Team:NUDT CHINA/Contribution

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CONTRIBUTION

Wet-Lab
New Documentations to an Existing Part

1.1 Overview

Cryptochromes (CRY) are blue-light receptors that mediate various light responses in plants. The photoexcited CRY molecules undergo several biophysical and biochemical changes, including electron transfer, phosphorylation and ubiquitination, resulting in conformational changes to propagate light signals. The cryptochrome-interacting basic-helix-loop-helix 1 (CIB)-dependent CRY2 regulation of transcription is one of the modes of CRY signal transduction.

1.2 Corresbonding Parts

CRY2(BBa_K2980000): This part, submitted by team iGEM19_Tsinghua, gives a brief description that CRY2 is a blue light-stimulated photoreceptor and can interact with CIB1 under blue light stimulation. However, the in vitro functional validation of CRY2-CIB1 interaction is lacking.

CIB1(BBa_K2980002): This part, submitted in 2019 by team iGEM19_Tsinghua, gives a brief description that it can interact with CRY2 under blue light stimulation. The in vitro validation of CRY2-CIB1 interaction is lacking.

1.3 Experiment & Results

To characterize the interaction between CIB1 and CRY2, we inserted the CRY2-CIB1 pairs into a tetR-based system. CRY2 was linked to a tetR DNA binding domain, and the CIB1 was linked to a nuclear localized transcriptional activation domain VP64. Upon co-transfection of these plasmids with a tetO7 promoter driven SEAP reporter, we would be able to evaluate the binding affinity and light responsiveness of the CRY2-CIB1 pairs by measuring SEAP activity in the culture medium.

As we expected, HEK-293T cells were co-transfected with tetR-CRY2, CIB1-VP64 and tetO7-SEAP expressing plasmids showed significantly increased SEAP production upon 24h or 48 h of blue light illumination comparing to control cells transfected with the same plasmids placed in a dark incubator (p < 0.001, Fig.1). these results demonstrated that CIB1 can directly interact with CRY2 and therefore initiate SEAP transcription.


Figure 1. New data of those two parts we collected in this year's project: SEAP activity in the culture medium collect from cells transfected with tetR-Cry2, CIB1-VP64 and tetO7-SEAP expressing plasmids under either blue light illumination or dark condition.

Hardware
New Illumination System for Optogenetic Research

In this project, we intend to achieve blue light-mediated control of cell cycle, the CycleBlue system. Therefore a blue light illumination device which can manually adjust the light intensity and illumination time is needed. The device was built with 4 sets of blue light LEDs (6 LEDs each, 405nm, 1-3W) glued on an aluminum heatsink. The illumination device was covered by an acrylic light shield, with non-transparent surroundings and a clear top. Four standard cell culture plates could be placed on the top of the illumination device. Each LED sets were controlled independently with electromagnetic relays under control of a Raspberry Pi single-board computer. The Raspberry Pi was loaded with ubuntu system and a python-based control script, allowing us to control the on/off time of each set of LEDs conveniently (Fig.2).

For more information, please check it at our Hardware page.


Figure 2. Construction of blue light illumination system. (A) Schematic representation of the system design. (B) Pictures of the blue light illumination system installed in the incubator.