting up and
down 3x and
transfer 100 μl into A7...
❏Mix A7 by pipetting up and down 3x and transfer 100 μl
into A8...
❏Mix A8 by pipetting up and down 3x and transfer 100 μl into A9...
❏Mix A9 by
pipetting up
and down 3x and transfer 100 μl into A10...
❏Mix A10 by pipetting up and down 3x and
transfer
100 μl into A11...
❏Mix A11 by pipetting up and down 3x and transfer 100 μl into
liquid waste
TAKE CARE NOT TO CONTINUE SERIAL DILUTION INTO COLUMN 12.
❏ Repeat dilution series for rows B, C, D
❏ IMPORTANT! Re-Mix (Pipette up and down) each row of your plate immediately before
putting in the
plate reader! (This is important because the beads begin to settle to the bottom of the
wells within
about 10 minutes, which will affect the measurements.) Take care to mix gently and avoid
creating
bubbles on the surface of the liquid.
❏ Measure Abs600 of all samples in instrument
❏ Record the data in your notebook
❏
Import data into Excel sheet provided (particle standard curve tab)
The following table is our measurement data of particle standard curve.
Table2 particle standard curve tab
| Number of Particles | 2.35E+08 | 1.18E+08 | 5.88E+07 | 2.94E+07 | 1.47E+07 | 7.35E+06 | 3.68E+06 | 1.84E+06 | 9.19E+05 | 4.60E+05 | 2.30E+05 | 0 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Replicate 1 | 0.931 | 0.427 | 0.230 | 0.140 | 0.086 | 0.062 | 0.051 | 0.044 | 0.041 | 0.040 | 0.038 | 0.038 |
| Replicate 2 | 1.098 | 0.413 | 0.244 | 0.126 | 0.085 | 0.060 | 0.046 | 0.044 | 0.041 | 0.038 | 0.037 | 0.037 |
| Replicate 3 | 1.085 | 0.403 | 0.223 | 0.132 | 0.080 | 0.058 | 0.048 | 0.042 | 0.041 | 0.039 | 0.036 | 0.038 |
| Replicate 4 | 0.909 | 0.462 | 0.290 | 0.146 | 0.088 | 0.063 | 0.049 | 0.044 | 0.039 | 0.039 | 0.039 | 0.038 |
| Arith. Mean | 1.006 | 0.426 | 0.247 | 0.136 | 0.085 | 0.061 | 0.049 | 0.044 | 0.041 | 0.039 | 0.038 | 0.038 |
| Arith. Std.Dev. | 0.100 | 0.026 | 0.030 | 0.009 | 0.003 | 0.002 | 0.002 | 0.001 | 0.001 | 0.001 | 0.001 | 0.001 |
| Arith. Net Mean | 0.968 | 0.389 | 0.209 | 0.098 | 0.047 | 0.023 | 0.011 | 0.006 | 0.003 | 0.001 | 0.000 |
2.1.3Calibration 3: Fluorescence standard curve
We prepared a dilution series of fluorescein in four replicates and measured the fluorescence in a 96 well plate in our plate reader. By measuring these in our plate reader, we generated a standard curve of fluorescence for fluorescein concentration. We will be able to use this to convert our cell based readings to an equivalent fluorescein concentration.
We use the small molecule fluorescein, which has similar excitation and emission properties to GFP, but is cost-effective and easy to prepare. (The version of GFP used in the devices, GFP mut3b, has an excitation maximum at 501 nm and an emission maximum at 511 nm; fluorescein has an excitation maximum at 494 nm and an emission maximum at 525nm).
Fluorescein (provided in kit) 10ml 1xPBS pH 7.4-7.6 (phosphate buffered saline; provided by team) 696 well plate, black with clear flat bottom (provided by team)
Prepare the fluorescein stock solution:
❏ Spin down fluorescein kit tube to make sure pellet is at the bottom of tube.
