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Team:Colombia Uniandes/Project
From 2013.igem.org
Contents |
Overall project
Stress Sensor
Background
Glucocorticoids are hormones present in mammals that undergo changes in concentrations when the animal is under stressful stimuli. These hormones can be measured in blood plasma, urine, saliva and hair. Chemical methods are used to measure these hormones for veterinary and agricultural use, but these methods require expensive equipment and a capacitated technician which makes it uncomfortable and useless in the field.
Objective
Our aim is to create a glucocorticoid sensor that is able to discern between basal levels and stress levels of glucocorticoid hormones in a sample with an easily recognizable signal, such as color, to allow the sensor to be used in the field, household or the laboratory.
Nickel
Parts
Glucocorticoid sensor
Our construct
We plan to use the baker's yeast, Saccharomyces cerevisiae, as a vector for a plasmid which will contain a chimeric protein used as a transactivating factor in a biosensor with a colored reporter.
The Vector
We chose S. cerevisiae as a vector because one of the most important parts of our fusion protein, the glucocorticoid receptor hormone binding domain (GCR HBD) is eukaryotic, therefore we wanted an easy to use, easy to grow, eucaryotic vector to express our protein and build our biosensor.
The Chimera
The glucocorticoid receptor (GCR) from mammals contains three domains necessary for stress hormone related gene transcription, the hormone binding domain (HBD), the DNA binding domain (DNA-BD) and the gene transactivating domain (GTD).
However, for our construct's performance we used a chimeric protein. Just as the mythological creature made from fused parts from a lion, a goat and a snake, we created a chimeric protein using three domains from different organisms.
We used the glucocorticoid receptor hormone binding domain (GCR HBD) which came from a rat to recognize our hormones of interest. However, we replaced the other two domains with the herpesvirus gene transactivating domain (HV-GTD) and the yeast's DNA binding domain from GAL4. These two new domains have the advantage of being already used, characterized and being highly efficient. The HV-GTD is a highly efficient transactivating domain, recognized to be several orders of magnitude better than the GCR-GTD.
Tester Construct
| pBAP2 | Extracted |
| GAL4 | Extracted |
| VP16 | Extracted |
| GCR | Extracted |
| Yeast Terminator | In progress |
| pGAL1 | Stand by |
| mCherry | Extracted |
| pBAP2 + GAL4 | In progress |
| VP16 + GCR | Fused |
| VP16 + GCR + yeast Terminator | In progress |
| pBAP2 + GAL4 + VP16 + GCR + yeast Terminator | Stand by |
| pGAL1 + mCherry | Stand by |
*Updated as of July 11th.
