Difference between revisions of "Part:BBa K5107009"

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6xHIS-hAR

Open reading frame of human Androgen receptor(hAR) for expresssion and purification in yeast

Usage and Biology

we showcased that our biosensor using the human androgen receptor can detect testosterone. Although this is a favorable outcome, we used a factory-produced human androgen receptor for these tests. To make the testing of our biosensor more affordable, we aimed to produce human hormone receptors in-house in Cycle 3. In the first iteration of this Cycle, the goal was to produce the human androgen receptor hAR in yeast.

Desing phase

In the design phase, we identified what properties we wanted for the production process and the produced receptor itself:

1. The receptor must be able to be purified
2. The produced receptor should be exported outside of the cell to make the purification process easier
3. The produced human androgen receptor (HAR) should have the same properties as the androgen receptor from the human body
4. The production should preferably use the same TRP deficient S. cerevisiae strain as we used in the in cell system and we have experience with it.

To make the receptor purifiable we decided to use a 6xHIS tag at the N-terminal of the receptor. To make the yeast excrete the protein we included an alfa-tag at the N-terminal of the HIS tag.

We assumed the following:

1. The HAR produced in S. cerevisiae mimics the properties of the androgen receptor from the human body.
2. The HIS-tag doesn’t affect the properties of the receptor.

The Alfa-6xHIS-HAR sequence was retrieved using the online toolvectorbuilder We decided to use services from Genscript for synthesizing the construct, as they offer the pESC-TRP backbone, which is compatible with the S. cerevisiae trp1 strain we want to use. This backbone has a GAL1,10 promoter which we decided to use for inducing expression of our construct. Therefore we could use the TRP1 deficiency of the yeast strain to select for successful transformants.. Outsourcing the synthesis and assembly of this part would also save us very valuable lab time.

We used the codon optimization tool from Genscript to optimize the sequence for expression in S. cerevisiae and DNA synthesis. We also used it to make the sequence legal for the iGEM part repository standards.

Build phase

After receiving the plasmid from Genscript, we transformed the plasmid in E. coli. Then, after making a glycerol stock of the transformed E. coli and purifying the plasmid from an overnight culture of the E. coli using miniprep, we used 10uL of the purified plasmid for S. cerevisiae transformation. All the protocols are described Experiments.

After failing to produce colonies from the yeast transformation the first couple of attempts, we finally got 2 colonies. This was achieved with 1 uL of the plasmid stock from Genscript using our yeast transformation protocol.

After getting 2 colonies from the yeast transformation, we made 5 mL overnight cultures in yeast nitrogen base (YNB) media with 2% glucose. The next day we inoculated 250 mL YNB + 2% galactose media with the 5 mL overnight culture for one of the colonies.

After incubating the flask in a shaker at 180 rpm 30C for 2 days, we span down the culture to separate the cells from the broth. We performed SDS-PAGE and a Western blot to analyze the proteins found in the culture as described onExperiments.

Learn phase

From the SDS-PAGE and the Western blot, we concluded that we failed to produce any HAR - both in the cells (pellet) and out of the cells (culture/crude protein). This lead us to propose the following two explanations:

1. The induction and expression of the GAL1 promoter failed.
2. The colony was a false positive and does not contain our construct.

As we had a lot of trouble performing a successful transformation, we believe that it is much more likely that the second option is true. To confirm this, a colony PCR could be performed on the colonies, thus determining if the colonies contain our construct or are false positives. However, due to time limitations, we couldn’t perform this analysis, thus we left this cycle as is after this first failed iteration.

Sequence and Features

References