Team:Vilnius-Lithuania/Measurement

Description

This year Vilnius iGEM team is also participating in the iGEM Measurement InterLab study. This is the second year that iGEM holds an InterLab study. This is one massive amazing experiment which involves multiple teams all around the world working for the same scientific goal.

This year the aim of this study is to see how different laboratories perform the same experiment and how results between different laboratories can differ or resemble each other. The ultimate goal of InterLab study is to develop new methods of interpreting data, which would take into account the differences that might arise whilst performing similar experiments in different conditions.

All teams had to construct three different GFP expressing devices and subsequently measure their fluorescence output. Received fluorescence data reflects the strength of gene (GFP) expression.

Three devices had to be constructed:

  1. J23101 + I13504
  2. J23106 + I13504
  3. J23117 + I13504

J23101, J23106 and J23117 parts are different promoters characteristic of different transcription levels. I13504 is ribosome binding sequence (RBS) + GFP + two terminators.

Experiments

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Cloning

Cloning was performed using Biobrick Standard Assembly method. All cloning procedures were carried out in pSB1C3 vector.

Firstly, plasmids, containing three promoters and GFP gene, were transformed into E. coli DH5α strain. Bacteria, containing the plasmids, were grown overnight and then DNA extraction ensued. The extracted DNA was cut using EcoRI and SpeI restriction enzymes for the promoters, and with EcoRI and XbaI for plasmids, containing the GFP gene. Later, plasmids with incorporated GFP gene were purified using PCR cleaning kit and the promoters were extracted from gel using freeze and squeeze protocol. Then the promoters were ligated into vectors, containing GFP gene, ligation products were again transformed into E. coli DH5α strain. The validity of our constructs was tested by restriction mapping – using StyI restrictase.

Results

Under construction.

Discussion

Under construction.

References

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