❏ Prepare 10x fluorescein stock solution (100 μM) by resuspending fluorescein in 1
mL of 1xPBS. [Note: it is important that the fluorescein is properly dissolved. To
check this, after the resuspension you should pipette up and down and examine the
solution in the pipette tip – if any particulates are visible in the pipette tip
continue
to mix the solution until they disappear.]
❏ 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
Prepare the serial dilutions of fluorescein:
Accurate pipetting is essential. Serial dilutions will be performed across columns 1-11. COLUMN 12 MUST CONTAIN PBS BUFFER ONLY. Initially you will setup the plate with the fluorescein stock in column 1 and an equal volume of 1xPBS in columns 2 to 12. You will perform a serial dilution by consecutively transferring 100 μl from column to column with good mixing.
❏ Add 100 μl of PBS into wells A2, B2, C2, D2....A12, B12, C12, D12
❏ Add 200 μl of fluorescein 1x stock solution into A1, B1, C1, D1
❏ Transfer 100 μl of fluorescein stock solution from A1 into A2.
❏ Mix A2 by pipetting up and down 3x and transfer 100 μl into A3…
❏ 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...
❏ Mix A5 by pipetting up and down 3x and transfer 100 μl into A6...
❏ Mix A6 by pipetting up and down 3x and transfer 100 μl into A7...
❏ Mix A7 by pipetting up and down 3x and transfer 100 μl into A8...
❏ Mix A8 by pipetting up and down 3x and transfer 100 μl into A9...
❏ Mix A9 by pipetting up and down 3x and transfer 100 μl into A10...
❏ Mix A10 by pipetting up and down 3x and transfer 100 μl into A11...
❏ Mix A11 by pipetting up and down 3x and transfer 100 μl into liquid waste
TAKE CARE NOT TO CONTINUE SERIAL DILUTION INTO COLUMN 12.
❏ Repeat dilution series for rows B, C, D
❏ Measure fluorescence of all samples in instrument
❏ Record the data in your notebook
❏ Import data into Excel sheet provided (fluorescein standard curve tab)
The following table is our measurement data of fluorescein standard curve.
Table3 fluorescein standard curve tab
| Fluorescein uM | 10.00 | 5 | 2.5 | 1.25 | 0.625 | 0.313 | 0.156 | 0.078 | 0.039 | 0.0195 | 0.0098 | 0 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Replicate 1 | 2.317E+05 | 1.573E+05 | 8.550E+04 | 4.689E+04 | 2.524E+04 | 1.263E+04 | 6.538E+03 | 3.390E+03 | 1.867E+03 | 1.050E+03 | 5.860E+02 | 6.100E+01 |
| Replicate 2 | 2.421E+05 | 1.498E+05 | 8.826E+04 | 5.172E+04 | 2.828E+04 | 1.406E+04 | 7.248E+03 | 4.023E+03 | 1.977E+03 | 1.069E+03 | 5.890E+02 | 6.300E+01 |
| Replicate 3 | 2.403E+05 | 1.527E+05 | 9.328E+04 | 4.981E+04 | 2.610E+04 | 1.302E+04 | 6.629E+03 | 3.472E+03 | 1.905E+03 | 8.630E+02 | 4.410E+02 | 6.200E+01 |
| Replicate 4 | 2.405E+05 | 1.548E+05 | 8.154E+04 | 4.317E+04 | 2.320E+04 | 1.172E+04 | 6.389E+03 | 2.866E+03 | 1.610E+03 | 8.270E+02 | 4.370E+02 | 6.500E+01 |
| Arith. Mean | 2.387E+05 | 1.537E+05 | 8.714E+04 | 4.790E+04 | 2.570E+04 | 1.286E+04 | 6.701E+03 | 3.438E+03 | 1.840E+03 | 9.523E+02 | 5.133E+02 | 6.275E+01 |
| Arith. Std.Dev. | 4.689E+03 | 3.204E+03 | 4.933E+03 | 3.727E+03 | 2.103E+03 | 9.682E+02 | 3.778E+02 | 4.736E+02 | 1.598E+02 | 1.250E+02 | 8.576E+01 | 1.708E+00 |
| Arith. Net Mean | 2.386E+05 | 1.536E+05 | 8.708E+04 | 4.783E+04 | 2.564E+04 | 1.279E+04 | 6.638E+03 | 3.375E+03 | 1.777E+03 | 8.895E+02 | 4.505E+02 |
2.2Cell measurement protocol
We used E. coli K-12 DH5-alpha for our cell measurement as other teams were required. For all of these cell measurements, we used the same plates and volumes that we used in our calibration protocol. We also used the same settings (e.g., filters or excitation and emission wavelengths) that we used in our calibration measurements. These settings guaranteed the consistency and repeatability of our experiments.
