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| − | <p class=MsoNormal><b><span lang=EN-US>Background</span></b></p> | + | <p><span style="color:#D3D3D3"><strong><span style="font-family:comic sans ms,cursive"><span style="font-size:28px">BackGround</span></span></strong></span></p> |
| | | | |
| − | <p class=MsoNormal><span lang=EN-US>Yogurt, as one of the oldest and most | + | <hr /> |
| − | ordinary fermented foods, is known all of the world. However, the yogurt
| + | <p><span style="font-family:arial,helvetica,sans-serif">As one type of the oldest fermented food, yogurt is very popular around the world. Different from other traditional dairy products (cottage cheese, sour cream and etc.), yogurt wins its popularity due to the health benefits it can offer. Primarily, yogurt comes from milk and is nutritionally rich in protein, calcium, riboflavin, vitamin B6 and vitamin B16. Additionally, Lactose-sensitive individuals may tolerate yogurt better than other dairy products due to the conversion of lactose to glucose and galactose, and the fermentation of lactose to lactic acid carried out by the bacteria in the yogurt. Most importantly, it generally possesses a certain amount of probiotics, which is necessary in our digestive tracts.</span></p> |
| − | section practically taken over the dairy aisle of most grocery stores. Differ
| + | |
| − | from other traditional dairy products(cottage cheese, sour cream and etc.), | + | |
| − | yogurt wins itself the most popularity due to the many health benefits that it | + | |
| − | can gives.</span></p> | + | |
| | | | |
| − | <p class=MsoNormal><span lang=EN-US>And, what kinds of health benefits of | + | <p><img alt="" src="https://static.igem.org/mediawiki/2015/3/31/NEFU_China_E48C74FA-5D41-4E45-AE7E-4987FF76E186.png" style="height:270px; width:1000px" /></p> |
| − | yogurt?</span></p>
| + | |
| | | | |
| − | <p class=MsoNormal><span lang=EN-US>Primarily, everyone needs to have a healthy | + | <p style="text-align:center"><span style="font-family:arial,helvetica,sans-serif">Fig1. Delicious yogurt</span></p> |
| − | amount of ''good'' bacteria in the digestive tract, and many yogurts are made
| + | |
| − | using active, good bacteria. And then, let us notice that yogurt comes from
| + | |
| − | milk and is as well nutritionally rich in protein, calcium, riboflavin, vitamin
| + | |
| − | B6 and vitamin B16, but it has nutritional benefits beyond those of milk, namely
| + | |
| − | due to its probiotics. In fact, the health benefits of yogurt are so impressive
| + | |
| − | that many health-conscious people make it a daily habit. Here are five possible
| + | |
| − | health benefits of having a yogurt a day: </span></p>
| + | |
| | | | |
| − | <p class=MsoNormal><span lang=EN-US> (1) Yogurt may help prevent | + | <p><span style="font-family:arial,helvetica,sans-serif">In spite of these health benefits, yogurt is easy to go bad and spoiled yogurt has more harm than good. Last semester, one of our teammates was absent for class due to eating some spoiled yogurt, although it was still within the expiration date. Generally, yogurt within expiration date is safe for eating if it is always kept cold, which was neglected by this teammate who consequently suffered from diarrhea. As you can see, yogurt may cause food poisoning even though it has not expired. This is one of the reasons why we came up with an idea of developing a yogurt guarder.</span></p> |
| − | Osteoporosis, </span></p>
| + | |
| | | | |
| − | <p class=MsoNormal><span lang=EN-US> (2) Yogurt may reduce the risk of | + | <p><span style="font-family:arial,helvetica,sans-serif">We searched the Internet for relevant information and found varieties of news about yogurt spoilage within shelf-life. Bacteria contamination in yogurt cannot be recognized by our naked eyes. Thus, many people may suffer from that.</span></p> |
| − | high blood pressure.</span></p>
| + | |
| | | | |
| − | <p class=MsoNormal><span lang=EN-US> (3) Yogurt with active cultures helps | + | <p><span style="font-family:arial,helvetica,sans-serif">To understand why yogurt may not be safe for eating even before the expiration date, we need to know how yogurt is produced. First, raw milk is treated with pasteurization to kill most microorganisms inside without destroying its nutritional components. Second, <em>Lactobacillus</em> is inoculated for fermentation. Then, yogurt is made. Unlike sterilization, pasteurization is unable to kill all microorganisms in the food. Instead, it intends to reduce the number of pathogens without significantly affecting nutrient. So yogurt must be kept in cold for a limited time period; otherwise pathogenic bacteria that survive from pasteurization can quickly grow and cause yogurt spoilage.</span></p> |
| − | the gut</span></p> | + | |
| | | | |
