Team:Shanghaitech/InterLab
Abstract
Repeatability is one of the most important part of biological experiment, however, it’s difficult to repeat the measurement data in different labs. As for the measurement of fluorescence, it can be influenced by various factors: bacterial strain, culture medium, plate reader and so on. Therefore, following a standard protocol strictly is one of the guarantee of the repeatable experimental conclusion. This year, we use plate reader rather than flow cytometry in 2016 (click here to view). Except for individual settings, we follow the standard protocol by iGEM authority completely, the data is reliable.
Plasmids and the prediction of results
All the eight plasmids are shown below:
From the table above, there are three different constitutive promotors (pcons) and two different ribosome binding sites (RBSs), which form six different combinations as Test Device 1-8.
According
to the data of iGEM parts, we know that the strength of these three pcons: J23101＞J23106＞
J23117. However, there lacks the
reliable data of the strength of pcon (J23151) and the comparison of three RBSs
(B0032, B0034, J364100). In conclusion, we predict the result of the Interlab fluorescence
measurement:
Test Device 1 ＞ Test Device 2 ＞ Test Device 3
Test Device 4 ＞ Test Device 5 ＞
Test Device 6
All the plasmid sequences (.dna file) can be download from: https://drive.google.com/file/d/0Bxb6U-RiSYsgQ1pTU0FsTG1SY1k/view?usp=sharing
Component cell strain & 96 well plate & Plate reader
Component cell strain
Brand: 康为世纪 (cwbiotech)
Strain: E.coli K-12 DH5α
96 well plate
Brand: Corning
-Black plate
-Flat-bottomed wells
Plate reader
Brand: BioTek
Instrument model: Cytation 5
Serial number: 1511021F
Pathlength correction: No
Number of flashes per well: 10
Orbital averaging (mm): 3.5
Fluorescence reading: top optic
Filter: Yes
Excitation wavelength (nm): 485/9
Emission wavelength (nm): 528/9
Protocol
(Some of the following steps are different
from standard protocol from iGEM authority, the changes and the reasons are
shown as red.)
Calibration ——
OD600 reference
point
Materials
1ml LUDOX
ddH2O
96 well plate
Method
◻
Add 100 µl LUDOX into wells A1, B1, C1, D1
◻
Add 100 µl of H2
O into
wells A2, B2, C2, D2
◻
Measure absorbance 600 nm of all samples in all standard measurement modes in instrument
◻
Record the data in the table below
◻
Import data into Excel (OD600 reference point tab
) Sheet_1 provided
Calibration ——
fluorescein
fluorescence standard curve
Materials
fluorescein
10mL 1X PBS
96 well plate
Method
——
Prepare
the fluorescein stock solution
◻
Spin down fluorescein stock tube to make sure pellet is at the bottom of tube.
(5000rpm, 5min)
◻
Prepare 2X fluorescein stock solution (100 µM) by resuspending fluorescein in 1 mL of 1X PBS.
◻
Dilute the 2X fluorescein stock solution with 1X PBS to make a 1X fluorescein solution and resulting concentration of fluorescein
stock solution 50 µM.
——
Prepare
the serial dilutions of fluorescein
◻
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...
◻
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
◻
Repeat dilution series for rows B, C, D
◻
Measure fluorescence of all samples in all standard measurement modes in
instrument
◻
Record the data in your notebook
◻
Import data into Excel (fluorescein standard curve tab
) Sheet_1 provided
Cell measurement
Materials
E.coli K-12 DH5α
component cells
LB (Luria Bertani) medium
Chloramphenicol (stock concentration 30 mg/mL dissolved in EtOH - working stock 30 ug/mL)
(This concentration
refers to one of our PIs, and it’s the common concentration in my lab.)
(The file can be download
here:
https://drive.google.com/open?id=0Bxb6U-RiSYsgbjEzU0o4eVNYNWc
)
14 mL polypropylene
round-bottom tube with aluminum foil cover
(Comparing with 50mL
falcon tube, it is much more suitable for incubation, the reason is that its cover
isn’t completely closed which can make the oxygen entry freely, so the E.coli can be incubated in an aerobic environment.)
