Difference between revisions of "Team:SCAU-China/Model"

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<div class="p_font_size"><small>Figure 10 The biosynthesis pathway of astaxanthin formation in transgenic rice endosperm. The dotted arrows indicate pathway limitations in rice endosperm. The solid arrows indicate the existence of carotenogenic reactions. The red arrows indicate the reactions catalaysed by four exogenous transgenes Psy, CrtI, BHY and BKT.</small></div>
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<div class="p_font_size" style="margin-bottom:20px"><small>Figure 10 The biosynthesis pathway of astaxanthin formation in transgenic rice endosperm. The dotted arrows indicate pathway limitations in rice endosperm. The solid arrows indicate the existence of carotenogenic reactions. The red arrows indicate the reactions catalaysed by four exogenous transgenes Psy, CrtI, BHY and BKT.</small></div>
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<div class="p_font_size"><small>Figure 11 Correlation analyses between expression levels of four genes involved in astaxanthin biosynthesis and astaxanthin content.</small></div>
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<div class="p_font_size" style="margin-bottom:20px"><small>Figure 11 Correlation analyses between expression levels of four genes involved in astaxanthin biosynthesis and astaxanthin content.</small></div>
 
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<div class="p_font_size">The change in concentration of protein Y is due to the difference between its production and degradation/dilution, as described by a dynamic equation:<div>
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<div class="p_font_size">The change in concentration of protein Y is due to the difference between its production and degradation/dilution, as described by a dynamic equation:</div>
 
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<div class="p_font_size">In the first step of our modeling, we support that all genes have the same expression way in the cell. We support that all the input signals appear at time t=0. Because we don’t have enough time to measure transcription and translation rates of the genes, we assume that all the proteins have the same production rate and this rate are supported to be 200 nM/h. To measure the degradation rate of protein, αdeg, T1/2 is used in the following calculation, in which period of time the protein decrease by 50%.T1/2 of each protein is determined by their Instability index (II). Degradation rates of protein can be calculated by the aquation, T1/2 =log(2)/α.Yst means steady-state concentration of the proteins.</div>
 
<div class="p_font_size">In the first step of our modeling, we support that all genes have the same expression way in the cell. We support that all the input signals appear at time t=0. Because we don’t have enough time to measure transcription and translation rates of the genes, we assume that all the proteins have the same production rate and this rate are supported to be 200 nM/h. To measure the degradation rate of protein, αdeg, T1/2 is used in the following calculation, in which period of time the protein decrease by 50%.T1/2 of each protein is determined by their Instability index (II). Degradation rates of protein can be calculated by the aquation, T1/2 =log(2)/α.Yst means steady-state concentration of the proteins.</div>
 
 
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<table class="table">
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<thead>
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<th>Protein names</th>
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<th>Gene names</th>
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<th>Organism</th>
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<th>Instability index (II) </th>
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<th>β</th>
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<div class="p_font_size">As our project is to use the rice as a bioreactor and produce astaxanthin,and we also hope that aSTARice can serve as a kind of food in human’s daily lives,so we are very concerned about the safety of aSTARice.</div>
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<th>αdeg</th>
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<div class="p_font_size">We believe that risks are mainly embodied in the following two aspects: one is that the encoding products of marker genes might be toxic and allergenic potentially for human or livestock. Antibiotic gene might be transferred into gut microbes’ genome of humans or animals , increasing the resistance of the microbes of the antibiotic, thus result in reducing the effectiveness of antibiotics in clinical treatment;the other one is that marker genes could be spread through pollens or other methods into other organisms,which is called genetic drift. Genetic drift can transfer antibiotic genes to weeds or other plants,which is threatening the ecological environment.</div>
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<th>Yst</th>
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<div class="p_font_size">In order to reduce the effects of antibiotic marker gene, we use a technique called selectable marker-free technique in the project, so the final aSTARice is an marker-free transgenic plants(MFTPs).We design a Cre-loxP based side-direct recombinant system to knock out hygromycin phosphotransferase gene,minimizing the effect of the selectable maker gene.</div>
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<div class="p_font_size">In order to reduce the risk of genetic drift as well as the threat to the ecological environment, we add isolation area to surround the experimental fields.By isolating the transgenic rice,we reduce the possibility of genetic drift duel to the transfer of the pollen,sufficiently guaranteeing the safety of the project.Whats more,we are also analyzing  coding products by HPLC and trying to make sure that there is no harmful products in our rice.</div>
