Team:UCL/Project/Detection

OXIDATIVE STRESS PROMOTER

For Plaque Specific Expression

In order to direct microglia activity to senile plaques , we needed to find a way to detect these plaques. Several possible routes were explored; initial focus was on a plaque binding protein, such as the B10 antibody (Haupt et al. 2011). However, there was no easy way for plaque binding to transduce changes in gene expression. Therefore, alternatives were explored, where plaque proximity could be indirectly detected via a proxy. One such proxy is oxidative stress - free radical production (Colton et al., 2000) which is generated by plaques. Microglia are naturally attracted to plaques, and upon reaching plaques, a standard immune response follows, which includes free radical production. Therefore, we have designed a promoter which will start transcription in response to oxidative stress generated by the native microglia and plaques.

This promoter is an improvement of a yeast minimal promoter cyc1001 already in the registry. Although from yeast, parts of this promoter show homology to the consensus sequences of mammalian core promoter elements, notably the TATA box and initiator element (Sandelin et al. 2007). NF-κB is a transcription factor which translocates to the nucleus under oxidative stress (Shi et al., 2003), and binds to the sequence GGGAATTT (Park et al., 2009). Thus, by placing this site upstream of the yeast minimal promoter, we created a novel mammalian promoter which initiates transcription in response to oxidative stress.

To construct this, we firstly created a BioBrick which consists of 5 copies of the NF-κB binding site (5NFKB). This was done using linkers - short overlapping primers were ordered, and allowed to anneal, and then ligated together. We were unable, however, to confirm that the annealing process had worked with the equipment we had (though technically we had a new BioBrick) because the size of the linkers was so small and due to time constraints were forced to move on to other areas of our project.