Team:UCopenhagen/InterLab

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).

Experiments

We amplified the aroG and trpE genes using genomic DNA from E.coli strain MG1655. The amplification was done using two sets of primers per gene, with overhangs allowing for insertion of a point mutation in each gene and for cloning with USER assembly.

The mutations were designed in accordance with previous work by Gu et al. (2012). In TrpE, position 293 is changed from methionine to threonine, and for AroG position 150 is changed from proline to leucine.




Figure 2 Point mutation in trpE, and insertion in vector with USER assembly. To perform the point mutation, we designed two sets of primers for each gene. The outer primers are the same as used for amplification from the genomic DNA. The central primers have overhangs containing the point mutations. This way, the genes are split in two, and the two parts are combined when inserting in the USER cassette in the expression vector - and now containing a point mutation in the middle.

Fluorescence


Some transformed cells continue to increase fluorescence despite a decrease in OD in the same samples. Devices 3 and 6 are very close to the negative control in fluorescence. Click on figures for enlarged images:

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Conclusion

Our data indicate which plasmid elements induce the highest production of GFP. Plasmids containing J23117 (Test devices 3 and 6) does not express fluorescence to a higher degree than the negative control. Plasmids with J23101 (Device 1 and 4) induced the highest fluorescence, and plasmids containing J23106 (Test devices 2 and 5) were somewhere in between. Combining the J23101 or J23106 with I13504 (Test devices 1-3) gave a higher fluorescence than adding BCD2.E0040.B0015 (Test devices 4-6).

These results are not reliable on their own, but will be more robust and reliable when combined with data from the other teams participating in the interlab study.

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