Difference between revisions of "Part:BBa K3886003"
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<p>In our project, we plan to use the Hidro system as a delivery device for the oral cavity or intestinal tract to achieve the expression of drug molecules or health regulatory molecules by referencing common beverages such as coffee. In these scenarios, the caffeine-sensing switch needs to be able to respond effectively to caffeine and eventually turn off the switch after caffeine metabolism is complete.</p> | <p>In our project, we plan to use the Hidro system as a delivery device for the oral cavity or intestinal tract to achieve the expression of drug molecules or health regulatory molecules by referencing common beverages such as coffee. In these scenarios, the caffeine-sensing switch needs to be able to respond effectively to caffeine and eventually turn off the switch after caffeine metabolism is complete.</p> | ||
<p>The results of our mathematical simulations meet these expectations, and the model developed here will have the potential to guide actual dietary inputs to achieve the desired health modulation effects.</p> | <p>The results of our mathematical simulations meet these expectations, and the model developed here will have the potential to guide actual dietary inputs to achieve the desired health modulation effects.</p> | ||
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| + | </html> | ||
| + | ===Application of the caffeine–controlled genetic switch based in Hidro system=== | ||
| + | <html> | ||
| + | <p>In the project of NDNF_China 2021, we hope to help engineered strains work beyond the laboratory in a safe, stable and traceable way. So we present Hidro: a hydrogel system enclosing engineered bacterial strains. The outer layer of Hidro is a compact shell, offering both protection and containment, preventing the strains from escaping into the wild; the inner core of Hidro provides a supportive environment for them under harsh conditions, thus enabling their stable function; A genome-integrated Tracing and Control system offers tracking and specific killing of engineered strains in case of emergencies. We have experimentally demonstrated that Hidro can be implemented in diverse scenarios, such as heavy metal sensing, food-quality detection, drug secretion, etc. The Hidro system has the great potential to promote synthetic biology applications beyond the laboratory.</p> | ||
| + | <p>We first tested whether our caffeine–controlled genetic switch could be integrated into the Hidro system. After encapsulating engineered bacteria into Hidro beads and incubating with and without caffeine molecules respectively, we slice the beads at a thickness of roughly 0.5 mm. The sliced samples are then imaged with a fluorescence microscope under green light. It was confirmed visually that Hidro exposed to caffeine exhibited strong red fluorescence.</p> | ||
| + | <div style="text-align: center;"> | ||
| + | <img src="https://2021.igem.org/wiki/images/4/42/T--NDNF_China--part_caffeine_hidro.png" alt="" width="500"> | ||
| + | <h6 style="text-align:center">Figure 7: (A) The schematic of caffeine–controlled genetic switch encapsulated in the Hidro system; (B) The image of Hidro taken by a fluorescence microscope under green light.</h6> | ||
| + | </div> | ||
| + | <p>The Hidro system has potential for safe drug delivery in food and the human gut (See more information in NDNF Proposed Implementation). So we then designed a caffeine–controlled L-Dopa production system based on the switch designed here (Figure 8A).</p> | ||
| + | <p>Derived from the biosynthesis of L-tyrosine (Figure 8B), L-Dopa is a naturally occurring amino acid that acts as a precursor to a number of neurochemicals such as adrenaline and dopamine. L-Dopa is an amino acid that is made and used as part of normal biological functions in humans.</p> | ||
| + | <p>We replace the output gene with L-Dopa metabolic pathway HpaB and HpaC, each with a specifically designed RBS (Figure 8A). After 24 hours of incubation in a tube, we removed the Hidro beads, centrifuged the liquid and took out the supernatant for L-dopa concentration measurement. From the result shown in Figure 8C, we can see that the Hidro group showed L-dopa production compared to those in the control group. It means that Hidro can realize the sensing and screting process. It further proves that Hidro has a great potential to be applied in Health.</p> | ||
| + | <div style="text-align: center;"> | ||
| + | <img src="https://2021.igem.org/wiki/images/6/63/T--NDNF_China--part_caffeine_L_Dopa.png" alt="" width="500"> | ||
| + | <h6 style="text-align:center">Figure 8: (A) The schematic of caffeine–controlled genetic switch encapsulated in the Hidro system to produce L-dopa; (B) The metabolic pathway from L-tyrosine to L-Dopa. (C) L- Dopa concentration measurement in the caffeine–controlled L-Dopa production Hidro system.</h6> | ||
| + | </div> | ||
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Revision as of 11:46, 20 October 2021
Caffeine Sensor
Trace and Control System was used in Hidro Project by NDNF_China 2020.