Incubator at 37℃
1.5 mL eppendorf tubes for sample storage
Ice bucket with ice
Pipettes
Method
Day
1: Transform E.coli DH5α
with eight kinds of plasmids from 2017 iGEM kit, plate 7.
Day
2: Pick 2 colonies from each of plate and inoculate
it on 5 mL LB medium + chloramphenicol in 14mL
polypropylene round-bottom tube. Grow the cells overnight for 18 hours at 37°C and 220 rpm.
Day
3: Cell growth, sampling, and assay.
◻
Set the instrument to read OD600 (as OD calibration setting)
◻
Measure OD600 of the overnight cultures
◻
Record data in the notebook
◻
Import data into Excel (Dilution Calculation) Sheet_1 provided
◻
Dilute the cultures to a target OD600 of 0.02 in 5 mL LB medium + Chloramphenicol 14mL
polypropylene round-bottom tube with aluminum foil cover.
(Overmuch cultures in
tube leads to anaerobic environment. Therefore, about 1/3 cultures here.)
◻
Incubate the cultures at 37°C and 220 rpm.
◻
Take 500 µL samples of the cultures at 0, 2, 4, and 6 hours of incubation.
◻
Place samples on ice.
◻
At the end of sampling point, measure the samples (OD and Fl measurement).
◻
Record data in the notebook.
◻
Import data into Excel (cell measurement tab) Sheet_1 provided.
Results
Calibration —— OD600 reference point
Raw data link: https://drive.google.com/open?id=0Bxb6U-RiSYsgTFlyeVRGeXlscGs
Calibration —— fluorescein fluorescence standard curve
Raw data link: https://drive.google.com/open?id=0Bxb6U-RiSYsgVGl6RG1aQ1dxVFE
According
to the diagram above, we can notice that when the concentration of fluorescein
is high (＞10μM), the curve
doesn’t follow linear relation. As for this problem, we have communicated with
other iGEM team, as expected, this is a common question. Referring to some materials,
we give two possible explanations:
——When concentration is too high, light cannot
pass through the sample to cause excitation, thus very high concentrations can
have very low fluorescence.
——The surface portion of sample nearest the
light absorbs too much light, little is available for the rest portion of the
sample; thus the readings will not be linear, though the measurement will be
within the range of a calibration curve.
Cell measurement
Raw data link: https://drive.google.com/open?id=0Bxb6U-RiSYsgbnNJRVpOaUJLZVU
All the raw data
and following tables refer to the wells arrangement of iGEM protocal below:
Results:
OD600 —— 0h
OD600 —— 2h
OD600 —— 4h
OD600 —— 6h
Fluorescence —— 0h
Fluorescence —— 2h
Fluorescence —— 4h
Fluorescence —— 6h
The curve are
shown below:
OD600 0——6 h
We find that
Positive Control and Test Device 1 have a lower OD600 value at 2h and 4h.
According to the fluorescence curve, we suppose the more efficient GFP
expression (strong promotor and RBS) leads to the less OD600 value. Generally
speaking, all the eight kinds of bacteria have the similar growth condition
during 0——6 h.
Fluorescence 0——6 h
According to the
diagram above, we can find distinctly that Positive Control、 Test Device 1 and Test Device 4 have more efficient GFP expression
than the others.
Fluorescence/OD600 0——6 h
Generally speaking,
the fluorescence/OD600 value means the amount of GFP in each bacterium, thus,
this value can represent the GFP expression strength than fluorescence value.
the diagram shows the fluorescence/OD600
results among the six devices:
Test Device 1 ＞ Test
Device 2 ＞
Test Device 3
Test Device 4 ＞ Test Device 5 ＞
Test Device 6
The results correspond
to the prediction, which means that our data is strict and reliable.
