Team:BIT-China/Design
After literature research, we found that yeast was widely used in aging research for the following reasons:
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● It has the process of apoptosis, which can accumulate ROS.
Like mammalian cells, yeast cells (Saccharomyces cerevisiae) undergoing apoptosis show characteristic markers such as DNA cleavage, apoptosis-typical chromatin condensation, externalization of phosphatidylserine, ROS accumulation, and Cytochrome C release from mitochondria.[1][2][3] - ● As unicellular organism and an excellent model of genetics, yeast is useful for oxidation research.[4]
- ● It has a short growth cycle and relatively simple culture conditions. The protocols of yeast are mature, too.
Thus, S. cerevisiae constitutes an interesting model for studies of the oxidation process, which makes possible experimentation not easily available in higher eukaryotes.
Therefore, we choose it as our host cell. [5]
As mentioned before, we need to keep yeast under the stress of oxidation. Reactive oxygen and nitrogen species (ROS and RNS), known as free radicals, play a key role in the aging and disease by creating vascular oxidative stress. Particularly, ROS account for more than 95% of the total free radicals in the human body. In hence we simulated cell oxidation state through accumulate ROS to finish the test work. As we mentioned in Background part, ROS are mainly divided into four categories: hydrogen peroxide(H2O2), superoxide anion(O2-), hydroxyl radicals(OH-), and single-line oxygen(1[O2]).
All of them are eventually transformed into hydrogen peroxide, in that case we simplify our work to accumulate hydrogen peroxide.
As one of the important apoptosis-mediated factors, ROS could be propose under a variety of stress, such as cold induction, metal ion induction, high pressure induction. However, considering the feasibility, time period, operational difficulty, and the cause of ROS accumulation, we finally accumulated ROS through overexpressing yeast endogenous genes.
It's been established that overexpress some genes can accumulate ROS in yeast cells previously, in hence we selected two genes, yno1 (encodes a genuine NADPH oxidase, which is located in the endoplasmic reticulum (ER) and produces superoxide in a NADPH-dependent fashion) [6] . and ndi1 (codes for the internal mitochondrial ubiquinone oxidoreductase, which transfers electrons from NADH to ubiquinone in the electron transport chain) [7] to achieve our goal of ROS accumulation.
Higher ROS content comes higher mortality rate. In order to prevent the yeast from dying for oxidation, simultaneously, maintain a high level of ROS accumulation in vivo, we need to knock out the gene yca1, which encodes synthesis of metacaspase, a homologue of the mammalian caspase, and is known to play a crucial role in the regulation of yeast apoptosis, for avoiding the death induced by high concentration ROS. [8]
Up to now, we complete the construction of a yeast stain that can accumulate ROS endogenous alive.
Let's see how other parts are built step by step.
When we have already placed the yeast cells in an oxidative state, next step is to control the accumulation of ROS. So we set up the feedback part.
We supposed that the reason of ROS accumulation in our project is the overexpression of related genes. If so, the ROS accumulation can be reduced through repressing gene expression. There are two common global-repressors to inhibit the gene expression, RNAi and dCas9 technology. We compared two technologies and asked our advisor, Dr. Ying Wang for advice. She suggested that we chose dCas9 for it got a higher success rate. We followed her advice and continued other designs.
After choosing the inhibition method, we started to find a ROS sensor with low sensitivity to ROS, since we hoped the sensor would only work under high level of ROS. But it didn’t exist, the only thing we could find in literature was protein Yap1, a basic leucine zipper (bZip) transcription factor from yeast. [9]
Yap1 can sense the cytoplasmic H2O2 produced in mitochondria, which is a kind of signal meaning cells were at a high oxidant condition through an intramolecular disulfide bond generation. There two methionine closed to each other locates on the surface of protein, so when the ROS concentration increases significantly, the two thiol would be oxidized and generate an intramolecular disulfide bond generation leading change of protein structure. Then it will entry the nucleus to active some antioxidants enzymes transcription. Yap1 responds rapidly to cytoplasmic H2O2 due to a enzyme, Gpx3 (Orp1), a kind of catalase which can identify hydrogen peroxide efficiently, through the transformation of intermolecular disulfide bond. [10]
Thus instead of using a sensor that is less sensitive to ROS, we tried to find some weak downstream promoters so that the ligated dCas9 protein would only have significant inhibitory effect when ROS accumulation is forcing yeast into death.
