Difference between revisions of "Team:CityU HK/Experiments"
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| + | |||
| + | <!----------- sidebar html ------------> | ||
| + | <div class="area"></div><nav class="main-menu"> | ||
| + | <ul> | ||
| + | <li> | ||
| + | <a class="scroll" href="#laczy"> | ||
| + | <i class="sidebar-fa"></i> | ||
| + | <span class="nav-text" id="first-item"> | ||
| + | <i>LacZY</i> plasmid | ||
| + | </span> | ||
| + | </a> | ||
| + | </li> | ||
| + | |||
| + | <li class="has-subnav"> | ||
| + | <a class="scroll" href="#lactose"> | ||
| + | <i class="sidebar-fa fa fa-th-list fa-2x"></i> | ||
| + | <span class="nav-text"> | ||
| + | Lactose inducible promoter | ||
| + | </span> | ||
| + | </a> | ||
| + | </li> | ||
| + | |||
| + | <li class="has-subnav"> | ||
| + | <a class="scroll" href="#lysis"> | ||
| + | <i class="sidebar-fa"></i> | ||
| + | <span class="nav-text"> | ||
| + | Lysis plasmid | ||
| + | </span> | ||
| + | </a> | ||
| + | </li> | ||
| + | |||
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| + | <a class="scroll" href="#characterization"> | ||
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| + | <span class="nav-text"> | ||
| + | Characterization | ||
| + | </span> | ||
| + | </a> | ||
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<div class="container"> | <div class="container"> | ||
<div class="content-wrap"><div id='wsite-content' class='wsite-elements wsite-not-footer'> | <div class="content-wrap"><div id='wsite-content' class='wsite-elements wsite-not-footer'> | ||
| + | |||
<h2 class="wsite-content-title" style="text-align:left;"><span style=""><span style="">Module Description</span></span><br /></h2> | <h2 class="wsite-content-title" style="text-align:left;"><span style=""><span style="">Module Description</span></span><br /></h2> | ||
| − | <div | + | <div id="laczy" class="paragraph" ><font size="3"><span "font-size:12.0pt;mso-bidi-font-size:="" 11.0pt;font-family:"calibri","sans-serif";mso-ascii-theme-font:minor-latin;="" mso-fareast-font-family:新細明體;mso-fareast-theme-font:minor-fareast;mso-hansi-theme-font:="" minor-latin;mso-bidi-font-family:"times="" roman";mso-bidi-theme-font:minor-bidi;="" mso-ansi-language:en-us;mso-fareast-language:zh-tw;mso-bidi-language:ar-sa"="" style="">In the belief that everyone can enjoy dairy products, our team engineered an <em style="">E. coli </em>strain to help relieve of those people with lactose intolerance. The bacteria carry two recombinant genes which can synthesize </span><span "font-size:12.0pt;mso-bidi-font-size:11.0pt;font-family:symbol;="" mso-fareast-font-family:新細明體;mso-fareast-theme-font:minor-fareast;mso-bidi-font-family:="" "times="" roman";mso-bidi-theme-font:minor-bidi;mso-ansi-language:en-us;="" mso-fareast-language:zh-tw;mso-bidi-language:ar-sa"="" style="">β</span><span "font-size:12.0pt;mso-bidi-font-size:11.0pt;font-family:"calibri","sans-serif";="" mso-ascii-theme-font:minor-latin;mso-fareast-font-family:新細明體;mso-fareast-theme-font:="" minor-fareast;mso-hansi-theme-font:minor-latin;mso-bidi-font-family:"times="" roman";="" mso-bidi-theme-font:minor-bidi;mso-ansi-language:en-us;mso-fareast-language:="" zh-tw;mso-bidi-language:ar-sa"="" style="">-galactosidase, the enzyme for breaking down lactose and lactose permease, allowing lactose to enter the bacteria faster. Lysis plasmid was constructed with features to allow the release of lactase quickly once the bacteria sense the presence of lactose in the surrounding.</span></font><br /><br /></div> |
| − | <h2 class="wsite-content-title" style="text-align:left;">< | + | <h2 class="wsite-content-title" style="text-align:left;"><span style=""><span style="">A. <i>lacZY</i> plasmid</span></span><br /></h2> |
