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                 <b>Tab. 2:</b> Fluorescein fluorescence measurement with gain 48 & 70
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                 <b>Tab. 2:</b> Fluorescein fluorescence plate reader measurement with gain 48 & 70 in a 96 well plate. The concentration cuts in half from pure fluorescein in line 1 to 1:1024 in line 11 and dH<sub>2</sub>O only in line 12.
 
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Revision as of 20:39, 1 November 2017

INTERLAB MEASUREMENT STUDY


Reliable and repeatable measurement is key to compare and analyse data from different labs all over the world. To support the effort to create detailed protocols to measure fluorescence and improve the possibility of comparing data, the iGEM Team NAWI_Graz 2017 decided to participate in the Fourth International InterLaboratory Measurement Study in synthetic biology. The goal is to establish a GFP measurement protocol based on engineering principles.

Our tasks were to follow exactly the iGEM Plate Reader Protocol , fill in the prepared excel sheets and send the results to the iGEM headquarter.

Results

For this study we needed to transform 8 devices into the presupposed bacteria strain E.coli DH5 alpha. This included a Positive Control I20270, the constitutive TetR repressible promoter as Negativ Control R0040, Test Device 1 J364000, Test Device 2 J364001, Test Device 3 J364002, Test Device 4 J364003, Test Device 5 J364004 and Test Device 6 J364005. They are all stored in pSB1C3 and were cultured on LB-Agar plates with a chloramphenicol concentration of 100 µg/ml. As we had grown colonies the day after the transformations, we could start with the cell measurement protocols.

Calibration Protocols

1. OD 600 Reference point Results of OD 600 measurement of 1 ml LUDOX and 1 ml H 2O with dH 2O as blank.

LUDOX Table

Tab. 1: OD 600 measurement

2. Protocol fluorescein fluorescence standard curve: fluorescein Table

Tab. 2: Fluorescein fluorescence plate reader measurement with gain 48 & 70 in a 96 well plate. The concentration cuts in half from pure fluorescein in line 1 to 1:1024 in line 11 and dH2O only in line 12.

The task was to find the optimal plate reader settings for the cell measurement on Day 3 through a fluorescein dilution with PBS and a 485 excitation & 531 emission fluorescence measurement. We came up with the problem, that the same gain settings could not be used for both, fluorescein and cell measurement as in each case one of the measurement only displayed 50% usable outcomes. Additionally, only a gain of 48 or less displayed a complete set of fluorescein values. But the producing company of our plate reader only recommends a gain range for 50-150. So not even gain 48 displays “usable" values. We found the possibility for wider detection ranges in the product help documents, but our plate reader model Synergy MX from BioTek did not offer this option. Problems with the dilution by pipetting could be excluded.

[Fluorescein standard curve]
[Standard curve (log)]
Diagram 1 & 2: This diagrams display the amount of fluorescence according to the fluorescein µM concentration in 100 µl LB media per well.

As values bigger than 100,000 are labeled as OVERFLOW and don’t represent a usable number, the excel algorithm can’t display them in a usable curves.


Cell Measurement Protocol

  • Day 1 OD600 Measurement
  • Day 2 Transformation

The standard dilution sheet only calculates volumes for a target of 10 ml. The official InterLab protocol wants a target volume of 12 ml but also the use of the dilution calculation sheet, so we made a decision and sticked clearly to the protocol and did not decide to change the sheet algorithm.

  • Day 3 Cell growth, sampling and assay. Preparing and taking probes at T0h, T2h, T4h, T6h accordingly to the dilution sheet and plate reader protocol.
Flourescence table
Tab. 3: Raw readings of fluorescence measurement of the day 2 ONC at 0 hours, 2 hours, 4 hours and 6 hours.
As you see Device 1 had the strongest fluorescence during the experiment. Even though we tried several different gain settings. The actual gain we should use, according to our results following the protocol, would be a gain of 48, but most of the devices did not fluorescent enough to be detected with this low gain. Even though we started with a T0 OD 600 of 0.5 by accident, our cells did not grow fast enough to get usable values of the fluorescence measurement. The trouble shooting did not bring up good possibilities because the growth settings were not comparable with our project experiments.
Flourescence table

Even though device 2 has the strongest fluorescence, device 4 grew better resulting in a higher OD 600.


Measurement Procedure Details

96 well plate, black, flat, transparent bottom Read: Fluorescence Endpoint Excitation: 485 Emission: 531 Band width: 20.0 Optics: Top, Gain: 70 Read speed: Normal, Delay: 100 ms, Measurements/Data Point: 10 Read Height: 8 mm Temperature: 28,8 °C (no preset)

Protocols used