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

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       <li><a href="#vecter" class="on">Safe Project Design</a></li>
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       <li><a href="#vecter" class="on">Vecter Constraction</a></li>
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       <li><a href="#tissue">Safe lab work</a></li>
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       <li><a href="#tissue">Tissue Culture</a></li>
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       <li><a href="#pcr">Marker free</a></li>
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       <li><a href="#pcr">PCR and RT-PCR analysis</a></li>
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      <li><a href="#phenotype">Phenotype of seeds</a></li>
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      <li><a href="#hplc">HPLC</a></li>
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      <li><a href="#modeling">Modeling</a></li>
 
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       <div class="img"><img src="https://static.igem.org/mediawiki/2016/3/31/T--SCAU-China--img_1.jpg"></div>
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       <div class="text">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.<br><br>
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      <div class="text">Fig.1 Not I digestion of constructs cotaining different numbers of genes.
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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.<br><br>
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Not I is the acceptor vector (pYLTAC380GW plasmid) and lanes 3-6 are intermediate products created during the construction of our delivering vecter,pYLTAC380MF-BBPC. Bands with arrows are the trangenes release from vector backbone and length of corresponding genes are indicated at the paretheses.
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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.<br><br>
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  We also employed the cre-loxp system to remove selected maker.particular information please check in the part----maker free.
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       <li><a href="#tissue" class="on">Safe lab work</a></li>
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       <li><a href="#tissue" class="on">Tissue Culture</a></li>
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       <li><a href="#pcr">Marker free</a></li>
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       <li><a href="#pcr">PCR and RT-PCR analysis</a></li>
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      <li><a href="#phenotype">Phenotype of seeds</a></li>
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      <li><a href="#hplc">HPLC</a></li>
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      <li><a href="#modeling">Modeling</a></li>
 
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      <div class="text"><p>1. Training</p>
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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: <br>
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       <img src="https://static.igem.org/mediawiki/2016/6/61/T--SCAU-China--img_2.jpg"><br>
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I.Non-biological operations:<br>
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       <img src="https://static.igem.org/mediawiki/2016/4/4c/T--SCAU-China--img_3.jpg"><br>
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I.1 Handling toxic chemicals<br>
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       <img src="https://static.igem.org/mediawiki/2016/7/75/T--SCAU-China--img_4.jpg">
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I.2 Emergency measures(such as how to tackle fire, electric leakage and negligent wounds)<br>
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I.3 Instruments and facilities operation principles<br><br>
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II Biological operations:<br>
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II.1 Potential threats of our engineering bacterium (Escherichia coli)<br>
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II.2 Effective protection during organism operations<br>
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II.3 Waste materials handling measures<br>
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II.4 Emission rules
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       <img src="https://static.igem.org/mediawiki/2016/0/07/T--SCAU-China--safetu_01.JPG"><br><br>
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<div class="text"><p>2. Rules and Regulations</p>
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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:<br><br>
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1.All laboratories must be specially design and should set up strict management systems, standard operation procedures and rules.<br>
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2.Each staff should be equipped with personal safety equipment to avoid direct contact with the pathogenic microorganism or toxic chemicals.<br>
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3.New staffs should be well trained and should pass the experiment test before performing experiments themselves.<br>
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4.One person at least in each laboratory should take charge of biosafety and establish a continuous biosafety training program.<br>
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5.Laboratories should establish emergency handling procedures and have routine inspection for all of the equipment.
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       <li><a href="#vecter">Safe Project Design</a></li>
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       <li><a href="#vecter">Vecter Constraction</a></li>
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       <li><a href="#tissue">Safe lab work</a></li>
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       <li><a href="#tissue">Tissue Culture</a></li>
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       <li><a href="#pcr" class="on">Marker free</a></li>
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       <li><a href="#pcr" class="on">PCR and RT-PCR analysis</a></li>
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      <li><a href="#phenotype">Phenotype of seeds</a></li>
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      <li><a href="#hplc">HPLC</a></li>
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      <li><a href="#modeling">Modeling</a></li>
 