| − | <div><div class="wsite-image wsite-image-border-none " style="padding-top:10px;padding-bottom:10px;margin-left:0;margin-right:0;text-align: | + | <div><div class="wsite-image wsite-image-border-none " style="padding-top:10px;padding-bottom:10px;margin-left:0;margin-right:0;text-align:left"> |
<a> | <a> | ||
| − | <img src=" | + | <center> |
| + | <img src="https://static.igem.org/mediawiki/2015/2/23/2015CityU_HK_experiment_lacZY.png" alt="Picture" style="width:auto;max-width:70%" /> | ||
| + | </center> | ||
</a> | </a> | ||
| − | <br/><div style="display:block;font-size: | + | <br/><div style="display:block;font-size:90%"><font size= "3"><center><b>Figure 1. The design of <i>lacZY</i> plasmid </b></center></em>.</font></div><br /> |
</div></div> | </div></div> | ||
| − | <div class="paragraph | + | <div class="paragraph" ><font size="3"><span "font-size:12.0pt;mso-bidi-font-size:="" 11.0pt;font-family:"calibri","sans-serif";mso-ascii-theme-font:minor-latin;="" mso-fareast-font-family:新細明體;mso-fareast-theme-font:minor-fareast;mso-hansi-theme-font:="" minor-latin;mso-bidi-font-family:"times="" roman";mso-bidi-theme-font:minor-bidi;="" mso-ansi-language:en-us;mso-fareast-language:zh-tw;mso-bidi-language:ar-sa"="" style="">The <i>lacZ</i> gene encodes β-galactosidase, which is an enzyme that digests lactose into glucose and galactose. A strong ribosome binding site (BBa_B0034) is added in front of the <i>lacZ</i> gene to increase the binding affinity of ribosomes to RBS. The <i>lacY’</i> gene, preceded by a weak ribosome binding site (BBa_B0033), encodes for a mutated lactose permease that cannot be inhibited by glucose, and allows efficient transport of lactose into the cells. The constitutive promoter BBa_J23100 provides a constant and strong transcription for the two genes, which ensue sufficient production of β-galactosidase and allows transport of lactose for activating lysis plasmid.</span></font><br /><br /></div> |
| − | <div><div style="height: | + | |
| + | <div id="lactose"><div style="height: 100px; overflow: hidden; width: 100%;"></div> | ||
<hr class="styled-hr" style="width:100%;"></hr> | <hr class="styled-hr" style="width:100%;"></hr> | ||
<div style="height: 30px; overflow: hidden; width: 100%;"></div></div> | <div style="height: 30px; overflow: hidden; width: 100%;"></div></div> | ||
| + | <h2 class="wsite-content-title" style="text-align:left;"><span style=""><span style="">B. Construction of a tightly regulated lactose inducible promoter</span></span><br /></h2> | ||
| − | < | + | <div class="paragraph" ><font size="3"><span "font-size:12.0pt;mso-bidi-font-size:="" 11.0pt;font-family:"calibri","sans-serif";mso-ascii-theme-font:minor-latin;="" mso-fareast-font-family:新細明體;mso-fareast-theme-font:minor-fareast;mso-hansi-theme-font:="" minor-latin;mso-bidi-font-family:"times="" roman";mso-bidi-theme-font:minor-bidi;="" mso-ansi-language:en-us;mso-fareast-language:zh-tw;mso-bidi-language:ar-sa"="" style="">Building a tightly regulated lactose inducible promoter<br />This biobrick is a designed for engineering a tightly regulated LacI inducible promoter. This biobrick consists of three parts: the <em style="">lacI</em><sup>Q</sup>promoter (P<em style="">lacI</em><sup>Q</sup>), wild type <em style="">lacI </em>gene and the <em style="">PL8-UV5 </em> promoter, a modified glucose-independent LacI control promoter (Figure 2).</span></font><br /></div> |
| − | < | + | <div><div class="wsite-image wsite-image-border-none " style="padding-top:10px;padding-bottom:10px;margin-left:0;margin-right:0;text-align:left"> |
| + | <a> | ||
| + | <center> | ||
| + | <img src="https://static.igem.org/mediawiki/2015/7/7d/2015CityU_HK_pL8_UV5.png" alt="Picture" style="width:auto;max-width:60%" /> | ||
| + | </center> | ||
| + | </a> | ||