Competent cells (Escherichia coli strain DH5α) LB (Luria Bertani) media Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH) 50 ml Falcon tube (or equivalent, preferably amber or covered in foil to block light) Incubator at 37°C 1.5 ml eppendorf tubes for sample storage Ice bucket with ice Micropipettes and tips 96 well plate, black with clear flat bottom preferred (provided by team) Devices (from Distribution Kit, all in pSB1C3 backbone):
Day 1: transform Escherichia coli DH5α with these following plasmids (all in pSB1C3):
Day 2: 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.
Day 3: Cell growth, sampling, and assay
◻ Make a 1:10 dilution of each overnight culture in LB+Chloramphenicol (0.5mL of culture into 4.5mL of
LB+Chlor)
◻ Measure Abs600 of these 1:10 diluted cultures
◻ Record the data in your notebook
◻ 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 (amber, or covered with foil to block light).
◻ Take 500 µL samples of the diluted cultures at 0 hours into 1.5 ml eppendorf tubes,
prior to incubation. (At each time point 0 hours and 6 hours, you will take a sample
from each of the 8 devices, two colonies per device, for a total of 16 eppendorf tubes
with 500 µL samples per time point, 32 samples total). Place the samples on ice.
◻ Incubate the remainder of the cultures at 37°C and 220 rpm for 6 hours.
◻ Take 500 µL samples of the cultures at 6 hours of incubation into 1.5 ml eppendorf
tubes. Place samples on ice.
◻ At the end of sampling point you need to measure your samples (Abs600 and
fluorescence measurement), see the below for details.
◻ Record data in your notebook
◻ Import data into Excel sheet provided (fluorescence measurement tab)
Samples should be laid out according to the plate diagram below. Pipette 100 µl of each sample into each well. From 500 µl samples in a 1.5 ml eppendorf tube, 4 replicate samples of colony #1 should be pipetted into wells in rows A, B, C and D. Replicate samples of colony #2 should be pipetted into wells in rows E, F, G and H. Be sure to include 8 control wells containing 100uL each of only LB+chloramphenicol on each plate in column 9, as shown in the diagram below. Set the instrument settings as those that gave the best results in your calibration curves (no measurements off scale). If necessary you can test more than one of the previously calibrated settings to get the best data (no measurements off scale). Instrument temperature should be set to room temperature (approximately 20-25 C) if your instrument has variable temperature settings.
Workflow
The following table is our measurement data of cell measurement..
