Difference between revisions of "Team:NEFU China/fit"

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mso-hansi-font-family:"Arial Unicode MS";color:#323232'>As one type of the
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oldest fermented food, yogurt is very popular around the world. Different from
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other traditional dairy products (cottage cheese, sour cream and etc.), yogurt
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wins its popularity due to the health benefits it can offer. Primarily, yogurt
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comes from milk and is nutritionally rich in protein, calcium, riboflavin,
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vitamin B6 and vitamin B16. Additionally, Lactose-sensitive individuals may
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tolerate yogurt better than other dairy products due to the conversion of
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lactose to glucose and galactose, and the fermentation of lactose to lactic
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acid carried out by the bacteria in the yogurt. Most importantly, it generally
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color:#323232'>Fig1. Delicious yogurt</span><span lang=EN-US style='font-size:
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+
  
            </div>
+
<p class=a><span lang=EN-US style='font-size:13.0pt;font-family:Arial;
            </div>
+
mso-hansi-font-family:"Arial Unicode MS";color:#323232'>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><span lang=EN-US style='font-size:13.0pt;
 +
font-family:"Heiti SC Light";mso-hansi-font-family:"Heiti SC Light";mso-bidi-font-family:
 +
"Heiti SC Light";color:#323232'><o:p></o:p></span></p>
  
            <div class="row-fluid">
+
<p class=a><span lang=EN-US style='font-size:13.0pt;font-family:Arial;
                <div class="span12">
+
mso-hansi-font-family:"Arial Unicode MS";color:#323232'>We searched the
                    <div id="contentInnerSeparator"></div>
+
Internet for relevant information and found varieties of news about yogurt
                </div>
+
spoilage within <span class=GramE>shelf-life</span>. Bacteria contamination in
            </div>
+
yogurt cannot be recognized by our naked eyes. Thus, many people may suffer
    </div>
+
from that.</span><span lang=EN-US style='font-size:13.0pt;font-family:"Heiti SC Light";
 +
mso-hansi-font-family:"Heiti SC Light";mso-bidi-font-family:"Heiti SC Light";
 +
color:#323232'><o:p></o:p></span></p>
  
    <div class="contentArea">
+
<p class=a><span lang=EN-US style='font-size:13.0pt;font-family:Arial;
 +
mso-hansi-font-family:"Arial Unicode MS";color:#323232'>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, </span><i><span lang=ES-TRAD style='font-size:13.0pt;font-family:Arial;
 +
mso-hansi-font-family:"Arial Unicode MS";color:#323232;mso-ansi-language:ES-TRAD'>Lactobacillus</span></i><span
 +
lang=EN-US style='font-size:13.0pt;font-family:Arial;mso-hansi-font-family:
 +
"Arial Unicode MS";color:#323232'> 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><span
 +
lang=EN-US style='font-size:13.0pt;font-family:"Heiti SC Light";mso-hansi-font-family:
 +
"Heiti SC Light";mso-bidi-font-family:"Heiti SC Light";color:#323232'><o:p></o:p></span></p>
  
        <div class="divPanel notop page-content">
+
<p class=a><span lang=EN-US style='font-size:13.0pt;font-family:Arial;
 +
mso-hansi-font-family:"Arial Unicode MS";color:#323232'>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</span><span lang=EN-US style='font-size:13.0pt;
 +
font-family:Arial;mso-ascii-font-family:Helvetica;color:#323232'>¡æ</span><span
 +
lang=EN-US style='font-size:13.0pt;mso-hansi-font-family:Arial;color:#323232'> </span><span
 +
lang=EN-US style='font-size:13.0pt;font-family:Arial;mso-hansi-font-family:
 +
"Arial Unicode MS";color:#323232'>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</span><span lang=EN-US
 +
style='font-size:13.0pt;font-family:Arial;mso-ascii-font-family:Helvetica;
 +
color:#323232'>¡æ</span><span lang=EN-US style='font-size:13.0pt;mso-hansi-font-family:
 +
Arial;color:#323232'> </span><span lang=EN-US style='font-size:13.0pt;
 +
font-family:Arial;mso-hansi-font-family:"Arial Unicode MS";color:#323232'>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 </span><span lang=EN-US><a
 +
href="https://2015.igem.org/Team:NEFU_China/Result"><span class=Hyperlink0><span
 +
style='font-size:13.0pt;font-family:Arial;mso-hansi-font-family:"Arial Unicode MS"'>Result</span></span></a></span><span
 +
lang=EN-US style='font-size:13.0pt;font-family:Arial;mso-hansi-font-family:
 +
"Arial Unicode MS";color:#323232'> for more details)</span><span lang=EN-US
 +
style='font-size:13.0pt;font-family:"Heiti SC Light";mso-hansi-font-family:
 +
"Heiti SC Light";mso-bidi-font-family:"Heiti SC Light";color:#323232'><o:p></o:p></span></p>
  