| + | <br/><div style="display:block;font-size:60%"><font size= "3"><b>Figure 2. Gene construct of BBa_K1695000.</b> The biobrick consist three parts, the P<em style="">lacI</em><sup>Q</sup> promoter, <em style="">E. coli </em> wild type <em style="">lacI </em> gene linked with RBS (BBa_B0034) and the LacI regulated promoter <em style="">L8-UV5 </em>.</font></div><br /> | ||
| + | </div></div> | ||
| − | < | + | <h2 class="wsite-content-title" style="text-align:left;"><font size="5"><em style="">PlacI </em><sup>Q</sup></font></h2> |
| − | < | + | <div class="paragraph" ><font size="3"><span style="">The <em style="">PlacI</em><sup>Q</sup> is a mutated promoter of the <em style="">lacI</em> gene with a C --> T conversion in the -35 region (Calos, 1978) (Figure 2). According to Calos (1978), the LacI protein expression level is 10-fold higher in the <em style="">PlacI</em><sup>Q</sup> system than the <em style="">PlacI</em> system.</span></font><br /><br /></div> |
| − | <div class=" | + | <div><div class="wsite-image wsite-image-border-none " style="padding-top:10px;padding-bottom:10px;margin-left:0;margin-right:0;text-align:left"> |
| + | <a> | ||
| + | <center> | ||
| + | <img src="https://static.igem.org/mediawiki/2015/4/48/2015CityU_HK_lacIlacIQ.png" alt="Picture" style="width:auto;max-width:60%" /> | ||
| + | </center> | ||
| + | </a> | ||
| + | <br/><div style="display:block;font-size:90%"><font size= "3"><b>Figure 3. The DNA sequences of the <em style="">lacI </em> and <em style="">lacI</em><sup>Q</sup> promoters.</b> Only the top strands of the promoters are depicted and the -10 and -35 sequences are shown in boxes. The <em style="">lacI</em><sup>Q</sup> mutation is highlighted in red.</font></div><br /> | ||
| + | </div></div> | ||
| − | < | + | <div class="paragraph" ><font size="3"><span style="">Linking the constitutive promoter P<em style="">lacI</em><sup>Q</sup> renders LacI being expressed constitutively and the extra LacI protein provides a stronger inhibition on the <i>PL8-UV5</i> promoter.</span></font></div><br /> |
| − | <div class="paragraph" style="text-align:left;"><span style=""><font size="3"> | + | <h2 class="wsite-content-title" style="text-align:left;"><font size="5"><i>PL8-UV5</i> (BBa_K1695000)</font></h2> |
| + | |||
| + | <div class="paragraph" ><font size="3"><span style="">The <i>PL8-UV5</i> promoter is a mutated LacI controlled promoter. The two single base-pair mutations (C --> T at positions -66 and -55) at the CAP-binding site inactivate the binding of CAP protein (Hirschel, Shen, & Schlessinger, 1980), leading to the promoter expression being independent of the cyclic AMP level, which are produced under poor glucose supply. In addition, a two-base pair mutation (GT --> AA at -9 and -8) converts the sequence at the -10 region back to the consensus sequence (TATAAT), which allows the σ factor to bind to the -10 element without the help of CAP protein (Figure 4).</span></font><br /><br /></div> | ||
| + | |||
| + | <div><div class="wsite-image wsite-image-border-none " style="padding-top:10px;padding-bottom:10px;margin-left:0;margin-right:0;text-align:left"> | ||
| + | <a> | ||
| + | <center> | ||
| + | <img src="https://static.igem.org/mediawiki/2015/4/43/2015CityU_HK_WT_L8UV5.png" alt="Picture" style="width:auto;max-width:60%" /> | ||
| + | </center> | ||
| + | </a> | ||
| + | <br/><div style="display:block;font-size:90%"><font size= "3"><b>Figure 4. Sequence of wild type Lac promoter and <i>L8-UV5</i> Lac promoter. </b>The CAP-binding site is underlined, the -10 element is boxed, and the two LacI binding sites (O3, O1) are highlighted gray. The mutations in <i>L8-UV5</i> Lac promoter and the -10 region are highlighted red.</font></div><br /><br /> | ||
| + | </div></div> | ||
