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;

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.

Modelling of a caffeine–controlled genetic switch

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 4444
  Illegal BamHI site found at 1214
  Illegal BamHI site found at 3313
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1049
  Illegal AgeI site found at 3148
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 1031
  Illegal SapI site found at 3130