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 HOME HUMAN PRACTICES SAFETY NOTE RESULTS PARTS PROJECT TEAM

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:

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.

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 is, and the dilution rate is, giving a total degradation/dilution rate of

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.

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.

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.

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.

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.

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.

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.

Design for marker free

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. You can read more details, please click here!

Figure 1 The schematic diagram of the marker-free element. PV4 is an anther-specific promoter that drives Cre gene expression in anther.

Laws&regulations

The credibility of the policy about “aSTARice” transgenic rice project:

The world health organization

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"

The United Nations

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.

The European

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.

The United

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.

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.

China:

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.

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.

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.

3. Technical support system, safety evaluation, detection, monitoring and technical standards.

Lab work

Training

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:

1. Non-biological operations:

  1.1 Handling toxic chemicals

  1.2 Emergency measures(such as how to tackle fire, electric leakage and negligent wounds)

  1.3 Instruments and facilities operation principles

2.Biological operations:

  2.1 Potential threats of our engineering bacterium (Escherichia coli)

  2.2 Effective protection during organism operations

  2.3 Waste materials handling measures

  2.4 Emission rules

Rules and Regulations

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:

1. All laboratories must be specially design and should set up strict management systems, standard operation procedures and rules.

2. Each staff should be equipped with personal safety equipment to avoid direct contact with the pathogenic microorganism or toxic chemicals.

3. New staffs should be well trained and should pass the experiment test before performing experiments themselves.

4. One person at least in each laboratory should take charge of biosafety and establish a continuous biosafety training program.

5. Laboratories should establish emergency handling procedures and have routine inspection forall of the equipment.