| − | <p class=MsoNormal><span lang=EN-US> (4) Yogurt with active cultures may | + | <p><span style="font-family:arial,helvetica,sans-serif">We did a simple experiment to test the appearance and contents of yogurt under different conditions. We bought fresh yogurt from the same batch and kept some of them in fridge and some at 35℃ for half an hour. Then, we took pictures of them. The yogurt kept at both conditions looked the same. However, our subsequent experiments revealed that yogurt kept at 35℃ had a higher number of coliform bacteria than that from the fridge and the pathogens significantly exceeded the qualify standard. This means the pathogen contents of yogurt may significantly increase when we carrying yogurt from a supermarket to home, since yogurt will be exposed to ambient temperature for a while, especially in summer. (Visit <a href="https://2015.igem.org/Team:NEFU_China/Result">Result</a> for more details)</span></p> |
| − | discourage vaginal infections </span></p>
| + | |
| | | | |
| − | <p class=MsoNormal><span lang=EN-US> (5) Yogurt may help you feel fuller </span></p> | + | <p><span style="font-family:arial,helvetica,sans-serif"><img alt="" src="https://static.igem.org/mediawiki/2015/7/74/NEFU_China_E02C5302-5EE4-42CC-8D28-0E54DA55C306.jpg" style="height:450px; width:600px" /></span></p> |
| | | | |
| − | <p class=MsoNormal><span lang=EN-US> </span></p> | + | <p style="text-align:center"><span style="font-family:arial,helvetica,sans-serif">Fig2. Yogurt treated with different temperature. (A) Fresh yogurt kept in 4℃ for 0.5hour (B) Fresh yogurt kept in 35℃ for half an hour (C) Yogurt kept in 4℃ until one-day past expiration</span></p> |
| | | | |
| − | <p class=MsoNormal><span lang=EN-US>In spite of this health benefits, spoiled | + | <p><span style="font-family:arial,helvetica,sans-serif">Needless to say, no one would like to eat yogurt if he or she knows it may contain such a high amount of coliform. However, the current methods to test pathogenic bacteria generally contain multiple cumbersome steps, are time consuming and require special equipment. They obviously cannot be used in our daily life. Therefore, a handy detecting approach for pathogenic bacteria in yogurt is urgently needed.</span></p> |
| − | yogurt is more harm than good if you carelessly eat it. Firstly, let us talk | + | |
| − | about the shelf-life of yogurt. Yogurt provides two significant barriers to
| + | |
| − | pathogen growth (heat and acidity) to ensure a safe product. Discard batches
| + | |
| − | that fail to set properly, especially those due to culture errors. Yogurt
| + | |
| − | generally has a 10-21 day self-life when made and stored properly in the
| + | |
| − | refrigerator below 40¡ãF. Then let we look into yogurt manufacturing process:</span></p>
| + | |
| | | | |
| − | <p class=MsoNormal><span lang=EN-US> (1) pasteurization</span></p> | + | <p> </p> |
| − | | + | <p class= "footer" style= "color:white; margin- top:20px; text- align:center; "><span> Generated by </span><a href="https:// 2015.igem.org/ Team:NEFU_China/ Software"> Fligth iGEM</a>.</p> |
| − | <p class=MsoNormal><span lang=EN-US> (2) combine ingredients</span></p> | + | </div> |
| − | | + | <div class="fixl" style="width:300px"> |
| − | <p class=MsoNormal><span lang=EN-US> (3) inoculate</span></p>
| + | <table width="100%" border="0" cellspacing="0" cellpadding="0"> |
| − | | + | <!--<tr> |
| − | <p class=MsoNormal><span lang=EN-US> (3) incubate</span></p>
| + | < td width=" 73"><a href=" Safety.html">< img onmousemove= "this. src=' images/ btla. png' " onmouseout= "this. src=' images/ btl. png' " src= "images/ btl. png" width= "73" height= "73" /></a></ td> |
| − | | + | <td align="left" class="fixtit1">Safety</td> |
| − | <p class=MsoNormal><span lang=EN-US> (4) refrigerate</span></p>
| + | </tr>--> |
| − | | + | </table> |
| − | <p class=MsoNormal><span lang=EN-US>The first step pasteurization is used for
| + | |
| − | sterilization, but the pathogenic bacteria like <i>E.coli</i>, <i>salmonella</i>
| + | |
| − | and<i> bacillus</i> still possibly survive from the first step that can spoil
| + | |
| − | the yogurt during prolonged storage. </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span lang=EN-US>However, from the view of the premise of
| + | |
| − | self-life is numerous and strict, is self-life really reliable? On the one
| + | |
| − | hand, there are too many reasons like long-time transport can not make sure
| + | |
| − | yogurt is stored in a 40¡ãF environment. On the other hand, when you do yogurt yourself,
| + | |
| − | you should also need to make sure the use of clean equipment and containers. To
| + | |
| − | make matter worse, if yogurt spoiled within expiration date you can not tell
| + | |
| − | them apart, for their appearances were almost the same.(illustrated by fig.1)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span lang=EN-US><img width=692 height=520
| + | |