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      <div class="text">1. introduction<br><br>
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In this part, we mainly introduce the work we used cre/loxp recombination，a site-specific recombinase technology to delete the selective marker.<br><br>
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       <img src="https://static.igem.org/mediawiki/2016/0/0f/T--SCAU-China--img_5.jpg"><br>
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Once “aSTARice”  is regenerated, antibiotic genes will serve no useful purprose in such plants but they continue to express these gene products. What’s more, the marker gene may cause some safety problems. As a qualified iGEMer, we attended to use the cre/loxp system to obtain the marker-free transgentic plants, which have been eliminated any marker gene DNA sequences in the final product.<br><br>
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       <img src="https://static.igem.org/mediawiki/2016/b/b3/T--SCAU-China--img_6.jpg">
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DNA recombination mediated by Cre recombinase has become an important tool to generate marker-free transgentic plants because it is time-specific, tissue-specific and site-specific.<br><br>
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The Cre/loxP system of bacterio-phage P1 is a site-specific recombination system that consists of two components:the recombinase(Cre)and its recognition sites(loxP).Cre mediates recombination events and causes the excision of the DNA segment between two directly adjacent loxP sites. Besides，The result of recombination depends on the orientation of the loxP sites. For two lox sites on the same chromosome arm, inverted loxP sites will cause an inversion of the intervening DNA, while a direct repeat of loxP sites will cause a deletion event. If loxP sites are on different chromosomes, it is possible for translocation events to be catalysed by Cre thus inducing the recombination.
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<div class="text">If we want to assemble an tissue-specific promoter ahead of the Cre gene, the process of delection could be regulated when we designed a direct repeat of loxP sites with the intervening marker gene. As a result, we can delete the gene wherever it is located on. By using tissue-specific promoter, we have got the marker-free pollens.
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We found Pv4, a rice anther specific promoter. When our transgentic plants grow up, the Cre gene will be expressed which should be drived by Pv4, thus only in the anther of stamens.  Hence,we got the marker-free pollens. Through selfing , we can get the homozygote of marker-free transgentic plants.
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  <div class="text">We designed primers to primary screen the marker-free transgentic plants by PCR. However，due to the complexity of transgentic plants，it is hard to detect the marker-free process. So we designed more than one pair of primers to ensure the marker-free result.
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<div class="text">Result</div> 
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<div class="img"> <img src="https://static.igem.org/mediawiki/2016/7/70/T--SCAU-China--safetu_07.png">
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      <a id="phenotype"></a>
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      <li><a href="#vecter">Vecter Constraction</a></li>
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      <li><a href="#tissue">Tissue Culture</a></li>
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      <li><a href="#pcr">PCR and RT-PCR analysis</a></li>
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      <li><a href="#phenotype" class="on">Phenotype of seeds</a></li>
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      <li><a href="#hplc">HPLC</a></li>
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      <li><a href="#modeling">Modeling</a></li>
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      <img src="https://static.igem.org/mediawiki/2016/9/94/T--SCAU-China--image014.jpg">&nbsp;&nbsp;
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  <img src="https://static.igem.org/mediawiki/2016/f/f6/T--SCAU-China--image016.png">&nbsp;&nbsp;
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  <img src="https://static.igem.org/mediawiki/2016/0/02/T--SCAU-China--image015.png"><br><br>
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      <img src="https://static.igem.org/mediawiki/2016/8/8a/T--SCAU-China--image020.jpg">&nbsp;&nbsp;
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  <img src="https://static.igem.org/mediawiki/2016/8/88/T--SCAU-China--image022.png">&nbsp;&nbsp;
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  <img src="https://static.igem.org/mediawiki/2016/9/93/T--SCAU-China--image018.png">
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      <a id="hplc"></a>
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      <li><a href="#vecter">Vecter Constraction</a></li>
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      <li><a href="#tissue">Tissue Culture</a></li>
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      <li><a href="#pcr">PCR and RT-PCR analysis</a></li>
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      <li><a href="#phenotype">Phenotype of seeds</a></li>
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      <li><a href="#hplc" class="on">HPLC</a></li>
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      <li><a href="#modeling">Modeling</a></li>
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      <div class="img"><img src="https://static.igem.org/mediawiki/2016/d/dc/T--SCAU-China--image024.png"></div>
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      <div class="text">Fig.n HPLC analysis diagram of standard samples of astaxanthin(a);
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HPLC analysis diagram of  samples extract  from trangenes rice HG-380MF-BBPC (b);HPLC analysis diagram of  samples extract  from  HG-WT rice (c);<br><br>
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Compared with the standard samples with HPLC analysis diagram, you can see the sample extract  from rice and standard samples  have the same peak at the same time, show that the  transgenic rice seed which carry the four genes  expressed astaxanthin. Results indicate  that we are successful synthesize astaxanthin by rice endosperm synthetic .</div> 
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<div class="img"><img src="https://static.igem.org/mediawiki/2016/0/0b/T--SCAU-China--image026.png"></div>
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<div class="img"><img src="https://static.igem.org/mediawiki/2016/4/42/T--SCAU-China--image028.png"></div>
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<div class="img"><img src="https://static.igem.org/mediawiki/2016/8/84/T--SCAU-China--image030.png"></div>   
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      <a id="modeling"></a>
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      <li><a href="#vecter">Vecter Constraction</a></li>
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      <li><a href="#tissue">Tissue Culture</a></li>
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      <li><a href="#pcr">PCR and RT-PCR analysis</a></li>
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      <li><a href="#phenotype">Phenotype of seeds</a></li>
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      <li><a href="#hplc">HPLC</a></li>
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      <li><a href="#modeling" class="on">Modeling</a></li>
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      <div class="img"><img src="https://static.igem.org/mediawiki/2016/2/2e/T--SCAU-China--image032.png"></div>
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      <div class="text">Fig.Relative transcript levels of isoprenoid and carotenoid biosynthetic genes in rice at filling stage.qPCR analysis of the expression of CrtI,BKT,BHY,PSY, PDS, ZDS, ISO,BLCY,ELCY,HYD and rPSY in the rice of HG-WT, its transformants HG-BBPC-8-6-1,HG-BBPC-8-6-2,HG-BBPC-13-5-2,HG-BBPC-13-5-3,HG-BBPC-13-9-1and HG-BBPC-13-9-6 .</div> 
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Revision as of 07:56, 6 October 2016

