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Cloning

Our cloning results were confounding initially, however, we were able to create what appeared to be successful clones of our parts in pTrc99a as indicated by diagnostic restriction digests

Clones screened in this manner were sent for sequencing and we received primarily positive sequencing results. Sequencing results can be found HERE.

This sequencing data led us to believe we had our parts successfully in pTrc99a allowing us to continue with our cloning as laid out on our Wet Lab page.

We continued our cloning using primers designed to amplify both our parts and the trc-promoter from the pTrc99a backbone as the part BBa_K2042004 did not have samples in the registry. We were able to successfully amplify our parts, and select potentially successful clones using diagnostic digests. Several of our positive clones were then sent for sequencing and the data can be found HERE.

We also confirmed the expression of our parts using a Native SDS PAGE Gel stained using Coomassie staining seen below.

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HRP Results

Our testing of Horseradish peroxidase (HRP) began with measuring the absorbance of HRP in hydrogen peroxide (H_2 O_2) at 408 nm using UV vis spectrometry. We added HRP in small aliquots to an excess of H_2 O_2. This test yielded very small absorbances, but nonetheless showed an upward trend, which was expected. We expected the solution to turn red, but that did not happen, so we transitioned to other testing.

The next phase of testing involved chemiluminescence. A 10% hydrogen peroxide solution was made volumetrically and brought up to volume with pH 7.9 1X PBS (phosphate buffered saline). A ~500X luminol solution was made using 0.0145g of luminol and 1 mL of DMSO. 2 mL of the hydrogen peroxide solution with 20 uL of the luminol solution served as a control because it showed how much light was emitted from that without any HRP. Tests were done with 1, 2, and 3 uL of 1 ug/mL HRP added to a control solution. Each sample was run for 30 minutes, and two replicates were done of each sample, to end with 3 trials for each set of data. A consistent max light emission was observed between 5 and 10 minutes, making this the optimal measuring range for use in a test kit.

Here is an example of a fluorometer graph from Trial 1 of 1 uL of HRP

Averages were taken every 150 second interval.

The next stage of testing involved using tetramethylbenzidine (TMB). TMB, used in conjunction with HRP, yields a colored product. When HRP is added, the solution turns blue, then green, and finally yellow. The blue product is measured at 652 nm and the yellow product is measured at 450 nm. 1 X TMB was diluted to a 5% solution with diH2O. The 100 ug/mL HRP solution was diluted to a 2% solution, and varying amounts of that were used. 600 mL of TMB was used for each trial with 2 uL, 5 uL, or 8 uL of HRP. The uv vis of the solution was taken every ~0.5 seconds. The graph below shows how an increase in HRP increases the speed of the reaction, allowing it to reach the maximum absorbance at 652 nm sooner.

Initial Proof of Concept Testing

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Prototype Testing

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