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

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 H_​2​ 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 OD​600 of 0.02 in ​5 m​L 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.