Contents
Characterization
Introduction
Recent advances in synthetic biology have required the design of application-specific control systems that are functionalized to perform the user-defined and precisely controlled regulation processes. Initially, some common inducers like IPTG, tetracycline were used for the control of gene expression, but these wildly used inducers raised issues such as antibiotic resistance and side effects, especially in long-term applications. Traceless inducers, such as light or temperature, have recently been developed, but ambient light and ambient temperature make them less orthogonal than would be desirable.
The ideal inducer would be inexpensive, would have no side effects, and would be present in only a specific set of known sources. It has been proposed that ideal trigger molecules would be natural, nontoxic, highly soluble, inexpensive, and perhaps even origin from daily life.
Caffeine is a strong candidate. The caffeine is non-toxic, cheap to produce, and present in specific beverages, such as coffee and tea. Every day, more than two billion cups of coffee are being consumed worldwide, making coffee one of the most popular beverages after water.
Design a caffeine–controlled genetic switch
Here, NDNF_China 2021 have developed a sensitive engineered genetic system in response to dietary intake of coffee or other caffeine-containing beverages and characterised them in Hidro, a hydrogel system. This beverage-derived caffeine–controlled gene circuit expands the synthetic biology toolbox available for constructing safe and clinically relevant cell functions and have the potential to substantially advance Hidro application in health like bacterial therapies.
We used two of these domains: the single-domain VHH camelid anti-caffeine antibody; (referred to as acVHH) that homodimerizes in the presence of caffeine. In two separated acVHH domains, each was fused with a 10-residue linker, into the contiguous M86 intein. The intein was already inserted between ECF20(1–101) and ECF20(102–193). The resulting constructs were bipartite proteins, with each part driven by a constitutively-expressed promoter J23110. So in the presence of caffeine, they could homodimerize and reconstitute a complete ECF20 with the promotion of M86 intein and activate the downstream promoter, pECF20.
Figure 2: The design scheme of a caffeine–controlled genetic switch;
Characterization of a caffeine–controlled genetic switch
In order to test the performance of caffeine–controlled genetic switch, we first chose fluorescence protein mScarlet to be our reporter gene. The engineered strain containing this switch is first incubated with different concentrations of caffeine molecules in a liquid medium. Plate Reader test results show that fluorescence intensity increased with the increase of caffeine concentration, which reveals the successful construction of the circuit.
Figure 3: The response curve of caffeine–controlled genetic switch. Samples prepared in triplicate, data represent the mean ± 1 s.d.
To analyze the performance of different designs, we fitted the sensors’ dose–response curves to a Hill function-based biochemical model to describe their input-output relationships.
- The Hill constant EC50 is the inducer concentration that provokes half-maximal activation of a sensor; EC50 is negatively correlated with sensitivity.
- KTop is the sensor’s maximum output expression level; KTop is positively correlated with output amplitude.
From the data, we determined the following parameters：
Modelling of a caffeine–controlled genetic switch
Modelling in synthetic biology is a powerful tool that allows us to get a deeper understanding of our system. It guided and assisted the design of our detection system which helped us save a lot of time by avoiding dead-end designs.
We have designed a genetic switch that can be induced by caffeine. We expected to describe the activation of the caffeine-inducible switch through mathematical modeling to give a quantitative understanding between signal input and output, and thus provide guidance for realistic scenario applications.
Figure 4: The abstract scheme of the caffeine–controlled genetic switch in a cell. The caffeine nanobody-intein-ECF20 (NIE) is split separately, and then divided into two parts for expression. Then the input of caffeine will induce the recombine of split caffeine nanobody-intein-ECF20(NIE) protein (NIE), and then under the action of the intein, the split ECF20 will be recombined and the completed ECF20 will be released in the cytoplasm. Then the ECF20 will activate the pECF promoter and the expression of corresponding output.
Chemical formulas
* It describes the process of combining NIEα(1/2) protein and NIEβ(1/2) protein into NIE complex, and the breakdown of the NIE protein back into two separate parts.