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<div class="p_font_size">Rice was chose as the chassis though it isn’t included in the white line,but we checked-in with the official website in the beginning.Because we are the first team who choose rice as chassis,we have to asses the safety for rice.</div>
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<div class="p_font_size">Rice is the self-pollinated plant so that the gemotype in the individuals is pure and homogeneous.The probabilities that two flowers take place cross-pollinated are just 1%，and the distance of the spread of rice pollen are hardly 1meter.</div>
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<td>Actin</td>
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<div class="p_font_size">The phenomena of pollen escape are the main ways which lead to the flow of exogenous gene of transgene plants.Recently,the reaches of transgene plants verified that the exogenous gene of transgene plants will flow to the same species or weeds and even the conventional species.As igemers,we took safety into consideration carefully so that we saw plentiful papers ,and that we found a conclusion that under the close distance less than 1% adjacent non-genetically modified plants take place gene flow.If we increase the distance to 5~10 meters,the probabilities decrease to 0.001%~0.0001%,which will not happen in theory.when planting rice,we follow seriously the safe regulations of transgene plantsSo we have faith that it is impossible for gene flow come up under this plant distance.</div>
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<td>OsAct1</td>
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<td>Oryza sativa subsp. japonica</td>
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<div class="h2_font_size">Design for marker free</div>
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<td>34.96</td>
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<div class="p_font_size">In this part, we used a site-specific Cre/loxP recombination to delete the selective marker. To delete the selective resistance gene in transgenic rice, a marker-free element was used to assemble into four-gene multigene vector. This marker-free element was placed between two loxP sites, and consists of a HPT (hygromycin) resistance gene expression cassette and a Cre gene expression cassette controlled by anther-specific promoter (Figure 4). When Cre gene was expressed in transgenic rice anther, the Cre enzyme deleted the marker-free element between two loxP sites. <a href="https://2016.igem.org/Team:SCAU-China/Design#Makerfree" text-decoration=underline>You can read more details, please click here! </a></div>
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<td>200 nM/h</td>
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<!-- 加链接到二级栏目Design的maker free,Design还没做，到时候在maker free上加个锚点 -->
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<td>5.26 </td>
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<td>1</td>
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<div class="p_font_size"><strong>Figure 1</strong> The schematic diagram of the marker-free element. PV4 is an anther-specific promoter that drives Cre gene expression in anther.</div>
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<div class="h2_font_size">Laws&regulations </div>
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<td>phytoene synthase</td>
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<div class="p_font_size">The credibility of the policy about “aSTARice” transgenic rice project:</div>
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<td>PSY</td>
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<div class="p_font_size"><strong>The world health organization</strong></div>
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<td>Zea mays L.</td>
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<div class="p_font_size">In 2002, the world health organization claimed in"20 questions about genetically modified food".All of the genetically modified products as so far in the international market have passed the risk assessment by the national authorities. These different assessments in general follow the same basic principles, including the environment and human health risk assessment. These assessments are transparent, they did not show any risk for human health"</div>
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<td>59.82</td>
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<div class="p_font_size"><strong>The United Nations</strong></div>
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<td>200 nM/h</td>
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<div class="p_font_size">The United Nations food and agriculture organization in 2003-2004,in "the food and agriculture condition report” clearly pointed out that the current existed modified crops and food is safe, the method used to test its safety is appropriate.So far, there is no safety accident caused by eating genetically modified crops. Millions of people eat the food derived from genetically modified crops, mainly maize, soybean and rape seed, but did not find any adverse effect.</div>
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<td>9.09 </td>
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<div class="p_font_size"><strong>The European</strong></div>
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<td>1.73</td>
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<div class="p_font_size">In 2009, the transgenic group of European food safety authority set out an authoritative scientific opinion about herbicide resistant and insect-resistantcrops that in terms of impact on the environment of human and animal health, gm is the same safe as non-gm, without any harm to health and the environment.</div>
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<div class="p_font_size"><strong>The United </strong></div>
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<div class="p_font_size">In 2010, the United States national academy of sciences claimed in “the influence on American agricultural sustainability ”. In general, compared to traditional agriculturaltechnology for American farmers, genetically modified has created a huge environmental benefits and economic benefits.</div>
