Team:Peking/InterLab

Peking iGEM 2017

Interlab


The Peking iGEM 2017 team is participating in the Fourth InterLaboratory Measurement Study in synthetic biology, along with teams from around the world. We introduced eight plasmids into E. coli K-12 DH5-alpha and measured the GFP expression levels using a plate reader.

Introduction

Reliable and repeatable measurement is a key component of synthetic biology. However, there have been few opportunities to repeat the same measurements in different labs. In order to quantify the degree of variability exhibited by engineered genetic constructs across different laboratories, the measurement committee invited all iGEM teams to participate in the Interlab study, which provides researchers with a detailed protocol and data analysis form, with the aim to produce common, comparable units for measuring GFP on different plate readers.

Materials


Plasmids

  1. Positive Control (BBa_I20270): well 21B
  2. Negative Control (BBa_R0040): well 21D
  3. Test Device 1 (BBa_J364000): well 21F
  4. Test Device 2 (BBa_J364001): well 21H
  5. Test Device 3 (BBa_J364002): well 21J
  6. Test Device 4 (BBa_J364003): well 21L
  7. Test Device 5 (BBa_J364004): well 21N
  8. Test Device 6 (BBa_J364005): well 21P

Strains

Escherichia coli strain DH5α

Reagent

  • 1ml LUDOX
  • ddH2O
  • Fluorescein
  • 10ml 1xPBS

Instruments

  • Pipettes
  • 96-well plate
  • 50 ml Falcon tube (or equivalent, preferably amber or covered in foil to block light)
  • 1.5-ml Eppendorf tubes for sample storage, ice bucket with ice

Methods

At first, we used LUDOX-S40 as a single-point reference to obtain a ratiometric conversion factor to transform our absorbance readings into standard OD600 data. Simultaneously, the ratiometric conversion factor accounts for instrument differences.

Secondly, we generated a standard curve of fluorescence as a function of fluorescein concentration. We can thus use the standard curve to convert the fluorescence of GFP into a concentration of GFP.

Last and most importantly, we measured the A600 and fluorescence of green fluorescent protein. The GFP is used as a measurement marker of promoter activity, and the fluorescence/OD600 ratio is used to give an adjustment of the relative expression per cell. Furthermore, we tested some RBS devices that are intended to make gene expression more precise and reliable.




Figure1. The 96 well plate of the a dilution series of fluorescein in 4 replicates.




Figure2. The cultures at 6 hours of incubation.




Figure3. The machine and materials of the interlab study.




Results

Samples were prepared according to the standard protocol.The results for E. coli DH5α are shown in tables 1-10.

OD600 reference point


Table1. OD600 reference point.




Fluorescence standard curve


Table2. Fluorescein standard curve.



Figure4. Fluorescein standard curve.




Figure5. Fluorescein standard curve (log scale).




Raw Plate Reader Measurements


Table3. Raw Plate Reader Measurements of Fluorescence Raw at 0 Hour.



Table4. Raw Plate Reader Measurements of Fluorescence Raw at 2 Hour.



Table5. Raw Plate Reader Measurements of Fluorescence Raw at 4 Hour.



Table6. Raw Plate Reader Measurements of Fluorescence Raw at 6 Hour.



Cell Measurement

Table7. Raw data of Abs600 measurement at 0 Hour.



Table8. Raw data of Abs600 measurement at 2 Hour.



Table9. Raw data of Abs600 measurement at 4 Hour.



Table10. Raw data of Abs600 measurement at 6 Hour.









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