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+
<p class=a><span lang=EN-US style='font-size:13.0pt;font-family:Arial;
                <a href="index.html">Home</a> &nbsp;/&nbsp; <span>Simple</span>
+
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</v:shape><![endif]--><![if !vml]><img border=0 width=462 height=283
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src="https://static.igem.org/mediawiki/2015/7/79/Yg.png" v:shapes="officeArt_x0020_object"><![endif]></span><span
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+
<p class=a align=center style='text-align:center'><span lang=EN-US
                <div class="span8" id="divMain">
+
style='font-size:13.0pt;font-family:Arial;mso-hansi-font-family:"Arial Unicode MS";
<h1>igem-2015实验总纲</h1>
+
color:#323232'>Fig2. Yogurt treated with different temperature. (A) Fresh
                    <p><p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">材料:</span></span></p>
+
yogurt kept in 4</span><span lang=EN-US style='font-size:13.0pt;font-family:
 +
Arial;mso-ascii-font-family:Helvetica;color:#323232'>¡æ</span><span lang=EN-US
 +
style='font-size:13.0pt;mso-hansi-font-family:Arial;color:#323232'> </span><span
 +
lang=EN-US style='font-size:13.0pt;font-family:Arial;mso-hansi-font-family:
 +
"Arial Unicode MS";color:#323232'>for 0.5hour (B) Fresh yogurt kept in 35</span><span
 +
lang=EN-US style='font-size:13.0pt;font-family:Arial;mso-ascii-font-family:
 +
Helvetica;color:#323232'>¡æ</span><span lang=EN-US style='font-size:13.0pt;
 +
mso-hansi-font-family:Arial;color:#323232'> </span><span lang=EN-US
 +
style='font-size:13.0pt;font-family:Arial;mso-hansi-font-family:"Arial Unicode MS";
 +
color:#323232'>for half an hour (C) Yogurt kept in 4</span><span lang=EN-US
 +
style='font-size:13.0pt;font-family:Arial;mso-ascii-font-family:Helvetica;
 +
color:#323232'>¡æ</span><span lang=EN-US style='font-size:13.0pt;mso-hansi-font-family:
 +
Arial;color:#323232'> </span><span lang=EN-US style='font-size:13.0pt;
 +
font-family:Arial;mso-hansi-font-family:"Arial Unicode MS";color:#323232'>until
 +
one-day past expiration</span><span lang=EN-US style='font-size:13.0pt;
 +
font-family:"Heiti SC Light";mso-hansi-font-family:"Heiti SC Light";mso-bidi-font-family:
 +
"Heiti SC Light";color:#323232'><o:p></o:p></span></p>
  
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">菌株:保加利亚乳杆菌、鼠伤寒沙门氏菌、哈氏弧菌BB152、MC1061(待买)</span></span></p>
+
<p class=a><span lang=EN-US style='font-size:13.0pt;font-family:Arial;
 +
mso-hansi-font-family:"Arial Unicode MS";color:#323232'>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>
 +
</div>
 +
<a id="returnTop" href="javascript:;">Back to Top</a>
 +
</body>
  
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">质粒:PNZ8148、PHY300PLK、PBBR1MCS-5、PNZ9530(是否购买看实验结果)</span></span></p>
+
</p>
 +
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<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">一:</span></span></p>
+
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+
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<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">基因克隆</span></span></p>
+
</div> <div class="visualClear"></div>
 