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<td>phytoene desaturase</td>
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<div class="p_font_size">The world health organization, the United Nations food and agriculture organization, the us food and drug administration and other international organizations and authorities have said the current approval of commercialization of transgenic food is safe and edible.</div>
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<td>CrtI/PDS</td>
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<div class="p_font_size"><strong>China:</strong></div>
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<td>Erwinia uredovora</td>
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<div class="p_font_size">The Chinese government attaches great importance to the management work of the safety of agricultural genetically modified organisms, has formed a set of law which is suitable for China's national situation and consistent with international convention of laws and regulations, technical regulations and management system. As a result,it has achieved remarkable resultsin accordance with the implementation of safety management.</div>
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<td>31.38</td>
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<div class="p_font_size">1. The legal system: the agricultural genetically modified organisms safety management regulations, the measures for the administration of agricultural genetically modified organisms safety assessment, the safety measures for the administration of import of agricultural genetically modified organisms, the identification measures for the administration of agricultural genetically modified organisms, the agricultural genetically modified organisms processing measures for examination and approval, the inspection and quarantine measures for the administration of entry and exit of genetically modified products.</div>
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<td>200 nM/h</td>
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<div class="p_font_size">2. The administrative management system: joint inter-ministerial meeting, agricultural genetically modified organisms safety management leading group, the provincial administrative department of agriculture, municipal (county) level administrative department of agriculture.</div>
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<td>4.69 </td>
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<div class="p_font_size">3. Technical support system, safety evaluation, detection, monitoring and technical standards.</div>
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<td>0.89</td>
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<div class="h2_font_size">Lab work</div>
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<div class="p_font_size"><strong>Training</strong></div>
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<td>β-carotene hydroxylase</td>
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<div class="p_font_size">Members in our team have read the established laboratory safety principles of our school and the topics in our safety training can be summarized as follows:</div>
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<td>BHY</td>
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<div class="p_font_size">1. Non-biological operations:</div>
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<td>Haematococcus pluvialis</td>
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<div class="p_font_size">&nbsp;&nbsp;1.1 Handling toxic chemicals</div>
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<td>52.32</td>
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<div class="p_font_size">&nbsp;&nbsp;1.2 Emergency measures(such as how to tackle fire, electric leakage and negligent wounds)</div>
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<td>200 nM/h</td>
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<div class="p_font_size">&nbsp;&nbsp;1.3 Instruments and facilities operation principles</div>
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<td>7.89 </td>
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<div class="p_font_size">2.Biological operations:</div>
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<td>1.50</td>
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<div class="p_font_size">&nbsp;&nbsp;2.1 Potential threats of our engineering bacterium (Escherichia coli)</div>
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<div class="p_font_size">&nbsp;&nbsp;2.2 Effective protection during organism operations</div>
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<div class="p_font_size">&nbsp;&nbsp;2.3 Waste materials handling measures</div>
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<td>β-carotene ketolase</td>
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<div class="p_font_size">&nbsp;&nbsp;2.4 Emission rules</div>
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<td>BKT</td>
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<td>Chlamydomonas reinhardtii</td>
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<td>40.90</td>
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<td>200 nM/h</td>
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<td>6.12 </td>
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<td>1.16</td>
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</tr>
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<tr>
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<td>phytoene desaturase</td>
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<td>PDS</td>
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<td>Oryza sativa cv.</td>
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<td>49.69</td>
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<td>200 nM/h</td>
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<td>7.5 </td>
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<td>1.43</td>
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</tr>
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<td>ζ-carotene desaturase</td>