| + | <div class="paragraph" ><font size="3"><span style="">The use of the <i>PL8-UV5</i> promoter can remove the inhibitory effect of glucose on lactose induction of the promoter. The expression of the gene downstream of this promoter is thus solely dependent on lactose concentration.</span></font><br /><br /></div> | ||
| + | |||
| + | <div id="lysis"><div style="height: 100px; overflow: hidden; width: 100%;"></div> | ||
| + | <hr class="styled-hr" style="width:100%;"></hr> | ||
| + | <div style="height: 30px; overflow: hidden; width: 100%;"></div></div> | ||
| + | |||
| + | <h2 class="wsite-content-title" style="text-align:left;"><span "font-size:12.0pt;mso-bidi-font-size:11.0pt;font-family:"calibri","sans-serif";="" mso-ascii-theme-font:minor-latin;mso-fareast-font-family:新細明體;mso-fareast-theme-font:="" minor-fareast;mso-hansi-theme-font:minor-latin;mso-bidi-font-family:"times="" roman";="" mso-bidi-theme-font:minor-bidi;mso-ansi-language:en-us;mso-fareast-language:="" zh-tw;mso-bidi-language:ar-sa"="" style="">C. Lysis plasmid</span></h2> | ||
| + | |||
| + | <div><div class="wsite-image wsite-image-border-none " style="padding-top:10px;padding-bottom:10px;margin-left:0;margin-right:0;text-align:left"> | ||
| + | <a> | ||
| + | <center> | ||
| + | <img src="https://static.igem.org/mediawiki/2015/0/06/2015CityU_HK_experiment_lysis.png" alt="Picture" style="width:636;max-width:70%" /> | ||
| + | </center> | ||
| + | </a> | ||
| + | <br /> | ||
| + | <div style="display:block;font-size:90%"><font size= "3"><center><b>Figure 5. The design of lysis plasmid</b></center></font></div> | ||
| + | </div></div> | ||
| + | |||
| + | <div class="paragraph" ><font size="3"><span style="">The lysis plasmid consists mainly of two parts: the lysis cassette and <i>lacI</i> repressor gene. </span><br /><span style=""></span><br /><span style=""></span> <span "font-size:12.0pt;mso-bidi-font-size:11.0pt;font-family:="" "calibri","sans-serif";mso-ascii-theme-font:minor-latin;mso-fareast-font-family:="" 新細明體;mso-fareast-theme-font:minor-fareast;mso-hansi-theme-font:minor-latin;="" mso-bidi-font-family:"times="" roman";mso-bidi-theme-font:minor-bidi;="" mso-ansi-language:en-us;mso-fareast-language:zh-tw;mso-bidi-language:ar-sa"="" style="">The lysis cassette is composed of holin (<em style="">S</em> gene), endolysin (<em style="">R</em> gene) and spanin (<em style="">Rz</em> gene). Two phage origins of the lysis cassette, phage lambda and phage 21, were cloned and compared. To speed up the cell lysis for releasing β-galactosidase, 2 modifications including codon optimization and mutations on the <em style="">S </em>gene were included. A missense mutation and a deletion of the trans-membrane domain were applied to the <em style="">S</em> gene of phage lambda and phage 21, respectively. Our team has constructed 9 lysis cassettes with different combinations (please refer to the <a href="https://2015.igem.org/Team:CityU_HK/Parts" style="color: #000000"><b><u>PARTS</u></b></a>). For all the cassettes, <i>PL8-UV5</i>, a LacI regulated promoter was used to turn the transcription on.</span></font></div> | ||
| + | |||
| + | <div id="characterization"><div style="height: 100px; overflow: hidden; width: 100%;"></div> | ||
| + | <hr class="styled-hr" style="width:100%;"></hr> | ||
| + | <div style="height: 30px; overflow: hidden; width: 100%;"></div></div> | ||
| + | |||
| + | <h2 class="wsite-content-title" style="text-align:left;"><span "font-size:12.0pt;mso-bidi-font-size:11.0pt;font-family:"calibri","sans-serif";="" mso-ascii-theme-font:minor-latin;mso-fareast-font-family:新細明體;mso-fareast-theme-font:="" minor-fareast;mso-hansi-theme-font:minor-latin;mso-bidi-font-family:"times="" roman";="" mso-bidi-theme-font:minor-bidi;mso-ansi-language:en-us;mso-fareast-language:="" zh-tw;mso-bidi-language:ar-sa"="" style="">Characterization of the biobricks/ parts</span></h2> | ||