+
    </div>
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">A:多一个碱基&rarr;初步采用SOE-PCR解决</span></span></p>
+
</div>
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">B:正常</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">C:正常(中间含一段碱基序列,但未造成移码)</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">D:多一个碱基&rarr;初步采用SOE-PCR解决</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">R:正常</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">K:未克隆出</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">二:</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif"><strong>质粒构建:</strong></span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">1)ABCDRK+ PNZ 8148(问题:连好T-5载体的质粒酶切切不开!?)</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">2)DRK+PBBR3MCS-5(初步采用PCR)</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">3)PET28b(含蓝光色素蛋白),启动子替换(酶切)&rarr;+PHY300PLK</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">4)PNZ 8148+蓝光色素蛋白&rarr;证明启动子好用</span></span></p>
+
 
+
<p>5)plsr+蓝光色素蛋白+ PNZ 8148&rarr;证明启动子好用</p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif"><strong>大肠杆菌(</strong><strong>MC1061</strong><strong>)感受态制备:</strong></span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">主要试剂</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">(1)0.1mol/L CaCl2溶液</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">(2)LB液体培养基</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">(3)30%甘油:30mL甘油溶于100mL蒸馏水,高压灭菌。</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">主要设备</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">(1)超净工作台</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">(2)冷冻离心机</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">(3)恒温摇床</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">(4)-70℃冰箱</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">(5)10mL移液管</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">(6)吸耳球</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">(7)1mL、200&mu;L移液枪(配套枪头)</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">(8)50mL 离心管</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">(9)1.5mL离心管</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">实验材料:大肠杆菌</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">实验步骤</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">(一)受体菌的培养</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">(1)从LB平板上挑取新活化的E. coli DH5&alpha;单菌落,接种于3~5mL LB液体培养基中,37℃下振荡培养过夜(12h左右)。</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">(2)将该菌种悬液以1:100的比例接种,取250&mu;L菌液转接到25mL LB液体培养基中,37℃振荡培养2~3h至OD600=0.5左右。</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">(二)感受态细胞的制备(注意:以下操作在超净工作台完成。)</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">(1)将菌液转入50mL离心管中,冰上放置10min。</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">(2)在4℃下,4000r/min离心10min。弃去上清,将管倒置1min以便培养液流尽。</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">(3)用冰上预冷的0.1mol/L的CaCl2 溶液10mL轻轻悬浮细胞,冰上放置30min。</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">(4)0~4℃ 4000r/min离心10min,弃去上清,加入2mL预冷的0.1mol/L的CaCl2 溶液,轻轻悬浮细胞,冰上放置(务必冰上放置)。(注意:以上操作完成了新鲜感受态细胞的制备)</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">(三)感受态细胞的分装与冻存</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">(1)在2mL制备好的感受态细胞中加入2mL30%甘油(即1:1体积,甘油终浓度15%)。</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">(2)将此感受态细胞分装成每份200&mu;L (1.5mL dorf管),液氮速冻,快速转入-70℃冰箱保存。(如果没有液氮,可以将分装的感受态细胞直接转入-70℃冰箱保存。)</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">注意事项</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">⑴、细胞的生长状态和密度</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">最好从-70℃或-20℃甘油保存的菌种中直接转接用于制备感受态细胞的菌液。不要用已 经过多次转接,及贮存在4℃的培养菌液。细胞生长密度以每毫升培养液中的细胞数在5&times;107个左右为佳。即应用对数期或对数生长前期的细菌,可通过测定培养液的OD600控制。对TG1菌株,OD600为0.5时,细胞密度在5&times;107个/ml左右。(应注意OD600值与细胞数之间的关系随菌株的不同而不同)。密度过高或不足均会使转化率下降。 此外,受体细胞一般应是限制-修饰系统缺陷的突变株,即不含限制性内切酶和甲基化酶的突变株。并且受体细胞还应与所转化的载体性质相匹配。</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">⑵、试剂的质量</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">所用的CaCl2等试剂均需是最高纯度的,并用最纯净的水配制,最好分装保存于4℃。</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">⑶、防止杂菌和杂DNA的污染</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">整个操作过程均应在无菌条件下进行,所用器皿,如离心管,移液枪头等最好是新的,并经高压灭菌处理。所有的试剂都要灭菌,且注意防止被其它试剂、DNA酶或杂DNA所污染,否则均会影响转化效率或杂DNA的转入。</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">⑷、整个操作均需在冰上进行,不能离开冰浴,否则细胞转化率将会降低。</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">三:</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">AI-2的化学检测:</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">标品:抗坏血酸</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">标准曲线:抗坏血酸标准曲线</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">试剂:</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">1、10 mM 1,10-phenanthroline/3.32 mM Fe(III) :1,10-phenanthroline(0.198 g)+去离子蒸馏水(50 mL),用1 M HCl调pH 2。