Team:TU-Eindhoven/Notebook/Week10

• Introduction
  • Home page
  • Background
    information
  • About us
  • Contact us
  • Our photos
• Project
  • Project description
  • Lab approach
  • Device information
  • Initial model
  • Yeast model
  • Lab results
  • Our BioBricks^TM
  • Our
    accomplishments
• Considerations
  • Public outreach
  • Safety questions
  • Other thoughts
• Future applications
  • Human interest
  • Further
    developments
• Notebook
  • Before week 1
  • Week 1
  • Week 2
  • Week 3
  • Week 4
  • Week 5
  • Week 6
  • Week 7
  • Week 8
  • Week 9
  • Week 10
  • Week 11
  • Week 12
  • After week 12
  • Lab protocols
• Partners
  • ICMS TU/e
  • Team collaborations
  • Attributions and Contributions
  • Our sponsors

Things we did this week

This week, some of us got a guided tour at the building of the Discovery Festival in Eindhoven. The organization is still progressing! Furthermore, everybody is working hard on writing the pieces for the wiki. The [http://www.cursor.tue.nl/nieuwsartikel/artikel/lichtgevende-afbeeldingen-maken-van-gistcellen/ Cursor], the news paper of the Eindhoven University of Technology, published an article on our project. You can find the English verion [http://www.cursor.tue.nl/en/news-article/artikel/lichtgevende-afbeeldingen-maken-van-gistcellen/ here].

Progression in the lab

The yeast seems to be complete, with the GECOs and both the Cch1 and Mid1 channel proteins, and is amplified for testing and long-term storage in -80 degrees Celcius. Unfortunately, the yeast has grown slowly on the bottom of the culture tubes only. This is caused by the rack we used to hold the tubes. We used a vertical rack instead of the usual tilted rack, because all the slots of the tilted racks were occupied. As soon as some slots of the tilted rack became vacant, we put our tubes in it. Furthermore, the yeast are induced in 2% galactose SC medium for another test. As an alternative to ‘eye balling’ the yeast for fluorescent activity, we tried a plate reader. It’s a very sensitive spectro-fluorometer. It took a long time to prepare the wells plates for the test, because it was the first time we had to do this for the project.

Since we need extra GECO protein for testing, we precultured some E. coli BL21 carrying the GECOs on the pET28a production vector. The precultures are transferred to 100ml production volumes and induced with IPTG. Because the cooling of the incubator had not been switched on, the temperature was higher than we wanted. It seemed that no protein had been produced in the Erlenmeyer flasks, so we discarded them in the appropriate waste box, waiting for sterilization. A couple of hours later, we discovered that our Erlenmeyer flasks had turned red and green. Therefore we decided to test their fluorescence anyway. Both the R- and the G-GECO are strongly fluorescent in the blue light. We would like to know why the culture was fluorescent after taking it out of the incubator and letting it stand at room temperature for a few hours! Our first guess is that the bacteria have sedimented, started dying of a lack of oxygen at the bottom of the flask and thus releasing their cell contents into the medium. When the released GECOs came into contact with any calcium present in the medium, they got activated. Furthermore, the cell bodies have all sedimented and the medium turned from turbid to clear, allowing fluorescent light to shine colorfully instead of scatter into a blurry white.

We continued working on the BioBricks. The colonies that seem to carry the BioBrick plasmid are amplified, isolated by miniprep and submitted for sequencing.