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<td>ZDS</td>
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<td>Oryza sativa cv.</td>
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<td>49.12</td>
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<td>200 nM/h</td>
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<td>7.32 </td>
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<td>1.39</td>
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</tr>
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<td>lycopene epsilon-cyclase</td>
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<td>LCY</td>
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<td>Oryza sativa cv.</td>
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<td>44.66</td>
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<td>200 nM/h</td>
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<td>6.67 </td>
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<td>1.27</td>
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</tr>
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<td>carotene 3-hydroxylase</td>
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<td>HYD</td>
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<td>Oryza sativa cv.</td>
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<td>53.70</td>
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<td>200 nM/h</td>
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<td>7.89 </td>
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<td>1.5</td>
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<div class="p_font_size">However, the experimental data show great difference from our prediction model. Some genes are supported to be strong expressed in our model while few expressions were detected actually. Therefore, we believe that there are relationships between different genes we mentioned. For example, the gene PSY control the expression of the phytoene synthase, which is determinant transformation from pre-phytoene diphosphate to phytoene and phytoene is substrate of the following reaction, which was catalyzed by phytoene desaturase(encoded b gene CrtI/PDS).In this way, gene PSY and CrtI/PDS have strong relationships so the expression of one gene affect the other one.</div>
 
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<div class="p_font_size"><strong>Rules and Regulations</strong></div>
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<div class="p_font_size">We have graduate students as our supervisors to ensure our operation correctness preventing safety problems. All of our team members have at least been working in a lab for 3 months and received biosafety training. The biosafety guidelines of our institution can be describe as follows:</div>
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<div class="p_font_size">1. All laboratories must be specially design and should set up strict management systems, standard operation procedures and rules. </div>
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<div class="p_font_size">2. Each staff should be equipped with personal safety equipment to avoid direct contact with the pathogenic microorganism or toxic chemicals.</div>
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<div class="p_font_size">3. New staffs should be well trained and should pass the experiment test before performing experiments themselves.</div>
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<div class="p_font_size">4. One person at least in each laboratory should take charge of biosafety and establish a continuous biosafety training program.</div>
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<div class="p_font_size">5. Laboratories should establish emergency handling procedures and have routine inspection forall of the equipment.</div>
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<div class="p_font_size" style="margin-bottom:20px"><small>Figure 12 qRT-PCR analyses of foreign and endogenous genes involved in carotenoids biosynthesis in aSTARice endosperm.</small></div>
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<div class="p_font_size"><small>The PDS, ZDS, ISO, BLCY, ELCY, HYD and rPSY ,all are rice endogenous genes for carotenoid biosynthesis.</small></div>
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<div class="p_font_size">The data show that there is a positive correlation between the expression of CrtI gene and PSY gene in the indica transgenic rice HG1. The expression of BKT and BHY are fluctuant. Some transgenic lines have low expression of BKT and high expression of BHY while some strains have low expression of BHY but high expression of BKT and CrtI. All endogenous genes involved in carotenoid biosynthesis are low or no expression in aSTARice endosperm. The production and accumulation of astaxanthin in aSTRice is determined by co-expression of PSY, Crtl, BHY and BKT.</div>
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<div class="p_font_size">Reference:</div>
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<div class="p_font_size">【1】Alon U. An introduction to systems biology: design principles of biological circuits [M]. CRC press, 2006.</div>
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<div class="p_font_size">【2】Ingalls B. Mathematical Modelling in Systems Biology: An Introduction[J]. Internet.[cited at p. 117], 2013.</div>
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Revision as of 07:32, 14 October 2016

SCAU

Modeling
Astaxanthin is becoming a more and more popular health care product, and our project is focusing on producing and accumulating astaxanthin in rice endosperm bioreactor, and, finally, getting the astaxanthin products. During our experiments, we have collected some data and used educated guesses that are biologically feasible, trying to figure out, 1.which foreign genes plays a more important role in the pathway; 2. The interaction between four foreign genes and other endogenous genes on expression level. Through these analyses, we wonder a better optimizing strategy to increase the production of astaxanthin in rice endosperm bioreactor.