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Fig.1 Not I digestion of constructs cotaining different numbers of genes. Not I is the acceptor vector (pYLTAC380GW plasmid) and lanes 3-6 are intermediate products created during the construction of our delivering vecter,pYLTAC380MF-BBPC. Bands with arrows are the trangenes release from vector backbone and length of corresponding genes are indicated at the paretheses.



     

     
Fig.n HPLC analysis diagram of standard samples of astaxanthin(a); HPLC analysis diagram of samples extract from trangenes rice HG-380MF-BBPC (b);HPLC analysis diagram of samples extract from HG-WT rice (c);

Compared with the standard samples with HPLC analysis diagram, you can see the sample extract from rice and standard samples have the same peak at the same time, show that the transgenic rice seed which carry the four genes expressed astaxanthin. Results indicate that we are successful synthesize astaxanthin by rice endosperm synthetic .
Fig.Relative transcript levels of isoprenoid and carotenoid biosynthetic genes in rice at filling stage.qPCR analysis of the expression of CrtI,BKT,BHY,PSY, PDS, ZDS, ISO,BLCY,ELCY,HYD and rPSY in the rice of HG-WT, its transformants HG-BBPC-8-6-1,HG-BBPC-8-6-2,HG-BBPC-13-5-2,HG-BBPC-13-5-3,HG-BBPC-13-9-1and HG-BBPC-13-9-6 .