From Saccharomyces cerevisiae UAP1/QRI1 is isolated. This is done by genomic DNA, using primers to amplify the gene of interest. The final amplicon of UAP1/QRI1 is 1 434 bp as observed in figure 1. Furthermore, we amplified the plasmid backbone used in testing pASG-IBA3 (IBA3) from Escherichia coli (E. coli) DH5α using primers. The final amplicon of this is 3 115 bp as observed from figure 1.
After successful transformation confirmed by growth on ampicillin agar plates, single colonies were picked and amplified using primers specific to the cloning site of IBA3. From these four clones, we expected a band around 1 500 bp to indicate successful insert of UAP1/QRI1. As expected, we did get amplified bands around 1 500 bp.
We quantified protein content using Bradford assay. From what we see in Figure 3:
From this, protein concentration is estimated by 5-degree polynomial regression of the standard, then unknown protein concentration is calculated and shown in Table 1.
| # | Induced (+; µg/µL) | Non-induced (-; µg/µL) |
|---|---|---|
| 1 | 1,368856 | 3,81238 |
| 2 | 1,528336 | 3,651112 |
| 3 | 1,164268 | 3,804776 |
| 4 | 0,479516 | 3,863736 |
From sequencing of the entire pASG-IBA3 plasmid, we got the following sequence as a vector map.
Confirmation of gene sequence was done using alignment with NCBI alignment of two or more sequences with standard settings for nucleotide-nucleotide BLAST. The sequenced file was compared with the known gene (sequence in part, BBa_K4450002). Alignment is found here:
| Sequence 1 |
| Sequence 2 |
| sequence.json |
To check for protein expression, we decided to run an SDS-page. Six cultures were prepared with the mutation, separated in induced and non-induced batches. From this, we got the following image which show clear expression in the induced cultures versus the non-induced cells. The protein is approximately 53.5 kDa large. Only batch 4 showed no expression of the protein.
From the dephosphorylation assay we found the following after incubation of plate.
This plate was measured at 655 nm, and we found the following absorbance values.
No statistical observable effect between induced and uninduced culture lysate. This might suggest low expression of UAP1 relative to other phosphatases expressed in the lysate. Another explanation could be denaturation of protein, inhibiting protein activity due to method of cell lysis.