| + | |||
| + | <h2 class="wsite-content-title" style="text-align:left;"><span "font-size:12.0pt;mso-bidi-font-size:11.0pt;font-family:"calibri","sans-serif";="" mso-ascii-theme-font:minor-latin;mso-fareast-font-family:新細明體;mso-fareast-theme-font:="" minor-fareast;mso-hansi-theme-font:minor-latin;mso-bidi-font-family:"times="" roman";="" mso-bidi-theme-font:minor-bidi;mso-ansi-language:en-us;mso-fareast-language:="" zh-tw;mso-bidi-language:ar-sa"=""></span><span "font-size:12.0pt;mso-bidi-font-size:11.0pt;font-family:"calibri","sans-serif";="" mso-ascii-theme-font:minor-latin;mso-fareast-font-family:新細明體;mso-fareast-theme-font:="" minor-fareast;mso-hansi-theme-font:minor-latin;mso-bidi-font-family:"times="" roman";="" mso-bidi-theme-font:minor-bidi;mso-ansi-language:en-us;mso-fareast-language:="" zh-tw;mso-bidi-language:ar-sa"="" style=""><font size="5">RNA levels of <i>lacZ</i> and <i>lacY</i></font><br /></span></h2> | ||
| + | |||
| + | <div class="paragraph" ><font size="3">To determine the levels of the <em style=""><span style="">lacZ </span></em>and <em style=""><span style="">lacY</span></em> transcripts, total RNA extraction was extracted, RNA was converted into cDNA by reverse transcription and the levels of the two transcripts were determined by real time PCR.<em style=""><span style=""></span></em><br /><br /> <strong>RNA extraction → Reverse transcription → Real time PCR</strong><br /> <br /><br /> <strong>RNA extraction:</strong> <a href="https://2015.igem.org/Team:CityU_HK/Protocol#cloning" style="color: #000000"><b><u>(Link to the protocol)</u></b></a><br /> To extract the total RNA from the engineered <em style="">E. coli</em><br /><br /> <em style=""><span style=""> </span></em><br /> <strong>Reverse transcription:</strong> <a href="https://2015.igem.org/Team:CityU_HK/Protocol#cloning" style="color: #000000"><b><u>(Link to the protocol)</u></b></a><br /> To convert the total RNA into cDNA for the subsequent real time PCR. Real time PCR can only quantify the levels of cDNA but not RNA.<br /><br /> <br /> <strong>Real time PCR:</strong> <a href="https://2015.igem.org/Team:CityU_HK/Protocol#cloning" style="color: #000000"><b><u>(Link to the protocol)</u></b></a><br /> <span "font-size:12.0pt;mso-bidi-font-size:11.0pt;font-family:="" "calibri","sans-serif";mso-ascii-theme-font:minor-latin;mso-fareast-font-family:="" 新細明體;mso-fareast-theme-font:minor-fareast;mso-hansi-theme-font:minor-latin;="" mso-bidi-font-family:"times="" roman";mso-bidi-theme-font:minor-bidi;="" mso-ansi-language:en-us;mso-fareast-language:zh-tw;mso-bidi-language:ar-sa"="" style="">To amplify our desired cDNA (</span><em style=""><span "font-size:12.0pt;mso-bidi-font-size:11.0pt;font-family:"calibri","sans-serif";="" mso-ascii-theme-font:minor-latin;mso-fareast-font-family:新細明體;mso-fareast-theme-font:="" minor-fareast;mso-hansi-theme-font:minor-latin;mso-bidi-font-family:"times="" roman";="" mso-bidi-theme-font:minor-bidi;mso-ansi-language:en-hk;mso-fareast-language:="" zh-tw;mso-bidi-language:ar-sa"="" style="">lacZ</span></em><span "font-size:12.0pt;mso-bidi-font-size:11.0pt;font-family:"calibri","sans-serif";="" mso-ascii-theme-font:minor-latin;mso-fareast-font-family:新細明體;mso-fareast-theme-font:="" minor-fareast;mso-hansi-theme-font:minor-latin;mso-bidi-font-family:"times="" roman";="" mso-bidi-theme-font:minor-bidi;mso-ansi-language:en-us;mso-fareast-language:="" zh-tw;mso-bidi-language:ar-sa"="" style=""> and <i>lacY</i>) if the RNA extract contains the <i>lacZ</i> and <i>lacY</i> transcript. It will be difficult to detect the small amount of RNA in the cell, if the RNA is not amplified.</span></font></div> | ||
| + | |||
