Part:BBa_K5415003
Nonuple HRE biosensor with RFP for severe hypoxia detection.
The composite part we have developed is a hypoxia biosensor designed to detect low hypoxic conditions, built in silico using Benchling and HRE gene sequences imported from the https://doi.org/10.1016/j.bbrc.2006.06.043, pMC3-yeGFP imported from Addgene (Plasmid #176181). Gibson and in vivo cloning methods were employed. The constructs were transformed into E. coli.
The biosensor construct features three key components: nine hypoxia response elements (HREs), restriction sites (NcoI), and a red fluorescent protein (RFP). The hypoxia response elements are short DNA sequences that respond to the binding of hypoxia-inducible factor (HIF) complexes, which are activated in low oxygen environments. These binded HRE sequences trigger the expression of the RFP, allowing the cell to fluoresce under hypoxic conditions. The use of two restriction sites, NcoI, facilitates further manipulation of the construct if needed.
Initially, this biosensor system was intended to operate in tandem with two other analogous constructs—HREx3 and HREx6, constituting low and moderate hypoxic conditions respectively. Each biosensor in the proposed system was designed to have its own distinct fluorescent protein, making it possible to differentiate between various constructs based on the fluorescent signal. Together, these three biosensors would create a semi-throughput hypoxia signalling and monitoring system, providing a more nuanced detection of oxygen levels in a given microenvironment. However, due to transformation limitations, only the HRE3x construct was successfully integrated into E. coli at this stage. The premise of such a biosensor complex holds promise as an effective tool for monitoring hypoxia in biological research or biotechnological applications.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 1
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Illegal XhoI site found at 103 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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