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Team:UST Beijing/Project
From 2014.igem.org
Revival of the magic.
PROJECT NAME
Vitamin C synthesis pathway in animals. The reactions are catalyzed by the following enzymes: 1, UDP-glucose pyrophosphorylase; 2, UDP-glucose dehydrogenase; 3, nucleotide pyrophosphatase; 4, UDP-glucuronosyltransferase; 5, UDP-glucuronidase; 6, phosphatase; 7, b-glucuronidase; 8, glucuronate reductase; 9, gulonolactonase; 10, L-gulonolactone oxidase. Three possible mechanisms for glucuronate formation (a, b and c) are shown. SMP30 KO mice, senescence marker protein 30 knockout mice; ODS rats, osteogenic disorder Shionogi rats; od ⁄ od pigs, mutant pigs deficient in L-gulonolactone oxidase; GLO KO mice, L-gulonolactone oxidase knockout mice. Source: FEBS Journal 274 (2007) 1–22.
Comparison between mouse and human GLO gene locus identified only 6 exons on human genomic DNA. These exons also carry a few nucleotide mutations. These remaining exons include sequences coding for both FAD binding and D-arabinono-1,4-lactone oxidase(ALO) domain of GLO enzyme protein. This evidence indicates that human GLO gene used to be active in the past. Source: Nishikimi M et al JBC (1994) 269:13685-88.
The capacity of vitamin C synthesis of the several common species in the evolutionary tree is compared. As we can see in the picture, the dark branches show the species that can self-produce VC, while the light branches represent the species lack the ability, including human and other primates. But some species which share the same origins with human still have the GLO gene to produce vitamin C like Galago and Lemur. So we tried to find the functional GLO gene from these species to complete the capacity of VC synthesis of human.
An Alternative hypothesis believes that the losing of GLO gene, production of which catalyze final production of vitamin C, could be advantageous because this Vitamin C (VitC) production is at the expanse of hydrogen peroxide formation[Free Radical Research, July 2005; 39(7): 671–686] and depletion of glutathione. However, some species of birds and bats have regained their capability of vitamin C synthesis after they lost it in the evolution process, which means such capability is still helpful. Another evidence is that GLO gene knock out mice show some disability in environment adaptation [Immune Network 2012;12(1):18-26]. We believe that reactivation of GLO in animal is more likely to be advantageous but the expression rate should be carefully controlled because the overwhelming amount of VitC could interfere the metabolism in cells. Since the promoter and other regulatory sequence of GLO Pseudogene have already lost its original function, new method was needed to determine appropriate expression rate. Considering the function of Vc as Antioxidant, proteorhodopsin as a light driven proton pump was designed and introduced to simulate oxygen species rich environment. Proteorhodopsin, when exposed to light, can contribute greatly to free radical production was used to titration the possible effect of different amount of GLO expression.
Some studies suggest that losing the ability to synthesize vitamin C might be advantageous because vitamin C over-production leads to the formation of hydrogen peroxide and the depletion of glutathione. There is also an alternative hypothesis. Since GLO gene reactivations have been documented in bat and bird species, the former hypothesis is no longer tenable. Some evidence of the evolutionary tree in the two pictures above shows that there are some species in the lack of the capacity branch regain the ability to synthesize vitamin C during the natural selection. So regaining the capacity to synthesize vitamin C might be beneficiary if the synthesis is properly controlled.
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Synthetic human GLO cDNA sequence was generated using GeneOptimizer software at life technology website.
We downloaded GLO gene sequences of several species from database and analyze these sequences with CLC sequence viewer software. Comparison results of promotor and other regulation sequences from different dietary animals are shown in this picture. The signature sequences conserved can be seen in boxes. The sequences between the black ball and the code ATG represent exons that are still found in the genome of these species. Translation initiation of translation starts from the codon ATG.
Mitochondrial proteorhodopsin (mPR) is a transmembrane protein which could make protons flow when irradiated by the light. When it was irradiated by the light, the small molecular within the protein would be changing, and when it replies, the protein would be changing, too. All of these will make protons finish the transmembrane transport and formation of protons flow. Source: UST_Beijing iGEM 2011
A new Biobrick device: Mitochondrial proteorhodopsin (humanized genetic code bias)
Partsregistry submission：
BBa_K603000 (pSB1AC3)
BBa_K603001 (pSB1C3)
Transient co-expression of mitochondrial proteorhodopsin (mPR) and GFP in human embryonic kidney cells HEK293.
As we can see from the pictures taken by fluorescence microscope, transient expression of mitochondrial proteorhodopsin (mPR) and GFP genes in human embryonic kidney cells HEK293 is obviously related to the light exposure. In the dark (None) GFP expression rate is lower than the weak light exposure (weak) . The flow cytometry result also shows that the light exposure increased level of transient GFP expression ( accumulative green fluorescence is intensity 195.84) in HEK293 cells co-transfected with mPR and GFP expression vectors compared with no light exposure (168.23). The culture condition is nutrient-poor, therefore energy harvest from light is positively correlated to GFP expression.
Under nutrient-rich culture conditions, GFP-expressing cells displayed light-induced toxicity. The images of GFP expression in HEK293 cells illustrate related apoptosis because of excessive free radical due to electron-chain overload, which is produced either in the exposure to light or presence of mPR , or both of them. Certainly, the existence of oxygen free radical can jeopardize the survival of cells.
(A) Transient GFP expression in HEK293 cells without mPR transfection but light exposure.
(B) Transient GFP expression in HEK293 cells without mPR transfection in darkness.
(C) Transient GFP expression in HEK293 cells transfected with mPR and light exposure.
(D) Transient GFP expression in HEK293 cells transfected with mPR in drakness.
The graph above shows that the amount of surviving cells is related to the change of Vitamin C (VitC) concentration. When the fold of dilution of Vit C is zero , the amount of surviving cells is relatively high (probably due to Vitamin C poor solubility). However, with the growth of fold of dilution, the number of surviving cells declines, because high concentration of VitC makes media acidic which can do harm to cells. When the fold of dilution comes to two, the cells are weakest. When higher than 2 fold of diluton, the VitC contributes the growth of amount of surviving cells. The higher the fold of dilution of VitC before seven, the higher the amount of surviving cells was. After the seven fold of dilution of VitC, the amount of surviving cells will decrease due to too low Vit C concentration.