</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">2、<a name="OLE_LINK2"></a><a name="OLE_LINK1">硫酸铁铵</a> (0.16 g)加到去离子蒸馏水(100 mL)中。</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">步骤:</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">标准:0.5 mM ascorbic acid</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">对于Fe(III)离子减少的检测 ,1 mL 样品(colony rinse in SNSS or cell free supernatant from a bacterial culture) +1 mL Fe(III)-1,10- phenanthroline 反应1 min ,用水稀释到5 mL,过0.2 &mu;m滤膜,510nm测吸光度。</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">Negative control:培养基without ascorbic acid (20 &mu;M)</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">positive control:培养基with ascorbic acid (20 &mu;M)</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">干扰因素:</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">0.5Mammonium acetate</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">0.5M sodium hydroxide and</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">0.5 M N-(&szlig;-ketocaproyl)-homoserine lactone (3-oxo-C6-AHL)</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">四:</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">保加利亚乳酸菌感受态的制备及电转化</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">实验前准备</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">1、 筛选转化子的抗生素浓度的确定</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">分别配制 含 1.0、2.0、3.0、4.0、5.0、6.0、7.0、8.0、9.0、10.0、11.0、12.0、13.0、14.0 &mu;g/mL 抗生素的 MRS 抗生素平板,涂布生长至对数期的保加利亚乳杆菌,37℃静止培养 36~48 h 观察结果, 根据抗生素平板上乳杆菌的生长情况确定抗生素的浓度。</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">2、保加利亚乳杆菌菌株在不同培养基中生长曲线的测定</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">将单菌落接入 10 mL MRS 培养液中,37 ℃静置培养过夜后, 以 2%接种量分别接入 100 mL含 2.5%甘氨酸的液体SMRS 培养基中,37 ℃静置培养,每隔 2 h 取样,于波长 600 nm处测 OD 值。得到对数中期的时间(文献中为8h)</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">乳杆菌感受态细胞的制备与电转化</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">1、将单菌落接入10 mL MRS 培养液中,37 ℃静置培养过夜</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">2、感受态细胞的培养:(以 2%接种量分别接入100 mL 2.5%甘氨酸的液体 SMRS 培养基中 37 ℃静置培养,冷却(将培养物置于冰上 20~30 min),离心洗涤与细胞重悬(6 000 r/min 离心 10 min,收集菌体,用 PB 缓冲液洗涤 3 次后,再用1 mL PB 缓冲液重悬菌体)。</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">3、感受态细胞与质粒 DNA 混合(吸取 50 &mu;L 新制备的感受态细胞悬液到预冷的无菌EP 管中,加入 1 &mu;L 质量浓度为 1 &mu;g/&mu;L 的质粒,冰浴 5 min, 将此混合液加入预冷的 2 mm 电转化杯中)高压脉冲电击转化(电场强度 12 kV/cm,电阻 200 &Omega;、电容 25 &mu;F)。</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">4、转化子复苏(电击完毕后迅速向电转化杯中加入950 &mu;L含有 20 mmol/L(MgCl2、CaCl2)的 SMRS 液体培养基,混匀后倒入无菌 EP 管中,37 ℃静置培养2 h)。</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">5、转化子培养与筛选 (取用再生液体培养基稀释后的电击转化细胞液 100 &mu;L,涂布到含相应浓度的抗生素的 MRS 平板上,37 ℃倒置培养 36~48 h 后,计数转化子并计算转化效率)。</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">五:</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">表达产物分析:</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">RNA水平:多重PCR</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">蛋白质水平:SDS-PAGE</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">制 胶</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">1、试 剂</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">1) 丙烯酰胺(电泳级)</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">2) 双丙烯酰胺(N,N&mdash;甲叉双丙烯酰肢)</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">3) Tris 絨</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">4) SDS (十二烷基磺酸钠)</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">5) TEMED</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">6) 过硫酸铵</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">7) &alpha;-巯基乙醇</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">8) 甘油</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">9) 溴酚蓝</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">10)甘氨酸</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">11)盐酸</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">12) DTT</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">2、储存液</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">(1) 2M Tris-HCl(pH8.8),100ml</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">称取 24.2g Tris 碱;</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">加50ml蒸馏水;</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">缓慢的加浓盐酸至pH8.8 (约加4ml);让溶液冷却至室温,pH将会升高;</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">加蒸馏水至100ml</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">(2)1M Tris-HCI(pH6.8),100ml</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">称取12.1g的Tris碱;</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">加50ml蒸馏水;</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">缓慢的加浓盐酸至pH6.8(约加8ml);让溶液冷却至室温,pH将会升高L;</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">加蒸馏水至总量100ml.