* It describes the process that NIE binds to caffeine, forming a NIE-caffeine complex, which gradually turns in to ECF20 molecule and NI-Caffeine complex.
* It describes the process that NIE-caffeine complex gradually turns in to NI complex and Caffeine molecule.
Abbreviations
- NIE: Complex of Nanobody(acVHH)-Intein-ECF20
- NI: Complex of Nanobody(acVHH)-Intein
ODE Equations
Based on the the above chemical reaction formulas, ODE equations were used to describe the process using diverse parameters:
* Describe the protein expression of caffeine nanobody - intein -ECF20 (NIE) part α.
* Describe the protein expression of caffeine nanobody - intein -ECF20 (NIE) part β.
* Describe the protein production changes of caffeine nanobody-intein-ECF20 complex（NIE）.
* Describe caffeine concentration changes in the environment.
* Describe the protein concentration changes of the NI-caffeine complex.
* Describe the protein concentration changes of the NIE-caffeine complex.
* Describe changes in the levels of ECF20 protein in the environment.
* Describe the process of ECF20 activating mCherry mRNA expression.
* Describe the expression of mCherry protein.
Parameters annotion
Simulation Results and analysis
As can be seen from the results of the following analysis, the output of the caffeine sensor undergoes a rising at the beginning and then shows a decreasing trend after caffeine is completely consumed. Eventually, the output of the caffeine sensor gradually converges to 0 as the caffeine molecule is degraded or metabolized.
Figure 5: Trends of consumption of Caffeine molecules.
Figure 6: Trend of relative fluorescence intensity of mCherry with time
In our project, we plan to use the Hidro system as a delivery device for the oral cavity or intestinal tract to achieve the expression of drug molecules or health regulatory molecules by referencing common beverages such as coffee. In these scenarios, the caffeine-sensing switch needs to be able to respond effectively to caffeine and eventually turn off the switch after caffeine metabolism is complete.
The results of our mathematical simulations meet these expectations, and the model developed here will have the potential to guide actual dietary inputs to achieve the desired health modulation effects.
Application of the caffeine–controlled genetic switch based in Hidro system
In the project of NDNF_China 2021, we hope to help engineered strains work beyond the laboratory in a safe, stable and traceable way. So we present Hidro: a hydrogel system enclosing engineered bacterial strains. The outer layer of Hidro is a compact shell, offering both protection and containment, preventing the strains from escaping into the wild; the inner core of Hidro provides a supportive environment for them under harsh conditions, thus enabling their stable function; A genome-integrated Tracing and Control system offers tracking and specific killing of engineered strains in case of emergencies. We have experimentally demonstrated that Hidro can be implemented in diverse scenarios, such as heavy metal sensing, food-quality detection, drug secretion, etc. The Hidro system has the great potential to promote synthetic biology applications beyond the laboratory.
We first tested whether our caffeine–controlled genetic switch could be integrated into the Hidro system. After encapsulating engineered bacteria into Hidro beads and incubating with and without caffeine molecules respectively, we slice the beads at a thickness of roughly 0.5 mm. The sliced samples are then imaged with a fluorescence microscope under green light. It was confirmed visually that Hidro exposed to caffeine exhibited strong red fluorescence.
Figure 7: (A) The schematic of caffeine–controlled genetic switch encapsulated in the Hidro system; (B) The image of Hidro taken by a fluorescence microscope under green light.
The Hidro system has potential for safe drug delivery in food and the human gut (See more information in NDNF Proposed Implementation). So we then designed a caffeine–controlled L-Dopa production system based on the switch designed here (Figure 8A).
Derived from the biosynthesis of L-tyrosine (Figure 8B), L-Dopa is a naturally occurring amino acid that acts as a precursor to a number of neurochemicals such as adrenaline and dopamine. L-Dopa is an amino acid that is made and used as part of normal biological functions in humans.
We replace the output gene with L-Dopa metabolic pathway HpaB and HpaC, each with a specifically designed RBS (Figure 8A). After 24 hours of incubation in a tube, we removed the Hidro beads, centrifuged the liquid and took out the supernatant for L-dopa concentration measurement. From the result shown in Figure 8C, we can see that the Hidro group showed L-dopa production compared to those in the control group. It means that Hidro can realize the sensing and screting process. It further proves that Hidro has a great potential to be applied in Health.
