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Revision as of 16:04, 28 October 2017

I N T E R L A B

Introduction

We participated in InterLitab, as we want to be contribute to the scientific progress made through this globe spanning project. In InterLab, 6 test devices are inserted in E.coli D5 α, and the growth and fluorescence is measured.

We used the following plasmids provided by iGEM HQ to transform E.coli:

  • Positive control
  • Negative control
  • Test Device 1: J23101+I13504
  • Test Device 2: J23106+I13504
  • Test Device 3: J23117+I13504
  • Test Device 4: J23101.BCD2.E0040.B0015
  • Test Device 5: J23106.BCD2.E0040.B0015
  • Test Device 6: J23117.BCD2.E0040.B0015

Calibrations

Before our measurements began, we performed some calibrations: First an OD600 reference point for our plate reader, performed with LUDOX according to the protocol. Here we found a correction factor which can be used to calculate OD from measured absorbance. Our correction fator is 3.11.

Secondly we made a fluorescence standard curve with a serial dilution of fluorescein (figure 1). We used the lower 5 data points to calculate a mean µM fluorescein pr a.u. We chose to use the lower concentration range due to two factors: 1) Linearity is better for the lower fluorescein concentrations, and 2) our measured data has a maximum fluorescence of 500, which makes it more important to have a good fit in the lower range.



Figure 1. - Standard curve of fluorescein fluorescence. Fluorescence in arbitraty units (a.u.), fluorescein concentration in µM.

Cell measurements

Two colonies from each transformation were picked, and grown in foil-covered 50 ml falcon tubes over night (18 hours).

Preparation OD was measured, and a dilution was calculated to achieve an OD of 0.02. Dilution calculations can be found in the table next to this. Here we used the calculated correction factor from our initial abs/OD calibration. From the absorption measurements taken at 0 hours, we see indications of pipetting errors, as the OD600 (average) ranges from 0.015 to 0.6 (table 1).

Title
Abs600 OD600 before Volume (ml) added to 12 ml media OD600 after dilution (0 hours)
Negative Control (Colony 1) 0,461 1,435 0,167 0,06483974
Negative Control (Colony 2) 0,463 1,439 0,167 0,02584249
Positive Control (Colony 1) 0,450 1,400 0,171 0,04467949
Positive Control (Colony 2) 0,474 1,475 0,163 0,01689103
Test Device 1: J23101+I13504 (Colony 1) 0,462 1,437 0,167 0,03043498
Test Device 1: J23101+I13504 (Colony 2) 0,472 1,469 0,163 0,01977106
Test Device 2: J23106+I13504 (Colony 1) 0,477 1,483 0,162 0,03175824
Test Device 2: J23106+I13504 (Colony 2) 0,454 1,410 0,170 0,018837
Test Device 3: J23117+I13504 (Colony 1) 0,507 1,577 0,152 0,03261447
Test Device 3: J23117+I13504 (Colony 2) 0,492 1,529 0,157 0,01471154
Test Device 4: J23101.BCD2.E0040.B0015 (Colony 1) 0,420 1,307 0,184 0,03759615
Test Device 4: J23101.BCD2.E0040.B0015 (Colony 2) 0,472 1,468 0,163 0,01860348
Test Device 5: J23106.BCD2.E0040.B0015 (Colony 1) 0,513 1,595 0,150 0,04234432
Test Device 5: J23106.BCD2.E0040.B0015 (Colony 2) 0,467 1,451 0,165 0,01494505
Test Device 6: J23117.BCD2.E0040.B0015 (Colony 1) 0,482 1,498 0,160 0,04452381
Test Device 6: J23117.BCD2.E0040.B0015 (Colony 2) 0,455 1,416 0,170 0,02008242

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