| + | <br><h2 class="wsite-content-title" style="text-align:left;"><span "font-size:12.0pt;mso-bidi-font-size:11.0pt;font-family:"calibri","sans-serif";="" mso-ascii-theme-font:minor-latin;mso-fareast-font-family:新細明體;mso-fareast-theme-font:="" minor-fareast;mso-hansi-theme-font:minor-latin;mso-bidi-font-family:"times="" roman";="" mso-bidi-theme-font:minor-bidi;mso-ansi-language:en-us;mso-fareast-language:="" zh-tw;mso-bidi-language:ar-sa"=""><font size="5">Characterization on LacZ</font></span></h2> | ||
| + | |||
| + | <div class="paragraph" ><font size="3"><span style="">To determine the level of β-galactosidase encoded by <em style="">lacZ</em>, ONPG assay was performed.</span><br /><br /><span style=""></span> <span style=""><strong>ONPG assay:</strong></span><span style=""> <a href="https://2015.igem.org/Team:CityU_HK/Protocol#characterization" style="color: #000000"><b><u>(Link to the protocol)</u></b></a></span><br /><span style=""></span><br /><span style=""></span> <span style="">Besides lactose, ONPG (ortho-Nitrophenyl-β-galactoside) is also a substrate of β-galactosidase. ONPG is digested into galactose and ortho-nitrophenol (ONP) which is yellow in colour. Chloroform was used to lyse the cells and the enzymes were released into the surrounding. By measuring the O.D. at 420 nm after lysing the cells, the level of the β-galatosidase was determined.</span></font><br /><span style=""></span><br /><span style=""></span></div> | ||
| + | |||
| + | <h2 class="wsite-content-title" style="text-align:left;"><span "font-size:12.0pt;mso-bidi-font-size:11.0pt;font-family:"calibri","sans-serif";="" mso-ascii-theme-font:minor-latin;mso-fareast-font-family:新細明體;mso-fareast-theme-font:="" minor-fareast;mso-hansi-theme-font:minor-latin;mso-bidi-font-family:"times="" roman";="" mso-bidi-theme-font:minor-bidi;mso-ansi-language:en-us;mso-fareast-language:="" zh-tw;mso-bidi-language:ar-sa"=""><font size="5"><i>PL8-UV5</i> characterization</font></span></h2> | ||
| + | |||
| + | <div class="paragraph" ><span style=""><font size="3"><i>L8-UV5</i> is a regulated promoter which can be induced by either lactose or the analog IPTG. To characterize the inducible property of the <i>L8-UV5</i> promoter, <i>PL8-UV5</i> was engineered upstream of the GFP gene. The GFP will be transcribed and translated after the promoter is induced. GFP fluorescent signal will be given by GFP. By measuring the GFP fluorescent signal after adding different concentration of IPTG to the bacteria, the inducible property of <i>L8-UV5</i> promoter can be determined.</font></span><br /><span style=""></span><br /><span style=""></span></div> | ||
<h2 class="wsite-content-title" style="text-align:left;"><font size="5">Lysis cassette characterization</font><br /><span style=""></span></h2> | <h2 class="wsite-content-title" style="text-align:left;"><font size="5">Lysis cassette characterization</font><br /><span style=""></span></h2> | ||
| − | <div class="paragraph | + | <div class="paragraph" ><font size="3"><span style="">To characterize the efficiency of the lysis cassette, the lysis cassette was put under the control of the T7 promoter in the pSNAP plasmid. By inducing the T7 promoter with lactose, the genes in the lysis cassette was transcribed and translated into the components of the lysis proteins, which cause cell lysis. </span><br /><br /><span style=""></span> <span style="">The O.D. and the colony forming units (CFU) were measured to determine the efficiency of the lysis cassette. </span><br /><span style=""></span><br /><span style=""></span> <span style=""> Inducing the promoter with lactose → </span><span style=""> Measure O.D. → </span><span style=""> Serial dilution → </span><span style=""> Spread plate → </span></font><span style=""></span><font size="3"><span style="">Incubate → </span></font><span style=""></span><font size="3"><span style="">Count the number of colonies</span><br /><span style=""></span><br /><span style=""></span> <span style="">The lower the CFU/O.D., the higher the efficiency of the lysis cassette after induction.</span><br /></font><span style=""></span><br /><span style=""></span></div> |