| − | src="https://static.igem.org/mediawiki/2015/5/56/NEFU_FI_TEST-yg.jpeg"> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span lang=EN-US>fig.1(those nine photos of yogurt with a
| + | |
| − | same production date were taken maintain a two days interval. Those yogurt were
| + | |
| − | kept in room temperature, and their packing were open only when took photos )</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span lang=EN-US> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span lang=EN-US>This is for sure, the pathogenic bacteria
| + | |
| − | can be detected. We then did an experiment based on our national food safety
| + | |
| − | standards. (<u>protocols click here</u>) </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span lang=EN-US> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span lang=EN-US>Needless to say, no one would willing to | + | |
| − | eat yogurt with such amount of coliform if he or she had see our results.
| + | |
| − | However, considering the test method containing cumbersome steps, costing much
| + | |
| − | time and requiring certain experimental apparatus, it on no condition can be
| + | |
| − | use in daily life. That is to say, if we don¡¯t do something we could still eat
| + | |
| − | yogurt that maybe contain much bacteria we don¡¯t want.</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span lang=EN-US> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span lang=EN-US>Now, a handy detector of pathogenic
| + | |
| − | bacteria is really needed.</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span lang=EN-US> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span lang=EN-US> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><b><span lang=EN-US>Design:</span></b></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='margin-left:0cm;text-indent:0cm'><span lang=EN-US>1.</span><span
| + | |
| − | lang=EN-US>Introduction of AI-2</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span lang=EN-US>2.Why we choose AI-2</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span lang=EN-US>3.Chassis(why we choose them)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span lang=EN-US>4.The system we will construct</span></p> | + | |
| − | | + | |
| − | <p class=MsoNormal><span lang=EN-US>5.NICE system</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span lang=EN-US>6.Plasmid construction</span></p> | + | |
| − | | + | |
| − | <p class=MsoNormal><span lang=EN-US>7.Quantification</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='margin-bottom:12.0pt'><span lang=EN-US> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='margin-bottom:12.0pt'><span lang=EN-US> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span lang=EN-US> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span lang=EN-US> Quorum sensing (QS) is a chemical communication
| + | |
| − | system among bacterium,and QS requires the production and detection of specific
| + | |
| − | signaling molecules,which are commonly referred to as
| + | |
| − | autoinducers.Autoinducer-2 (AI-2) molecules are one class of signaling
| + | |
| − | molecules involved in QS.AI-2 is a byproduct of the Activated Methyl
| + | |
| − | Cycle,which recycles S-adenosyl-l-methionine (SAM).As a main methyl donor,SAM
| + | |
| − | is converted to S-adenosyl-l-homocysteine (SAH).Then SAH is subsequently
| + | |
| − | hydrolyzed to adenine and 4,5-dihydroxy-2,3-pentanedione (DPD) by the nucleosidase
| + | |
| − | Pfs and the Luxs enzyme.Upon formation,DPD spontaneously cyclizes to form at
| + | |
| − | least two different interspecies communication molecules described as AI-2.</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span lang=EN-US> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span lang=EN-US> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span lang=EN-US> Why we chose AI-2 as signaling
| + | |
| − | molecule,that¡¯s because the production of AI-2 is the only quorum-sensing
| + | |
| − | mechanism shared by both Gram-positive and Gram-negative bacteria.There are
| + | |
| − | many <a name=" OLE_LINK7" ></a><a name=" OLE_LINK6"> pathogens< /a> in spoiled | + | |
| − | yogurt, Ecoli, Salmonella and Bacillus are the main part.Then AI-2 can be used
| + | |
| − | for a universal language among pathogens.Besides,AI-2 response pathway of our
| + | |
| − | chassis, lactobacillus and Lactococcus, is different from that kinds of
| + | |
| − | pathogens.</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span lang=EN-US> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='margin-bottom:12.0pt'><span lang=EN-US> We
| + | |
| − | choose lactobacillus and Lactococcus as our chassis.As beneficial bacteria,
| + | |
| − | they are food grade and are widely used in food production for thousands of
| + | |
| − | years.Lactobacillus can improve nutrient absorption and human gastrointestinal
| + | |
| − | function.They can also inhibit the reproduction of pathogenic bacteria in guts.
