Part:BBa_K2934008
pKatA-LacI-pLac-Glucose Oxidase-Histag for B. subtilis
Design
This part is made to produce the enzyme Glucose Oxidase (GOx) by Bacillus subtilis in a regulated manner. The part contains the Lac operon inhibitor (BBa_K143033), under the regulation of KatA promoter (BBa_K2934002). At growing concentrations of hydrogen peroxide, the suppression of KatA promoter is reduced, bringing to the production of LacI, which will suppress the production of GOx (under the regulation of pLac BBa_K143015). The GOx enzyme production in this circuit has negative feedback.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 1268
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1564
Illegal BsaI site found at 1895
Illegal BsaI.rc site found at 1841
Illegal BsaI.rc site found at 2666
Biological Assay
We planned an experiment in which we will be able to validate our “Honey Circuit” plasmid. We wanted to examine the behavior of “The Honey Circuit” over time, in the presence of glucose and under different levels of hydrogen peroxide, to determine whether the glucose oxidase (GOx) production is being regulated and reaches a steady state.
We constructed the “Honey Circuit” plasmid on a pBE-S commercial plasmid (TaKaRa) backbone and transformed it into B. subtilis. The bacteria were cultured in several Erlenmeyer flasks with LB medium, until the culture reached OD600 of 0.6. The following solutions were added to the flasks as detailed:
| 0 μM H2O2 | 50 μM H2O2 | 100 μM H2O2 | 16% glucose | Wild type (WT) bacteria |
We extracted the bacterial supernatant at five different time-points – 0, 1, 2, 5 hours, and O/N (all based on our modeling) and preformed GOx activity assay on each supernatant.
We hypothesized that GOx enzyme expression exhibits an underdamped oscillating behavior (the system oscillates with the amplitude gradually decreasing to zero): At first, we will see an oscillating concentration of GOx production, and at a certain point, the oscillations’ amplitude will decrease and reach a steady state. This behavior can be explained by our "Honey Circuit" – during its reaction, GOx produces hydrogen peroxide, which activates the KatA promoter and results in LacI production. Consequently, the increase in LacI leads to a decrease in GOx production. As a result, the catalase enzyme that is naturally expressed in B. subtilis breaking the hydrogen peroxide down, the remaining LacI proteins are degraded, and the expression of GOx continues.
At a certain point, the system will reach a steady state in which the levels of GOx, hydrogen peroxide, and LacI will remain balanced.
Our results are shown in figure 3:
Our results show behavior that fits our expectations: there is an altering activity of GOx that resembles an oscillation behavior and reaches a steady state after 26 hours. We assume that performing additional measurements will lead to a more clear oscillation display and to the revealing of the exact point of reaching the steady state.
As seen in the graph (Figure 3), the starting point of each sample is different. A possible explanation is that the primary production of GOx is happening immediately after the initial exposure to the added materials. With the addition of 16% glucose, the highest GOx production has observed, less than at 50 μM, 100 μM, and no H2O2, consistently.
When no H2O2 was added (0 μM, red graph), GOx activity levels reached zero quickly, probably since the bacteria’s GOx expression was already at its sinusoidal decrease at t=0 (while other bacteria were at its peak). After two hours at zero level of GOx activity, the enzyme activity raised to a final value of ~0.005 [U/(ml∙OD600)]. We assume that after a longer period, GOx activity will reach a constant H2O2 level.
At 50 μM H2O2 (green graph), as well as at 100 μM H2O2 (black graph), we can see a drastic decrease in the first two hours, followed by an increase after five hours of incubation to the activity of ~0.015 [U/(ml∙OD600)] and ~0.006 [U/(ml∙OD600)], in accordance. Finally, another decrease to a level of ~0.003 [U/(ml∙OD600)] was for both of the H2O2 concentrations added after 26 hours of incubation. These results can imply that H2O2 initial concentration activated the repression, at the first two hours, by the "Honey Circuit" as expected, and after 26 hours reacheds similar values of GOx activity.
At 16% glucose addition in the LB solution, similar behavior is shown (blue graph). The main difference is at the start and end points, which are considerably higher than other samples, reaching to GOx activity values of ~0.04 [U/(ml∙OD600)] and ~0.0145 [U/(ml∙OD600)], respectively. The higher GOx activity can be explained by the excess of GOx's substrate, glucose, comparing to the remaining samples that contain LB medium only (small amount of glucose). We assume that testing additional time-points of 16% glucose+LB samples, GOx activity value would decrease to ~0.003 [U/(ml∙OD600)] activity.
In conclusion, this assay supports that our "Honey Circuit" is responding to altered hydrogen peroxide levels in the solution, to a final approximal GOx activity value of ~0.003 [U/(ml∙OD600)].
Secretion Assay By SDS-PAGE
To ensure enzyme expression and secretion from our engineered bacteria, we performed SDS-PAGE. To determine whether the enzyme was secreted or expressed within the bacteria, we compared both lysate and supernatant samples of the engineered bacteria, as well as wild type bacteria samples. Methods are listed at the bottom of the page. The experiment results are shown in the following figures:
Western Blot
Western blot (WB) is a precise method compared to SDS-PAGE. By using antibodies to detect our protein's His-tag, it can be guaranteed that our protein had been expressed and secreted from the bacteria.
As seen in Figure 5, the WB test yields a definite result in the Honey Circuit strain's supernatant, meaning that our bacteria express and secrete the GOx protein.
Methods
For further reading of our full protocols (cloning, enzymatic assays and enzymatic assay under different pH levels) look at our wiki protocols page.
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