1. Genes and astaxanthin productiont
We have transferred four foreign genes, PSY, Crtl, BKT and BHY, into rice endosperm bioreactor. The enzymes encoded by these genes catalyze the following reactions:
image
Figure 10 The biosynthesis pathway of astaxanthin formation in transgenic rice endosperm. The dotted arrows indicate pathway limitations in rice endosperm. The solid arrows indicate the existence of carotenogenic reactions. The red arrows indicate the reactions catalaysed by four exogenous transgenes Psy, CrtI, BHY and BKT.
image
Figure 11 Correlation analyses between expression levels of four genes involved in astaxanthin biosynthesis and astaxanthin content.

The figure 11 showed us the correlations between astaxanthin concentration and expression of astaxanthin biosynthetic genes in rice endosperm bioreactor. OsActin1 gene was used to normalize expression. The encoded enzyme Crtl catalyzes the phytoene into lycopene, while enzyme BHY catalyzes the β-carotene into zeaxanthin. The expression of Crtl and BHY was significantly positively correlated with astaxanthin concentration in the rice endosperm bioreactor. The result suggests that the expression of Crtl and BHY are the rate-limiting factors to astaxanthin biosynthesis in rice endosperm bioreactor. Learning about this, in the future, we might increase the production of astaxanthin by enhancing the expression of Crtl and BHY.
2. Foreign genes and endogenous genesof
To synthesize astaxanthin in rice endosperm bioreactor, we have transduced four foreign genes, thus, we want to know the interplay between foreign genes and endogenous genes, or will their expression influence each other. Especially, we focus on the genes’ transcription network. The input signals usually change transcription factor activities on a sub-second timescale. Binding of the active transcription factor to its DNA sites often reaches equilibrium in seconds. Transcription and translation of the target gene takes minutes, and the accumulation of the protein can take minutes to hours.of
The production of the protein encoded by gene Y is balanced by two process, protein degradation( its specific destruction by specialized in the cell) and dilution( the reduction in concentration due to the increase of cell volume during growth). The degradation rate isimage, and the dilution rate isimage, giving a total degradation/dilution rate of
image
The change in concentration of protein Y is due to the difference between its production and degradation/dilution, as described by a dynamic equation:
image
At steady state, protein Y reaches a constant concentrationimage. The steady-state concentration can be found by solving forimage. This shows that the steady-state concentration is the ratio of the production and degradation/dilution rates:
image
If we take away the input signal, so that production of protein Y stops and there will be an exponential decay of Y concentration:
image
Similarly, if an unstimulated cell with Y=0 is provided with a signal, so that protein Y begins to accumulate. If an unstimulated gene becomes suddenly stimulated by a strong signal Sx, the dynamic equation approach to steady state:
image
T1/2 is defined as the time to reach halfway between the initial and final levels in a dynamic process:
T1/2 =log(2)/α
image
The concentration of Y rises from zero and gradually converges on the steady-state.
Note that at early times, whenimage,we can use a Taylor expansion to find a linear accumulation of a Y:
Y～βt13
early times, image
The concentration of protein Y accumulates at early times with a slope equal to its production rate. Later, as Y levels increase, the degradation term-image Y begins to be important and Y converges to its steady-state level.