| − | <h2 class="wsite-content-title" style="text-align:left;"><span "font-size:12.0pt;mso-bidi-font-size:11.0pt;font-family:"calibri","sans-serif";="" mso-ascii-theme-font:minor-latin;mso-fareast-font-family:新細明體;mso-fareast-theme-font:="" minor-fareast;mso-hansi-theme-font:minor-latin;mso-bidi-font-family:"times="" roman";="" mso-bidi-theme-font:minor-bidi;mso-ansi-language:en-us;mso-fareast-language:="" zh-tw;mso-bidi-language:ar-sa"=""><font size="5">Characterization of the lacY lacZ operon BBa_S04055 (from 2008 Caltech: Curing lactose intolerance)</font></span></h2> | + | <h2 class="wsite-content-title" style="text-align:left;"><span "font-size:12.0pt;mso-bidi-font-size:11.0pt;font-family:"calibri","sans-serif";="" mso-ascii-theme-font:minor-latin;mso-fareast-font-family:新細明體;mso-fareast-theme-font:="" minor-fareast;mso-hansi-theme-font:minor-latin;mso-bidi-font-family:"times="" roman";="" mso-bidi-theme-font:minor-bidi;mso-ansi-language:en-us;mso-fareast-language:="" zh-tw;mso-bidi-language:ar-sa"=""><font size="5">Characterization of the <i>lacY lacZ</i> operon BBa_S04055 (from <a href="https://2008.igem.org/Team:Caltech/Project/Lactose_intolerance" style="color: #000000"><u>2008 Caltech</u></a>: Curing lactose intolerance)</font></span></h2> |
| − | <div class="paragraph | + | <div class="paragraph" ><span "font-size:12.0pt;mso-bidi-font-size:="" 11.0pt;font-family:"calibri","sans-serif";mso-ascii-theme-font:minor-latin;="" mso-fareast-font-family:新細明體;mso-fareast-theme-font:minor-fareast;mso-hansi-theme-font:="" minor-latin;mso-bidi-font-family:"times="" roman";mso-bidi-theme-font:minor-bidi;="" mso-ansi-language:en-us;mso-fareast-language:zh-tw;mso-bidi-language:ar-sa"="" style=""><font size="3">Western blotting analysis <a href="https://2015.igem.org/Team:CityU_HK/Protocol#characterization" style="color: #000000"><b><u>(Link to the protocol)</u></b></a> was used to detect the expression level of β-galactosidase inside <em style="">E. coli</em> harboring <a href="http://parts.igem.org/Part:BBa_S04055" style="color: #000000"><b><u>BBa_S04055</u></b></a>.</font></span></div></div> |
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Latest revision as of 20:46, 18 September 2015
Module Description
In the belief that everyone can enjoy dairy products, our team engineered an E. coli strain to help relieve of those people with lactose intolerance. The bacteria carry two recombinant genes which can synthesize β-galactosidase, the enzyme for breaking down lactose and lactose permease, allowing lactose to enter the bacteria faster. Lysis plasmid was constructed with features to allow the release of lactase quickly once the bacteria sense the presence of lactose in the surrounding.
A. lacZY plasmid
The lacZ gene encodes β-galactosidase, which is an enzyme that digests lactose into glucose and galactose. A strong ribosome binding site (BBa_B0034) is added in front of the lacZ gene to increase the binding affinity of ribosomes to RBS. The lacY’ gene, preceded by a weak ribosome binding site (BBa_B0033), encodes for a mutated lactose permease that cannot be inhibited by glucose, and allows efficient transport of lactose into the cells. The constitutive promoter BBa_J23100 provides a constant and strong transcription for the two genes, which ensue sufficient production of β-galactosidase and allows transport of lactose for activating lysis plasmid.
