Difference between revisions of "Part:BBa K676002"

Revision as of 20:51, 9 October 2011

mNARK with YFP.LVA

A more sensitive hypoxia detector for bioreactor fermentation. Constructed with mNark promoter and enhanced YFP with LVA tag. This device is an improved and modified version of Part:BBa_K239011.

Usage and Biology

A very common issue faced in industrial grade bioreactor fermentations is the presence of dead spots inside the fermenter vessel where oxygen is not reaching in sufficient amount. Even though electronic probes can determine the amount of dissolved oxygen in the culture broth accurately, in large volumes the presence of hypoxic regions is inevitable and cannot be detected always by oxygen probes alone. Experiments carried out at the Univeristy of California have showed that NarK protein expression in E coli is upregulated 100-fold under anaerobic conditions and a further 8-fold in the presence of nitrate [13]. Therefore the Nark promoter can essentially be used as a sensory tool for detecting hypoxic regions inside a bioreactor.

Since then the nark promoter sequence has been studied and is now known to contain 2 Fnr (fumarate and nitrate reductase), 1 Fis, 4 NarL and 1 IHF binding sites that initiate transcription from the promoter by sigma factor 70. The chief protein in this sensory pathway is Fnr which is always present within the cell in a fairly large and constant quantity. In the presence of oxygen, the [4Fe-4S]2+ cluster of Fnr monomer is oxidised to a [2Fe-2S]2+ cluster and the protein therefore remains in their inactive monomer state [14]. But in the absence of oxygen, the [4Fe-4S]2+ cluster is retained and the Fnr protein dimerises and can then bind to DNA sequences and act as a transcriptional activator for various genes.

The optimal and conserved Fnr binding sequence has been determined as TTGATNNNNATCAA [15]. But the distal Fnr binding site in the E coli NarK promoter is slightly different from the consensus region and therefore has a lower affinity for Fnr. For this reason, a modified NarK promoter sequence was designed in which the distal binding site was re-written to the optimal sequence and also some of the excess nucleotides were removed. As a result, the final mNark promoter sequence is shorter and has a better affinity for activated Fnr and therefore, has a stronger activity under anaerobic condition.

Sequence and Features