Our project combines Optogenetics and photobiology to reach an unharmful control during the synthetic process. We invented a new blue light GAL4-motivated system. We built the basic CRY2 and SPA1 blue light control in the yeasts and compared that controlling system with the blue light CRY2/CIB1 controlling system.

1. Plasmid Construction

1.1 Polymerase Chain Reaction (PCR)

We performed PCR to obtain enough target gene fragments for further construction. Then, we used Electrophoresis testing and Gel Recovery. As shown in Fig 1.1, the gel indicates the success of our recovery of the gene fragments, GFP (781bp), UAS (114bp) and SPA1 (1635 bp).

Fig 1.1. Electrophoresis gel results of three essential genes.

1.2 Enzyme Digestion

We doubled the AD. Then, we mixed the reagents. After that, we used PCR to make reactions. In Fig 1.2A, the result of Gel Recovery shows that we have successfully operated double enzyme digestion for the AD gene. In Fig 1.2B, the development of Gel Recovery shows that we have successfully done the process of single enzyme digestion for the BD-CRY2 gene that the strip of AD is 7987 bp and the strp of BD-CRY2 is 6526 bp.

Fig 1.2. Electrophoresis gel results of AD, BD plasmid.

1.3 Homologous Recombination

We calculated the volume of DNA segments. Then, we mixed the materials. In Fig 1.3A, the Gel Recovery result showed our success in recombining AD-SPA1gene (1635bp) and BD-CRY2-N489-GFP (781 bp) gene. Fig 1.3B is our genetic map of the AD-SPA1 gene and BD-CRY2-N489-GFP gene.

Fig 1.3. Electrophoresis gel results of AD-SPA1, BD-CRY2-GFP plasmid.

To ensure that the plasmid construction is 100% correct, we sent them for sequencing and the results shown below in Fig 1.4 confirmed that SPA1 has been successfully attached to the plasmid AD (left) and the GFP has been successfully attached to the plasmid BD-CRY2 (right).

Fig 1.4. Sequencing result of AD-SPA1 and BD-CRY2-GFP

2. Yeast Hybrid

2.1 Yeast culture

We use PCR to heat the carrier DNA and then transfer it with another reagent into the clean bench. Then, we use the coated plate process and rocking inoculation. After that preparation, we culture those bacteria using a solution of bacteria and the defect medium. Figure 2.1 shows the defect medium in the experiment.

Fig 2.1. Yeast growing on the plate.

2.2 Blue Light Reactivity Verification

We culture the bacteria on -Trp-Leu-His-Ade defect medium and -Trp-Leu defect medium at 28℃ under dark and blue light condition irradiation for 48-96h. Here is our result of those two kinds of defect medium and the bacteria on those two kinds of medium. The results showed that yeast in the experimental and control groups grew successfully in the -Trp-Leu culture medium (Fig 2.2A and C) and -Trp-Leu-His-Ade culture medium under blue light conditions (Fig 2.2B). In the dark, the yeast grew successfully in the -Trp-Leu dish but not in the -Trp-Leu-His-Ade culture medium (Fig 2.2D). The results showed that the two plasmids were successfully combined.

Fig 2.2. Yeast growing condition on the different plates.

A and C show the target plasmids BD-CRY2-N489-GFP and AD-SPA1 successfully enter the yeast.

B and D show the yeast that has turned the blue light switch needs blue light to grow, and the plasmids BD-CRY2-N489-GFP and AD-SPA1-N545 have successfully fused.

2.3 GFP Fluorescence Expression Detection

The image below shows yeast under a fluorescence inverse microscope. It can be seen that yeast containing CRY2/CIB1-GFP shows green fluorescence, which GFP produces. When exposed to blue light, the experimental group could see green fluorescence of GFP, while the control group had a dark field of vision and no fluorescence. The experimental group fluoresces as evidence that the proteins can interact to make the GFP gene appear. This image also proves the successful attachment and expression of AD and BD plasmid from the experimental group.

Fig 2.3. Image under fluorescence inverse microscope.