</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">(3)10%(w/v)SDS,100ml 室温保存</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">&nbsp;称取10g的SDS;</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">加蒸馏水至总量为100ml。</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">(4)50%(v/v)甘油,100ml</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">到取 50ml 100%的甘油;</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">加人5ml蒸馏水。</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">(5)1% (w/v)溴酚蓝,10ml</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">称取100mg溴酚蓝;</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">加蒸馏水至10ml,搅拌直到完全溶解;</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">过滤除去聚合的染料。</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">3、 工作液</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">(1)&nbsp;&nbsp;&nbsp; A液:丙烯酰胺储存液,100ml</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">30% (w/v)丙烯酰胺,0.8%(w/v)双丙烯酰胺。</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">29.2g丙烯酰胺</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">0.8g双丙烯酰胺</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">(2)B液:4X分离胶缓冲液,100ml</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">75ml 2 mol/L Tris-HCl(pH8.8)&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 1.5 mol/L</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">4ml 10% SDS&nbsp;&nbsp; 0.4%</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">21ml蒸馏水</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">可在4&deg;C存放数月。</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">(3)C液:4X堆积胶缓冲液,100ml</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">50ml 1 mol/L Tris-HCI (pH6.8)&nbsp;&nbsp;&nbsp; 0.5mol/L</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">4ml 10%SDS&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 0.4%</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">46ml蒸馏水 可在41:存放数月。</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">(4)&nbsp;&nbsp;&nbsp; 10%过硫酸铵,5m丨 0.5g过硫酸铵</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">5mJ蒸溜水</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">可在密封的管内,4C存放数月。</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">(5)&nbsp;&nbsp;&nbsp; 电泳缓冲液,1L</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">3g Tris 碱&nbsp;&nbsp; 25mM</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">14.4g 甘氨酸&nbsp;&nbsp;&nbsp; 192mM</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">IgSDS&nbsp; 0-1%</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">加蒸馏水至1L</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">pH应该在8.3左右。也可以制成10x的储存液,在室 温下长期保存。</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">(6)5X样品缓冲液,10ml</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">0.6ml 1mol/L 的Tris-HCl(pH6.8)&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; &nbsp;&nbsp; 60mmol/L</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">5ml 50%的甘油&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;25%</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">2ml 10%的SDS&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;2%</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">0.5ml 2-巯基乙醇&nbsp;&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 14.4mmol/L&emsp;</span></span></p>
+
 
+
<p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">1ml 1%溴酚蓝&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 0.1%</span></span></p>
+
 
+
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Latest revision as of 17:52, 17 September 2015

Team:NEFU China/Description - 2015.igem.org

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Team:NEFU China/Description

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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.

 

Fig1. Delicious yogurt

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.

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.

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, Lactobacillus 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.

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¡æ 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¡æ 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 Result for more details)

Fig2. Yogurt treated with different temperature. (A) Fresh yogurt kept in 4¡æ for 0.5hour (B) Fresh yogurt kept in 35¡æ for half an hour (C) Yogurt kept in 4¡æ until one-day past expiration

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.

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