Microwell Temperature Control Using BcLOV4 Protein Variants
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Solution Overview
Problem
Current methods for remotely controlling proteins and cells, such as blue light-activatable systems and heat shock promoters, face limitations including inefficient tissue penetration, non-specificity, and potential tissue damage, with no means to achieve focused thermal control for post-translational modifications or signaling.
Innovation Solution
A device comprising a microwell plate with a temperature control assembly and a microcontroller to independently control temperature in each well, combined with a temperature-responsive protein variant (BcLOV4) that undergoes temperature-dependent membrane localization, allowing precise thermal control of proteins and cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Illumination intensity
If blue light is used to control cells remotely, then cell activation is achieved, but tissue penetration is limited to approximately 1 mm
Solution Approach 1:
The patent uses an upconverting nanoparticle intermediary that absorbs multiple low-energy infrared photons and converts them to a single high-energy visible photon. This mediator enables deep tissue penetration via infrared light while achieving the high-energy activation needed for cellular control, resolving the contradiction between penetration depth and activation efficiency.
2Adaptability or versatility
If heat shock promoters are used for thermal control, then transcription is induced, but the system responds non-specifically to other cell stresses and endogenous cellular programs
Solution Approach 1:
The patent employs a localized thermal control system where individual microwells can be heated independently to precise temperatures using integrated heating elements and thermistors. This localized approach ensures that only the intended target experiences the temperature change, eliminating non-specific responses to other cell stresses and achieving high spatial and temporal specificity.
3Productivity
If extended heating is applied to generate strong output from heat shock promoters, then transcriptional control is achieved, but surrounding tissue is damaged
Solution Approach 1:
The patent divides the heating system into independent microwell units, each with its own heating element and temperature control. This segmentation allows precise thermal control of individual wells at physiological temperatures, achieving the desired biological output without the extended high-temperature heating that causes tissue damage in conventional heat shock systems.
4Temperature
If conventional temperature control methods are used, then bulk temperature control is achieved, but focused thermal control for post-translational modifications is not possible
Solution Approach 1:
The patent segments the temperature control system into independent microwell units with individual heating elements and thermistors, enabling focused thermal control of specific wells. This modular segmentation achieves precise localized temperature control for post-translational modifications while maintaining manageable system complexity through standardized control architecture.
Solution Approach 2:
The patent implements feedback control using thermistors to monitor temperature in each microwell and adjust heating accordingly. This feedback mechanism enables precise temperature maintenance for post-translational modifications while the automated control system manages the complexity of multiple independent control loops.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Enables precise and focused thermal control of proteins and cells, overcoming the limitations of existing methods by allowing remote and on-demand control of protein activity and cellular processes without tissue damage, with the BcLOV4 protein variant providing a single-component solution for temperature-sensitive membrane localization and signaling.
Implementation Method 1
the heating thermistor comprises a resistive heating thermistor
Implementation Method 2
each of the at least one pair of thermistors comprises a heating thermistor and a measurement thermistor
Data Source
AI summary
Provided herein are a device for well plate temperature control, a method of independently controlling the temperature in individual wells of a microwell plate, and a temperature-responsive protein. The device includes a microwell plate with at least one well formed therein; a temperature control assembly including a printed circuit board with at least one pair of thermistors extending therefrom, each of the at least one pair of thermistors arranged and disposed to align with one of the at least one wells; and a microcontroller configured to individually control each of the at least one pair of thermistors. The method includes positioning the temperature control assembly adjacent to the well plate such that each pair of thermistors extends into one of the wells, and independently providing a current flow to each of the at least one pair of thermistors to separate heat each well. The temperature-responsive protein includes a BcLOV4 protein variant having a point mutation at Q355, the variant having at least 80% sequence homology with the wild-type BcLOV4 protein.