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<span class='h3bb'>Sequence and Features</span> | <span class='h3bb'>Sequence and Features</span> | ||
Revision as of 11:56, 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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 4444
Illegal BamHI site found at 1214
Illegal BamHI site found at 3313 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1049
Illegal AgeI site found at 3148 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 1031
Illegal SapI site found at 3130