Part:BBa_K3886003
Caffeine Sensor
Trace and Control System was used in Hidro Project by NDNF_China 2020.
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;
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
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