Project-Proof of Concept

Biological components

The following contents demonstrated the practicability of our biological components.

- Test levodopa yielding
- Test photoswitch efficiency
- Test levodopa yielding controlled by light
- Prove the existence of enzyme in bacteria

Test photoswitch efficiency

Here is the result of the photoswitches efficiency test, which is what we have mentioned before in the experiment part (Measure photoswitches efficiency). After we have harvested the cells, they are applied the fluorescence intensity measurement.
We divide fluorescence intensity by OD 600 and get a relative GFP expression condition. The values are applied to view the photoswitches efficiencies by ratio. To convey the result more directly, we illustrate the following column diagram. A truncation is made by us to put all data in one graph.

Only effective photoswitches are presented in the graph. In these groups, blue bars (Light) are higher than the black bars (Dark). This means GFP is expressed in a larger amount in Light groups. This indicates a successful construction of photoswitches.

Test enzyme efficiency

To support the model, the data of enzyme efficiency is necessary. In this test, leveled concentrations of the substrate, tyrosine, (0mM, 0.03mM, 0.3mM, 1.5mM, 3mM, 4.5mM, 6mM, 9mM) are prepared. We then inoculate Bacteria in 7 mL LB medium and take for 500μL each time. They are taken at specific points: 30h, 39h, 42h, 50h.
Cultures are observed turning dark:

We applied levodopa measurement on levodopa concentration by the method we have mentioned before.

The graphs here illustrate the levodopa yielding concentration at the initial substrate concentrations of 1.5mM, 3mM, and 6mM. In most groups, SMS yields the most, followed by WT (wildtype). Confusing results are in the group with 0mM substrate tyrosine. This is because we use LB medium in our experiment. LB medium is rich in nutrients and contained lots of tyrosine. If we need to get a more refined result, we should change it to M9. An interesting phenomenon is SMS samples have a burgeon after 30 hours but decline after 40 hours. This is more obvious at a lower substrate concentration. One assumption is that there may be an enzyme that decomposes levodopa. And in a low concentration, the levodopa productivity cannot meet the metabolic rate. As substrate concentration is increasing, the yielding increase. The most representative is the T7EA1 group. Since the enzyme expression level is low, a difference caused by enzyme saturation is magnified. A more detailed mathematical analysis is on the model page.

Test levodopa yielding under light regulation

Since the levodopa yielding needs to be regulated, we combine the systems and test the efficiency of the combined system. Samples are taken for 1mL each time in several points: 8h, 16h, 24h, 28h, 32h, 44h. We applied a levodopa measurement for each sample. VVD and pMagFast2-nMagHigh1 are efficient and picked. In the following graph, we illustrate the yielding condition of the two photoswitches under different culture conditions. In this graph, we can see our engineered E. coli produce levodopa at a relatively high rate in the Light group, but lower in the Dark group.

SDS-PAGE proves the existence of HpaB and HpaC

Though the cultures turn black and OD 400 meets expectations, we still want to confirm whether our bacteria express HpaBC. Therefore, we apply the SDS-PAGE to examine the sample. In the first gel, we find the difference in HpaB expression but obscure in the HpaC site. To identify HpaC, we redo SDS-PAGE on the same samples. Before the second gel, we make justification by measuring the concentration of protein. We carried measurement by a Beyontine protein concentration kit. In the second gel, though the samples are the same, we only find a difference in HpaC expression.

Hardware

The Bracelet

1.A gyroscope is installed in the bracelet; gyroscope is a commonly used part and with a diameter of only 16mm and a length of only 20.5mm, which is much smaller than the current component in the bracelet.

2.Our Bluetooth signal only needs to cover a short distance--from wrist to stomach--so disconnection or interference wouldn’t be a problem. Long-distance signaling is achievable as well, because the long-distanced signals are transmitted within familiar conditions, which means that we don’t need to modify modern technology to achieve our goal. The principle of transmission from bracelet to doctors is identical to that of people control their washing machine at home from their workplace.

The capsule

1.Lexan has already been proved to be both acid-proof and basic-proof. Therefore, we are convinced that using it as the outer shell of the lighting part wouldn’t do any harm to humans. Only ultraviolet and strong acid or basic could corrode Lexan.Fortunately, none of them would naturally exist inside the human body.

2.Though blue light indeed has some photo-toxicity, researches proved that only strong ultraviolet could really harm. The light needed to activate our engineered bacteria too slight to cause damage.

3.The battery’s durability is not a problem, because LED only consumes a small amount of light to ignite. With the existence of “button” cell, our hardware can sustain for a sufficiently long time..

Team:SMS Shenzhen/Proof Of Concept