Revision as of 16:52, 31 October 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. OD₆₀₀ Reference point

Results of OD₆₀₀ measurement of 1 ml LUDOX and 1 ml H₂O with dH₂O as blank.

Table 1: OD₆₀₀ measurement

Protocol Fluorescein Fluorescence Standard Curve

We measured the in PBS diluted fluorescein 96 well plate in a plate reader with the settings XY and turned of pathlength correction.

Table 2: Fluorescein fluorescence measurement with gain 48 & 70.

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.

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
Day 3 Overnight culture

InterLab

Bronze Medal Criterion #4

Standard Tracks: Participate in the Interlab Measurement Study and/or improve the characterization of an existing BioBrick Part or Device and enter this information on that part's Main Page in the Registry. The part that you are characterizing must NOT be from a 2017 part number range.

For teams participating in the InterLab study, all work must be shown on this page.

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 <font size="-2"><b>Diagram 1 & 2: This diagrams display the amount of fluorescence according to the fluorescein µM concentration in 100 µl LB media per well.</b> </font>

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