Part:BBa_K1328002
Part:BBa_K1328000 (HXT1-CMVmini-EGFP-tADH1) is a glucose-sensitive promoter of yeast origin and shown to function in both yeast and human cells. Exclusively activated by high glucose concentrations.
HXT-1+EGFP+ADH1 consists of HXT-1+CMV promoter, EGFP and terminator ADH1.
HXT-1+CMV promoter controls EGFP expression under the regulation of glucose.
EGFP is used here for testing of HXT1+CMV promoter and will be replaced by insulin gene.
HXT1-CMVmini-EGFP-tADH1
This is a composite part containing a glucose-sensitive promoter (BBa_K1328000) and a EGFP reporter with a tADH1 transcription terminator.
S. cerevisiae cells transformed with this part was cultured in glucose-free medium with glycerol as carbon-source for 8 hrs. They were then cultured for an additional hour with varying glucose levels (0, 5, 15, 25 mmol/L). A increasing level of GFP expression was observed with increasing levels of glucose concentration, in both microscopy and flow cytometry observations.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 818
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 827
- 1000COMPATIBLE WITH RFC[1000]
The HXT-1 Promoter
The HXT-1 promoter is a glucose-sensitive promoter derived from region -750 bp to -161 bp upstream of the S. cerevisiae Hexose Transporter 1(HXT-1) gene. In yeast, HXT-1 expression is positively regulated by glucose, and Ferrer-Martinez et al. characterized a minimal sequence between -750 bp and -161 bp upstream of the HXT-1 gene as the promoter region sufficient for glucose-sensitivity[1] (Henceforth referred to as the HXT-1 promoter).
The function of the HXT-1 promoter relies on a specific glucose-sensing pathway in yeast, and although there is no known mammalian homologue, HXT-1 promoter has been shown to retain its glucose sensitivity in human fibroblast cells[1].
In practice, the HXT-1 promoter works best as a hybrid promoter with a minimal-CMV (MIN) promoter attached downstream.
Constructing a Glucose Sensor
We were able to clone the HXT-1 promoter (586 bp) from the S. cerevisiae genome, and ligate it to a 206 bp minimal CMV promoter. Together they compose a glucose-sensitive promoter capable of functioning in both mammalian cells and S. cerevisiae cells.
We were surprised to learn there was no glucose-sensing promoter documented in the iGEM Parts Registry, therefore we submitted our glucose sensor as BioBrick BBa_K1328000 to the parts registry, hoping that other teams would benefit from our work.
Characterizing Glucose-Sensitivity
Before we put our system to producing insulin in mammalian cells, we first tested its glucose-sensitivity in yeast and mammalian cells by using EGFP as a reporter, by culturing transfected mammalian/yeast cells in culture media with different glucose concentrations. Fluorescence microscopy and flow cytometry analysis would then reveal any differences between the experiment groups.
Yeast
To fully characterize glucose-sensitivity, yeast cells transfected with the HXT1-CMVmini-EGFP plasmid was cultured in glucose-free media (with 3% glycerol as carbon source). They were then incubated with different concentrations of glucose (0, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM) for different durations (0.5 h, 1 h, 8 h).
Both fluorescence microscopy (Fig. 1) and flow cytometry analysis (Fig. 2) showed a positive correlation between EGFP fluorescence and glucose concentrations, demonstrating high glucose-sensitivity (lower than 5 mM). Time course studies revealed no significant difference between longer and shorter incubation durations, suggesting a fast response rate (less than 30 min).
HEK293 Cells HEK293 cells were transiently transfected with the HXT1-CMVmini-EGFP glucose-sensor/reporter plasmid, and cultured in glucose-deficient media. Different levels of glucose was then added to the cell cultures to induce EGFP expression. Cells were collected and subjected to FACS analysis. No significant EGFP signal change was observed along with increasing glucose levels, however GFP fluorescence was clearly visible under microscopic observation in all cases. We suspect this is due to HEK293 cells having a much lower glucose sensing threshold than yeast cells, and also caused by trace amount of glucose in the glucose-deficient media. This experiment will be repeated with glucose-free cell cultures and a weaker promoter than CMV-mini (which will be replaced with an even shorter 128 bp version.)
Glucose-Regulated Insulin Production
After demonstrating the function of our glucose-sensor system, we will proceed to testing its capabilities in regulating insulin production in human cell lines. An Sensor-insulin plasmid has been constructed and we will be detecting insulin production and secretion with western blots against insulin.
Reference
[1] Ferrer-Martı́nez, Andreu, et al. "A glucose response element from the S. cerevisiae hexose transporter HXT1 gene is sensitive to glucose in human fibroblasts." Journal of molecular biology 338.4 (2004): 657-667. [2] Strathdee, Craig A., Marilyn R. McLeod, and Jennifer R. Hall. "Efficient control of tetracycline-responsive gene expression from an autoregulated bi-directional expression vector." Gene 229.1 (1999): 21-29. [3] Baron, Udo, et al. "Co-regulation of two gene activities by tetracycline via a bidirectional promoter." Nucleic acids research 23.17 (1995): 3605.
Additional Documentation
For full characterization of this part, visit Team Tsinghua 2014's project wiki: http://2014.igem.org/Team:Tsinghua/Project/Microbe
Related Parts
HXT1-CMVmini (Part:BBa_K1328000) https://parts.igem.org/Part:BBa_K1328000
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