Table4 Fluorescence Raw Readings of Hour0 and Hour6
| Fluorescence Raw Readings: | |||||||||
|---|---|---|---|---|---|---|---|---|---|
| Hour 0: | Neg. Control | Pos. Control | Device 1 | Device 2 | Device 3 | Device 4 | Device 5 | Device 6 | LB + Chlor (blank) |
| Colony 1, Replicate 1 | 1200 | 1359 | 1664 | 1996 | 1240 | 1309 | 1244 | 1301 | 58 |
| Colony 1, Replicate 2 | 1215 | 1234 | 1719 | 1987 | 1290 | 1263 | 1301 | 1369 | 62 |
| Colony 1, Replicate 3 | 1234 | 1203 | 1742 | 2067 | 1275 | 1348 | 1302 | 1379 | 56 |
| Colony 1, Replicate 4 | 1279 | 1328 | 1748 | 2064 | 1281 | 1308 | 1281 | 1359 | 59 |
| Colony 2, Replicate 1 | 1286 | 1222 | 1323 | 1777 | 1220 | 1353 | 1083 | 1441 | 59 |
| Colony 2, Replicate 2 | 1223 | 1236 | 1285 | 1767 | 1252 | 1293 | 1254 | 1188 | 61 |
| Colony 2, Replicate 3 | 1264 | 1270 | 1308 | 1848 | 1283 | 1320 | 1232 | 1363 | 60 |
| Colony 2, Replicate 4 | 1205 | 1258 | 1260 | 1788 | 515 | 1279 | 1175 | 1281 | 59 |
| Fluorescence Raw Readings: | |||||||||
| Hour 6: | Neg. Control | Pos. Control | Device 1 | Device 2 | Device 3 | Device 4 | Device 5 | Device 6 | LB + Chlor (blank) |
| Colony 1, Replicate 1 | 1196 | 1396 | 5070 | 12217 | 1930 | 1329 | 2732 | 1262 | 60 |
| Colony 1, Replicate 2 | 1396 | 1399 | 5471 | 12751 | 1663 | 1354 | 2866 | 1463 | 57 |
| Colony 1, Replicate 3 | 1354 | 1463 | 5203 | 12685 | 1730 | 1334 | 2932 | 1396 | 62 |
| Colony 1, Replicate 4 | 1329 | 1530 | 5463 | 13085 | 1663 | 1399 | 2934 | 1363 | 61 |
| Colony 2, Replicate 1 | 1334 | 1262 | 1864 | 12417 | 1759 | 1463 | 3610 | 1432 | 61 |
| Colony 2, Replicate 2 | 1361 | 1463 | 1759 | 13019 | 1797 | 1412 | 3422 | 1369 | 62 |
| Colony 2, Replicate 3 | 1364 | 1062 | 1850 | 12818 | 1782 | 1396 | 3734 | 1354 | 60 |
| Colony 2, Replicate 4 | 1412 | 1592 | 2198 | 13085 | 1864 | 1469 | 3667 | 1464 | 59 |
Table5 Abs600 Raw Readings of Hour0 and Hour6.
| Abs600 Raw Readings: | |||||||||
|---|---|---|---|---|---|---|---|---|---|
| Hour 0: | Neg. Control | Pos. Control | Device 1 | Device 2 | Device 3 | Device 4 | Device 5 | Device 6 | LB + Chlor (blank) |
| Colony 1, Replicate 1 | 0.054 | 0.053 | 0.049 | 0.051 | 0.047 | 0.045 | 0.051 | 0.067 | 0.043 |
| Colony 1, Replicate 2 | 0.052 | 0.052 | 0.048 | 0.052 | 0.049 | 0.052 | 0.051 | 0.057 | 0.043 |
| Colony 1, Replicate 3 | 0.056 | 0.052 | 0.051 | 0.077 | 0.051 | 0.05 | 0.081 | 0.072 | 0.036 |
| Colony 1, Replicate 4 | 0.056 | 0.052 | 0.05 | 0.054 | 0.08 | 0.12 | 0.103 | 0.068 | 0.041 |
| Colony 2, Replicate 1 | 0.056 | 0.059 | 0.077 | 0.088 | 0.061 | 0.05 | 0.055 | 0.071 | 0.038 |