| + | |
| − | Lactococcus lactis is a Gram-positive lactic acid bacterium that is widely used
| + | |
| − | in food fermentations, such as in cheese and butter production. </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span lang=EN-US> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='margin-bottom:12.0pt'><span lang=EN-US> LuxS
| + | |
| − | converts SRH to the precursor of AI-2,DPD,in the cytoplasm.DPD then undergoes
| + | |
| − | spontaneous cyclization and expert to the culture supernatant.Depending on the
| + | |
| − | bacteria,response to AI-2 can follow one of the two currently identified
| + | |
| − | routes.In pathogens exemplified by Salmonella, AI-2 is bound to a homologue of
| + | |
| − | the periplasmic binding protein LsrB.LsrB is part of an ABC transporter(encoded
| + | |
| − | by the lsrACDBRK operon).This operon is regulated by the repressor LsrR,the
| + | |
| − | gene of which is located upstream. Immediately downstream of lsrR is a gene
| + | |
| − | (lsrK). LsrK phosphorylates AI-2, Phosphorylated AI-2 then causes LsrR to
| + | |
| − | relieve its repression of the lsr operon, allowing further AI-2 import. </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='margin-bottom:12.0pt'><span lang=EN-US> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='margin-bottom:12.0pt'><span lang=EN-US> The
| + | |
| − | nisin-controlled gene expression system NICE of Lactococcus lactis is one of
| + | |
| − | the most widely used expression systems in Gram-positive bacteria.Biosynthesis
| + | |
| − | of nisin is encoded by a cluster of 11 genes, NisR and NisK belong to the
| + | |
| − | family of bacterial two-component signal transduction systems.NisK is a
| + | |
| − | histidine</span><span style='font-family:ËÎÌå'>–</span><span lang=EN-US>protein
| + | |
| − | kinase that resides in the cytoplasmic membrane and is proposed to act as a
| + | |
| − | receptor for the mature nisin molecule. Upon binding of nisin to NisK, it
| + | |
| − | autophosphorylates and transfers the phosphate group to NisR, which is a
| + | |
| − | response regulator that becomes activated upon phosphorylation by NisK.