As a ROS detector, our system should not only sense the intracellular ROS content, but be able to output signal, so that we know what happen exactly in the little tiny cells. Time to select a signal output substance. Like what we mentioned in description , we considered glutathione, which could sense ROS in vivo, but could not output, so it’s incompetent. Chemical fluorescent probe was not good enough either since its toxicity and long processing time, we preferred to find something existing in intracellular environment as a signal output. Eventually we found roGFP2---a kind of green fluorescent protein sensitive to redox in the Registry.
The process of roGFP sensing ROS is similar to Yap1. RoGFP is a variant of GFP. Compared with the later one, roGFP has 8 amino acid mutations. Point of its sensitivity of redox states is two Mets which locate in the surface of protein. Once the cell is in a reduced state, the disulfide bond between two methionines will come into been, leading different absorption of light at two wavelengths. Specifically, the response of roGFP2 to H2O2 is reflected to its excitation spectrum (emission wavelength is 515 nm), which has a max absorbing peak at 405 nm and 490 nm. The peak value changes with the oxidation state of environment and this change is reversible. Measuring the ratio of the fluorescence intensity of the roGFP at two wavelengths can reflect the changes of intracellular redox state in real time. [11]
However, roGFP dosen’t only reacts with H2O2, but also other oxides, which means it gets oxidized generally, there’s a lot of interference. So according to information about Yap1, we found that compared with Yap1 and Grx (Saccharomyces cerevisiae gluten), Orp1 has a specificity to H2O2, instead of all kinds of ROS. [12]
Also there is similar transformation of intramolecular disulfide bond in Orp1-roGFP fusion protein to intermolecular transformation between Yap1 and Orp1.
With reference to the part designed by Cornell University in 2017: BBa_K229600 , Constitutive Promoter-RBS-roGFP2-Orp1 C82S, the roGFP2-Orp1 (C82S) fusion protein sensitive to the intracellular redox state response was selected as the signal output of our project.
We also optimized the codon for roGFP2, making it more suitable for translation system of Saccharomyces cerevisiae cells.
Having established each part of our system, we can achieve our original aim “Detect and measure the antioxidant in living cell”. Let’s review how we design the whole gene circuits.
Firstly, in regulator part, overexpression of two genes, ndi1 and yno1, stimulates ROS accumulation in H2O2 form. Through knocking gene yca1, improve the tolerance of yeast cell to high level of H2O2 .
Secondly, H2O2 sensor Yap1, will transfer into cell nuclear to start antioxidant-relative genes transcription once sense the over accumulation of ROS. If the Yap1 starts working, which indicates ROS level is so high that it is dangerous for our yeast cells. So the Yap1-dependent promoters selected from yeast genome will induce the dCas9 system to repress the translation process of ndi1 and yno1, maintaining a safe level of ROS amount in vivo.
Thirdly, through transforming the roGFP-Orp1 expression vector into host, we can easily know how everything going inside the yeast.
Eventually, final test part comes!
The testing antioxidant is added into culture, and the artificial accumulation of ROS will decrease through integrated cell reaction. Some dues to direct antioxidant reaction, other may be removed by cells natural anti-oxidant system which is activated by testing antioxidant. No matter which kind of working way the testing antioxidant is, its antioxidant capacity will be reflected by the remaining ROS level, reported through roGFP-Orp1. In a word, higher ROS remaining represent lower antioxidant capacity.
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