We considered the activation of transcription of a gene( mRNA production) and used a dynamical equation to describe the changes in the concentration of the gene product, the protein Y. The mRNA needs to be translated to form the protein, and mRNA itself is also degraded by specific enzymes.
Assuming that mRNA is produced at rateimage and degraded at rateimage, and that each mRNA produces on average p protein molecules over its lifetime. The protein is degraded/diluted at rateimage.
The dynamic equation for the concentration of mRNA of gene Y,image, is :
image①
The dynamical equation for the protein product is due to production of image copies per mRNA and degradation/dilution at rate :image
image
The steady-state mRNA level is found by settingimagein Equation ①, yielding
image
Using this forimagein Equation ① yields the following equation for the protein production rate:
image
In other words, the effective protein production rate is equal to the steady-state mRNA level times the number of proteins translated from each mRNA:
image
In the first step of our modeling, we support that all genes have the same expression way in the cell. We support that all the input signals appear at time t=0. Because we don’t have enough time to measure transcription and translation rates of the genes, we assume that all the proteins have the same production rate and this rate are supported to be 200 nM/h. To measure the degradation rate of protein, αdeg, T1/2 is used in the following calculation, in which period of time the protein decrease by 50%.T1/2 of each protein is determined by their Instability index (II). Degradation rates of protein can be calculated by the aquation, T1/2 =log(2)/α.Yst means steady-state concentration of the proteins.
Protein names Gene names Organism Instability index (II) β αdeg Yst
Actin OsAct1 Oryza sativa subsp. japonica 34.96 200 nM/h 5.26 1
phytoene synthase PSY Zea mays L. 59.82 200 nM/h 9.09 1.73
phytoene desaturase CrtI/PDS Erwinia uredovora 31.38 200 nM/h 4.69 0.89
β-carotene hydroxylase BHY Haematococcus pluvialis 52.32 200 nM/h 7.89 1.50
β-carotene ketolase BKT Chlamydomonas reinhardtii 40.90 200 nM/h 6.12 1.16
phytoene desaturase PDS Oryza sativa cv. 49.69 200 nM/h 7.5 1.43
ζ-carotene desaturase ZDS Oryza sativa cv. 49.12 200 nM/h 7.32 1.39
lycopene epsilon-cyclase LCY Oryza sativa cv. 44.66 200 nM/h 6.67 1.27
carotene 3-hydroxylase HYD Oryza sativa cv. 53.70 200 nM/h 7.89 1.5
However, the experimental data show great difference from our prediction model. Some genes are supported to be strong expressed in our model while few expressions were detected actually. Therefore, we believe that there are relationships between different genes we mentioned. For example, the gene PSY control the expression of the phytoene synthase, which is determinant transformation from pre-phytoene diphosphate to phytoene and phytoene is substrate of the following reaction, which was catalyzed by phytoene desaturase(encoded b gene CrtI/PDS).In this way, gene PSY and CrtI/PDS have strong relationships so the expression of one gene affect the other one.
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Figure 12 qRT-PCR analyses of foreign and endogenous genes involved in carotenoids biosynthesis in aSTARice endosperm.
The PDS, ZDS, ISO, BLCY, ELCY, HYD and rPSY ,all are rice endogenous genes for carotenoid biosynthesis.
The data show that there is a positive correlation between the expression of CrtI gene and PSY gene in the indica transgenic rice HG1. The expression of BKT and BHY are fluctuant. Some transgenic lines have low expression of BKT and high expression of BHY while some strains have low expression of BHY but high expression of BKT and CrtI. All endogenous genes involved in carotenoid biosynthesis are low or no expression in aSTARice endosperm. The production and accumulation of astaxanthin in aSTRice is determined by co-expression of PSY, Crtl, BHY and BKT.
Reference:
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【2】Ingalls B. Mathematical Modelling in Systems Biology: An Introduction[J]. Internet.[cited at p. 117], 2013.