| + | |
| − | Activated NisR* induces transcription from two of the three promoters in the
| + | |
| − | nisin gene cluster: PnisA and PnisF. In our parts,we use inducible promoter
| + | |
| − | PnisA to express gene A</span><span style='font-family:ËÎÌå'>¡¢</span><span
| + | |
| − | lang=EN-US>B</span><span style='font-family:ËÎÌå'>¡¢</span><span lang=EN-US>C</span><span
| + | |
| − | style='font-family:ËÎÌå'>¡¢</span><span lang=EN-US>D</span><span style='font-family:
| + | |
| − | ËÎÌå'>¡¢</span><span lang=EN-US>R</span><span style='font-family:ËÎÌå'>¡¢</span><span
| + | |
| − | lang=EN-US>K,the inducer nisin</span><span style='font-family:ËÎÌå'>¡¢</span><span
| + | |
| − | lang=EN-US>gene nisR</span><span style='font-family:ËÎÌå'>¡¢</span><span
| + | |
| − | lang=EN-US>nisK are necessary for PnisA.</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span lang=EN-US> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span lang=EN-US>Plasmid construction</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span lang=EN-US>¢ÙGene<i> A</i></span><i><span
| + | |
| − | style='font-family:ËÎÌå'>¡¢</span></i><i><span lang=EN-US>B</span></i><i><span
| + | |
| − | style='font-family:ËÎÌå'>¡¢</span></i><i><span lang=EN-US>C</span></i><i><span
| + | |
| − | style='font-family:ËÎÌå'>¡¢</span></i><i><span lang=EN-US>D</span></i><i><span
| + | |
| − | style='font-family:ËÎÌå'>¡¢</span></i><i><span lang=EN-US>R</span></i><i><span
| + | |
| − | style='font-family:ËÎÌå'>¡¢</span></i><i><span lang=EN-US>K </span></i><i><span
| + | |
| − | style='font-family:ËÎÌå'>¡¢</span></i><i><span lang=EN-US>plsr </span></i><span
| + | |
| − | lang=EN-US>was cloned from Salmonella typhimurium</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span lang=EN-US>¢ÚPlasmid pNZ8148 served for amplifying gene<i>
| + | |
| − | B</i></span><i><span style='font-family:ËÎÌå'>¡¢</span></i><i><span lang=EN-US>R</span></i><i><span
| + | |
| − | style='font-family:ËÎÌå'>¡¢</span></i><i><span lang=EN-US>K </span></i><span
| + | |
| − | lang=EN-US>respectively</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span lang=EN-US> Plasmid pBBR1MCS-5 served for
| + | |
| − | amplifying gene<i> A</i></span><i><span style='font-family:ËÎÌå'>¡¢</span></i><i><span
| + | |
| − | lang=EN-US>C</span></i><i><span style='font-family:ËÎÌå'>¡¢</span></i><i><span
| + | |
| − | lang=EN-US>D </span></i><span lang=EN-US>respectively</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='margin-bottom:12.0pt'><span lang=EN-US> We
| + | |
| − | replace promoter of pET-14b with <i>plsr</i>,The final construct called
| + | |
| − | pHY300PLK contains a blue pigment gene.</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='margin-bottom:12.0pt'><span lang=EN-US>¢ÛPlasmid | + | |
| − | pNZ8148(¡Á3)</span><span style='font-family:ËÎÌå'>¡¢</span><span lang=EN-US>pBBR1MCS-5(¡Á3)
| + | |
| − | and pHY300PLK were linearized and introduced by electroporation into our
| + | |
| − | chassis.</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='margin-bottom:12.0pt'><span lang=EN-US>¢ÜAs for
| + | |
| − | eletroporation,we have two different routes.One of which is that seven
| + | |
| − | linearized palsmid and pNZ9530(Low copy plasmid with pAM¦Â1 origin of
| + | |
| − | replication, which carries the nisR and nisK genes.) were introduced into lactobacillus.Or
| + | |
| − | seven linearized palsmid containing gene A</span><span style='font-family:ËÎÌå'>¡¢</span><span
| + | |
| − | lang=EN-US>B</span><span style='font-family:ËÎÌå'>¡¢</span><span lang=EN-US>C</span><span
| + | |
| − | style='font-family:ËÎÌå'>¡¢</span><span lang=EN-US>D</span><span style='font-family:
| + | |
| − | ËÎÌå'>¡¢</span><span lang=EN-US>R</span><span style='font-family:ËÎÌå'>¡¢</span><span
| + | |
| − | lang=EN-US>K</span><span style='font-family:ËÎÌå'>¡¢</span><span lang=EN-US>Blue
| + | |
| − | pigment were introduced into Lactococcus NZ9000(containing nisR and nisK)by
| + | |
| − | eletroporation.</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span lang=EN-US> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='margin-bottom:12.0pt'><span lang=EN-US> The
| + | |
| − | molecular structure of AI-2 is somewhat similar to ascorbic acid. It was
| + | |
| − | expected that AI-2 would also act as a reducing agent and reduce Fe(III) ions
| + | |
| − | in the presence of 1,10-phenanthroline to form the colored [(o-phen)3
| + | |
| − | Fe(II)]SO4 ferroin complex that could be quantified colorimetrically.</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span lang=EN-US> </span></p>
| + | |
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