| Colony 2, Replicate 2 | 0.056 | 0.089 | 0.052 | 0.087 | 0.052 | 0.053 | 0.053 | 0.05 | 0.038 |
| Colony 2, Replicate 3 | 0.056 | 0.055 | 0.056 | 0.052 | 0.051 | 0.05 | 0.069 | 0.049 | 0.037 |
| Colony 2, Replicate 4 | 0.073 | 0.051 | 0.052 | 0.049 | 0.06 | 0.052 | 0.078 | 0.048 | 0.039 |
| Abs600 Raw Readings: | |||||||||
| Hour 6: | Neg. Control | Pos. Control | Device 1 | Device 2 | Device 3 | Device 4 | Device 5 | Device 6 | LB + Chlor (blank) |
| Colony 1, Replicate 1 | 0.813 | 0.101 | 0.754 | 0.798 | 0.72 | 1.351 | 0.547 | 0.555 | 0.04 |
| Colony 1, Replicate 2 | 0.87 | 0.212 | 0.837 | 0.843 | 0.706 | 0.695 | 0.579 | 0.581 | 0.041 |
| Colony 1, Replicate 3 | 0.893 | 0.436 | 0.77 | 0.839 | 0.738 | 0.563 | 0.549 | 0.58 | 0.041 |
| Colony 1, Replicate 4 | 0.899 | 0.198 | 0.796 | 0.819 | 0.717 | 0.486 | 0.572 | 0.605 | 0.04 |
| Colony 2, Replicate 1 | 0.983 | 1.596 | 0.643 | 0.79 | 0.664 | 0.569 | 0.675 | 0.682 | 0.041 |
| Colony 2, Replicate 2 | 0.951 | 0.173 | 0.645 | 0.848 | 0.768 | 0.552 | 0.638 | 0.683 | 0.042 |
| Colony 2, Replicate 3 | 0.994 | 1.147 | 0.658 | 0.827 | 0.832 | 0.624 | 0.641 | 0.646 | 0.04 |
| Colony 2, Replicate 4 | 0.974 | 0.189 | 1.063 | 0.859 | 0.096 | 0.549 | 0.642 | 0.673 | 0.042 |
2.3 Colony Forming Units per 0.1 OD600 E. coli cultures
For the CFU protocol, we counted colonies for our two Positive Control (BBa_I20270) cultures and our two Negative Control (BBa_R0040) cultures. This procedure can be used to calibrate OD600 to colony forming unit (CFU) counts, which are directly relatable to the cell concentration of the culture, i.e. viable cell counts per mL. This protocol assumes that 1 bacterial cell will give rise to 1 colony
Step 1: Starting Sample Preparation
This protocol will result in CFU/mL for 0.1 OD600. Your overnight cultures will have a much higher OD600 and so this section
of the protocol, called “Starting Sample Preparation”, will give you the “Starting Sample” with a 0.1 OD600 measurement.
1. Measure the OD600 of your cell cultures, making sure to dilute to the linear detection range of your plate reader,
e.g. to 0.05 – 0.5 OD600 range. Include blank media (LB + Cam) as well.
2. Dilute your overnight culture to OD600 = 0.1 in 1mL of LB + Cam media. Do this in triplicate for each culture.
3. Check the OD600 and make sure it is 0.1 (minus the blank measurement).
Step 2: Dilution Series Instructions
Do the following serial dilutions for your triplicate Starting Samples you prepared in Step 1. You should have 12 total Starting
Samples - 6 for your Positive Controls and 6 for your Negative Controls.
For each Starting Sample (total for all 12 showed in italics in paraenthesis):