2.4 Sensitivity Evaluation of two Blue Light “Switches”

We test the two different switches during the Yeast Culture process. Here is the result of the traditional switch CRY2/SPA1 (in Fig 2.4) and CRY2/CIB1, our newly-developed switch (in Fig 2.4). The results showed that with the decrease in concentration, basically, the growth of yeast turns slower even with no growth under blue light. Under blue light activation conditions, CRY2 interacts with SPA1 or CIB1, forming a dimer, and in dark conditions, the dimer disassembles, resulting in slow or halted growth in yeast.

It is worth noting that at low concentrations (10^-4 v/vol to 10^-12 v/vol) of yeast, CRY2/SPA1 yeast continues to grow compared to CRY2/CIBI yeast, which essentially stops growing. This indicates that the binding ability of CRY2 and SPA1 under blue light is stronger, and they exhibit higher sensitivity to blue light responses.

Fig 2.4. Concentration gradient test, the volume proportion of yeast solution decreases from left to right

3. 3. β-Galactose Activity Test

3.1 Standard Curve

In order to evaluate the engineering of Gal4-activated gene in CRY2/SPA1 yeast, we conducted β-Galactose activity test with β-galactosidase (β-GAL) Activity Assay Kit from Sangon. We test the activity of β-Galactose for every concentration, from 200 nmol/ml to 0 nmol/ml. We calculate the average value and gain the absorbance value. Then, we formed a curve that perfectly fitted the linear model, like in Figure 3.1.

Fig 3.1. standard curve of β-Galactose Activity: the horizontal axis is the Absorbance (A) and the vertical axis is the concentration (nmol/ml) of β-Galactose

3.2 β-Galactose Activity with Different Time intervals (Dark and Blue Light)

The β-Galactose activity of CRY2/SPA1 was then tested from 0 to 3 hours, and the action was tested with an interval of 30 minutes. The result is shown below in a diagram. It can be seen that the β-Galactose activity of yeast containing novel blue-lights interaction under blue-light condition indicates an increasing trend through time. While in a dark state, the activity does not show a significant change which also proves the engineering success of the blue light “switch”.

Fig 3.2 β-Galactose activity (U) of the yeast (CRY2/SPA1) under blue light and dark light against hours, respectively

According to the literature and experiments, the transplantation of microorganisms is essential in preventing and treating diseases such as clostridium difficile infection, inflammatory bowel disease, inflammatory bowel disease, and diabetes [1].

Chimeric antigen receptor (CAR)-T cell therapy is a revolutionary new pillar in cancer treatment. CAR-T has already shown a significant benefit in clinical service for patients with terminal cancer. This artificial T cell can identify cancer cells and use their hinge to identify the antigen of the cancer cell. After that, the T cell will release cytokine to activate the immune system to fight against the cancer. However, this treatment faces various challenging problems. The over-release of cytokine can lead to a toxic level, thus causing several syndromes such as CRS, which refers to cytokine-release syndrome. CRS can be accompanied by cardiac dysfunction, system failure, and death. A recent method to ameliorate CAR-T toxicity is to use their gene strategies. However, this method is limited by its efficiency.

Our new blue light-dependent interaction can add an “off-switch” on CAR-T cells. Since the interaction is operated by light, the exchange can give an immediate response and prevent the T cell from expressing more cytokine in time. CAR-T is an emerging approach to tumor treatment, and in a vast recent scientific breakthrough, optogenetic technology wirelessly manipulates receptor T cells in vitro. And the switch of our blue light can be applied in this method [2].

[1] M. Cui et al., NIR light-responsive bacteria with live bio-glue coatings for precise colonization in the gut. Cell Rep 36, 109690 (2021).

[2] Nguyen, N.T., Huang, K., Zeng, H. et al. Nano-optogenetic engineering of CAR T cells for precision immunotherapy with enhanced safety. Nat. Nanotechnol. 16, 1424–1434 (2021). https://doi.org/10.1038/s41565-021-00982-5.