Appendix

Schedule of Construction

We have a schedule of ligations and transformations that we keep on googledocs to easily access information about the dna and cells in our freezer and fridge. This also organizes what ligations we plan to do in the future. In all there are about 40 ligations we want to do to construct the test constructs and first tristable system and based on the results of the tests, we will need to do as many as 20 more ligations.

Brown iGEM transformation database on googledocs

Brown iGEM ligation database on googledocs

Predicted Obstacles

1. Other iGEM teams (info via Patrick King) have found that the araC gene contains a promoter region. Grown in TOP10 cells the construct araC>RBS>GFP>Terminator was observed to give a strong florescent output. In our system, this would give a florescent output for one FP even if that construct was not on. One option would be to put AraC at the end of a construct so that it only promotes the end of the gene and then hits the terminator. This is not optimal, however.
2. AraC exhibits all or none gene expression in the presence of L-arabinose because the natural transporter, when induced, causes that cell to transport arabinose rapidly, depleting the culture. This may be overcome by adding a saturating level of arabinose. In the arabinose construct already made, we were not able to observe FP output even though there was a high level of arabinose. We used XL1Blue cells.
3. The degradation tags on the repressors may not give a long enough protein life time to yield strong repression. Thus, the tags may need to be removed to have a working device.

Email Correspondances about Tri-Stable Switch

Media:emailwithJasonLohmueller.txt

Media:emailwithNicolaNeretti.txt

Media:emailwithPatrickKing.txt