Team:Tongji China/InterLab

Tongji iGEM

InterLab

We partecipated in the 4^th iGEM Interlab study along with about 100 teams. We have focused on quantifying the expression of GFP in common, comparable or absolute units.

Background & Design

Our team took part in this study which aimed to standardize the measurements of fluorescence in different labs. The main task was to quantify expression of GFP in different units. In our case, we measured fluorescence using a plate reader.

It is very common to use fluorescence as a proxy for promoter activity, and the green fluorescent protein (GFP) is the most popular protein. Although detecting the intensity of fluorescence is an indirect measurement, we can use it as representative of the expression levels of GFP. This method has a significant advantage, which is that it allows to monitor continuously without disrupting cells.

Fluorescence/OD600 is routinely used to give an adjustment of the relative expression per cell.

We aim to proceed in the experiment using the supplied FITC as a standard reference material. The standard curve can be constructed by measuring the fluorescence of a dilution series. We have previously performed this standard curve on our own instrument alongside a standard curve for purified GFP. Using these standard curves alongside your own standard curve for FITC it is thus possible to transform a relative measurements of fluorescence into an absolute measurements of GFP.

However, we aim to contain instrument variability, at least to some degree, by measuring a standard scattering solution of a mono-dispersed silica suspension (LUDOX). The objective is to see if a simple, single fixed-point measurement can be used as a ratiometric adjustment to provide greater uniformity in fluorescence/OD600 measurements across sites.

Description

Plasmids containing promoters and GFP were taken from The 2017 DNA Distribution Kit 7 and all devices were transformed into E. coli.

3ml of liquid LB-M medium with chloramphenicol were inoculated with two chosen colonies of each device. Liquid cultures were incubated for 16~18 hours in HONOUR INCURATOR SHAKER placed in incubator. OD of these cultures was measured by MAPADA UV-3100PC SPECTROPHOTOMETER and diluted to 0.02. Fluorescence of biological and also technical replicates was measured using Thermo VARIOSKAN FLASH following our protocol.

Methods & Materials

Strain used: E. coli DH5-alpha

Plasmid DNA (100 pg/uL in 10uL of water)

Positive Control (BBa_I20270): J23151.B0032.E0040.B0010.B0012 in pSB1C3 Negative Control (BBa_R0040): R0040 in pSB1C3 Test Device 1 (BBa_J364000): J23101.B0034.E0040.B0010.B0012 in pSB1C3 Test Device 2 (BBa_J364001): J23106.B0034.E0040.B0010.B0012 in pSB1C3 Test Device 3 (BBa_J364002): J23117.B0034.E0040.B0010.B0012 in pSB1C3 Test Device 4 (BBa_J364003): J23101.J364100.E0040.B0010.B0012 in pSB1C3 Test Device 5 (BBa_J364004): J23106.J364100.E0040.B0010.B0012 in pSB1C3 Test Device 6 (BBa_J364005): J23117.J364100.E0040.B0010.B0012 in pSB1C3

Materials

FITC Standard: one tube with dried down FITC for creating a FITC standard LUDOX: one tube with 30% colloidal silica suspended in 1mL of water 1xPBS (phosphate buffered saline) LB (Luria Bertani) media Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH) 50 ml Falcon tube (or equivalent) or 250 ml shake flask for cell growth 1.5 ml eppendorf tubes for sample storage Ice bucket with ice Pipettes Black 96 well plate

Machines: SpectraMax M5, SPX-150B-Z biochemical incubator and THZ-312 Thermostatic oscillator Software: Microsoft Excel and pro5 Calibration: OD600 Reference point and FITC fluorescence standard curve Cell measurement: Transformation and Measurements

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