1. You will need 3 LB Agar + Cam plates (36 total).
2. Prepare three 2.0 mL tubes (36 total) with 1900 μL of LB + Cam media for Dilutions 1, 2, and 3 (see figure below).
3. Prepare two 1.5 mL tubes (24 total) with 900 μL of LB + Cam media for Dilutions 4 and 5 (see figure below)..
4. Label each tube according to the figure below (Dilution 1, etc.) for each Starting Sample.
5. Pipet 100 μL of Starting Culture into Dilution 1. Discard tip. Do NOT pipette up and down. Vortex tube for 5-10 secs.
6. Repeat Step 5 for each dilution through to Dilution 5 as shown below.
7. Aseptically spead plate 100 μL on LB + Cam plates for Dilutions 3, 4, and 5.
8. Incubate at 37°C overnight and count colonies after 18-20 hours of growth.
Step 3: CFU/mL/OD Calculation Instructions
Based on the assumption that 1 bacterial cell gives rise to 1 colony, colony forming units (CFU) per 1mL of an OD600 = 0.1 culture
can be calculated as follows:
1. Count the colonies on each plate with fewer than 300 colonies.
2. Multiple the colony count by the Final Dilution Factor on each plate.
Workflow
3. Result
3.1 Particle Standard Curve
Using the data mentioned above to draw the figure3_1 and figure3_2
figure3_1 Particle Standard Curve
figure3_2 Particle Standard Curve (log scale)
3.2Fluorescence standard curve
Using the data mentioned above to draw the figure3_3 and figure3_4
Figure3_3 fluorescein Standard Curve
Figure3_4 fluorescein Standard Curve (log scale)
3.3CFU
After speading plate 100 μL on LB + Cam plates for Dilutions 3, 4, and 5, we counted colonies and recorded the number of colonies. When the number of colonies was over 300, we thought it was too numerous to count so we recorded it as “TNTC”
| OD600 of starting sample dilutions | colonies of Dilution 3 | colonies of Dilution 4 | colonies of Dilution 5 | ||
|---|---|---|---|---|---|
| Neg. Control | Colony 1, Replicate 1 | 0.141 | TNTC | 45 | 5 |
| Colony 1, Replicate 2 | 0.103 | 178 | 8 | 1 | |
| Colony 1, Replicate 3 | 0.126 | TNTC | 56 | 5 | |
| Colony 2, Replicate 1 | 0.134 | TNTC | 105 | 6 | |
| Colony 2, Replicate 2 | 0.102 | TNTC | 33 | 3 | |
| Colony 2, Replicate 3 | 0.121 | TNTC | 41 | 4 | |
| Pos. Control | Colony 1, Replicate 1 | 0.135 | TNTC | 137 | 16 |
| Colony 1, Replicate 2 | 0.104 | 135 | 36 | 3 | |
| Colony 1, Replicate 3 | 0.123 | TNTC | 74 | 5 | |
| Colony 2, Replicate 1 | 0.141 | TNTC | 180 | 26 | |
| Colony 2, Replicate 2 | 0.101 | TNTC | 141 | 13 | |
| Colony 2, Replicate 3 | 0.132 | TNTC | 81 | 16 |
Using final dilution factor, we can calculate the CFU per 1 mL.
| OD600 of starting sample dilutions | CFU calculated by Dilution 3(x10^8) |
CFU calculated by Dilution 4(x10^8) |
CFU calculated by Dilution 5(x10^8) |
||
|---|---|---|---|---|---|
| Neg. Control | Colony 1, Replicate 1 | 0.141 | TNTC | 0.36 | 0.40 |
| Colony 1, Replicate 2 | 0.103 | 0.14 | 0.06 | 0.08 | |
| Colony 1, Replicate 3 | 0.126 | TNTC | 0.45 | 0.40 | |
| Colony 2, Replicate 1 | 0.134 | TNTC | 0.84 | 0.48 | |
| Colony 2, Replicate 2 | 0.102 | TNTC | 0.26 | 0.24 | |
| Colony 2, Replicate 3 | 0.121 | TNTC | 0.33 | 0.32 | |
| Pos. Control | Colony 1, Replicate 1 | 0.135 | TNTC | 1.10 | 1.28 |
| Colony 1, Replicate 2 | 0.104 | 0.11 | 0.29 | 0.24 | |
| Colony 1, Replicate 3 | 0.123 | TNTC | 0.59 | 0.40 | |
| Colony 2, Replicate 1 | 0.141 | TNTC | 1.44 | 2.08 | |
| Colony 2, Replicate 2 | 0.101 | TNTC | 1.13 | 1.04 | |
| Colony 2, Replicate 3 | 0.132 | TNTC | 0.65 | 1.28 |
4. Conclusion
After finishing all these measurements, we upload our data on time. And our data has been accepted. We are very delighted that we have completed the work of interlab and got accepted. Hoping that our work can make some contributions to the future work of Interlab measurement study.
