Team:Peking/Notebook

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        <li><a href="#010killing">Introduction</a></li>

                <li><a href="#0103killing">3.Immunity System</a></li>

        <li><a href="#020killing">Design</a></li>

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<h3 id="0102killing">2.Secretion</h3>

<p>In order to improve the efficiency of the hen egg lysozyme secretion, we also constructed a plasmid that contains both the lysozyme-linker-hlyA signal sequence and these 3 components. We transformed this plasmid, whose backbone is pET-21a, into the the <i>E. coli</i> BL21(DH3). This strain was defined as Stain B (Fig. 3B). We could further induce both Stain A and Stain B with IPTG and then tested the effect of the lysozyme secretion as well as the killing effect of the secreted lysozyme. </p>

<h3 id="0103killing">3. Immunity System</h3>

<p>Our construct contains the ykfE gene under control of T7 promoter in the pET-21a plasmid was designated Stain C. This pET-21a plasmid was transformed into <i>E. coli</i> BL21, and the resulting strain was designated as ykfE overexpression strain <B>(Fig. 5)</B>.</p>

<h3>1. Growth of algae and the killing efficiency of hen egg lysozymes</h3>

<p>The result indicates that 200ng/L lysozyme could kill the algae effectively within 72h. So the hen egg lysozyme, if could be properly expressed by our genetically engineered <i>E. coli</i>, would be a valid approach to kill the algae. </p>

<h3>2.The lysozyme immune system</h3>

<p>Considering the working mechanism of lysozyme that cleaving the peptidoglycan of bacterial cell wall can also wound the "manufacturer", our genetically engineered <i>E. coli</i>. Such a detrimental effect to <i>E. coli</i> was firstly measured<B> (Fig. 2A)</B>. To counteract this effect, the best solution to overcome the detrimental effect is to build an immune system for our genetically engineered <i>E. coli</i>. We utilized ykfE, a native inhibitor of lysozyme of <i>E. coli</i>. The circuit for the strain has been constructed <B>(Fig. 2B)</B>, and further experimental is expected coming soon. </p>

<h3>3. The killing efficiency of Lysozyme expressed by genetically engineered <i>E. coli</i></h3>

<p>We have constructed plasmids to express the hen egg lysozyme under the inducible promoter on the expression vector pET-21a. The expression of lysozyme in <i>E. coli</i> was verified by PAGE electrophoresis <B>(Fig. 3)</B>. We have verified that the hen egg lysozyme was successfully expressed in our <i>E. coli</i>. Although we have prepared the analysis that to use sonication lysed <i>E. coli</i> or purified protein to test whether the lysozyme is functionally expressed, the difficulty in the experimental protocol and time limit restrict our further trials. More data could probably be shown in our coming oral and poster presentations. </p>

<h3>4.Perspective experiments</h3>