B. Construction of a tightly regulated lactose inducible promoter
Building a tightly regulated lactose inducible promoter
This biobrick is a designed for engineering a tightly regulated LacI inducible promoter. This biobrick consists of three parts: the lacIQpromoter (PlacIQ), wild type lacI gene and the PL8-UV5 promoter, a modified glucose-independent LacI control promoter (Figure 2).
This biobrick is a designed for engineering a tightly regulated LacI inducible promoter. This biobrick consists of three parts: the lacIQpromoter (PlacIQ), wild type lacI gene and the PL8-UV5 promoter, a modified glucose-independent LacI control promoter (Figure 2).
Figure 2. Gene construct of BBa_K1695000. The biobrick consist three parts, the PlacIQ promoter, E. coli wild type lacI gene linked with RBS (BBa_B0034) and the LacI regulated promoter L8-UV5 .
PlacI Q
The PlacIQ is a mutated promoter of the lacI gene with a C --> T conversion in the -35 region (Calos, 1978) (Figure 2). According to Calos (1978), the LacI protein expression level is 10-fold higher in the PlacIQ system than the PlacI system.
Figure 3. The DNA sequences of the lacI and lacIQ promoters. Only the top strands of the promoters are depicted and the -10 and -35 sequences are shown in boxes. The lacIQ mutation is highlighted in red.
Linking the constitutive promoter PlacIQ renders LacI being expressed constitutively and the extra LacI protein provides a stronger inhibition on the PL8-UV5 promoter.
PL8-UV5 (BBa_K1695000)
The PL8-UV5 promoter is a mutated LacI controlled promoter. The two single base-pair mutations (C --> T at positions -66 and -55) at the CAP-binding site inactivate the binding of CAP protein (Hirschel, Shen, & Schlessinger, 1980), leading to the promoter expression being independent of the cyclic AMP level, which are produced under poor glucose supply. In addition, a two-base pair mutation (GT --> AA at -9 and -8) converts the sequence at the -10 region back to the consensus sequence (TATAAT), which allows the σ factor to bind to the -10 element without the help of CAP protein (Figure 4).
Figure 4. Sequence of wild type Lac promoter and L8-UV5 Lac promoter. The CAP-binding site is underlined, the -10 element is boxed, and the two LacI binding sites (O3, O1) are highlighted gray. The mutations in L8-UV5 Lac promoter and the -10 region are highlighted red.
The use of the PL8-UV5 promoter can remove the inhibitory effect of glucose on lactose induction of the promoter. The expression of the gene downstream of this promoter is thus solely dependent on lactose concentration.
C. Lysis plasmid
The lysis plasmid consists mainly of two parts: the lysis cassette and lacI repressor gene.
The lysis cassette is composed of holin (S gene), endolysin (R gene) and spanin (Rz gene). Two phage origins of the lysis cassette, phage lambda and phage 21, were cloned and compared. To speed up the cell lysis for releasing β-galactosidase, 2 modifications including codon optimization and mutations on the S gene were included. A missense mutation and a deletion of the trans-membrane domain were applied to the S gene of phage lambda and phage 21, respectively. Our team has constructed 9 lysis cassettes with different combinations (please refer to the PARTS). For all the cassettes, PL8-UV5, a LacI regulated promoter was used to turn the transcription on.
The lysis cassette is composed of holin (S gene), endolysin (R gene) and spanin (Rz gene). Two phage origins of the lysis cassette, phage lambda and phage 21, were cloned and compared. To speed up the cell lysis for releasing β-galactosidase, 2 modifications including codon optimization and mutations on the S gene were included. A missense mutation and a deletion of the trans-membrane domain were applied to the S gene of phage lambda and phage 21, respectively. Our team has constructed 9 lysis cassettes with different combinations (please refer to the PARTS). For all the cassettes, PL8-UV5, a LacI regulated promoter was used to turn the transcription on.
Characterization of the biobricks/ parts
RNA levels of lacZ and lacY
To determine the levels of the lacZ and lacY transcripts, total RNA extraction was extracted, RNA was converted into cDNA by reverse transcription and the levels of the two transcripts were determined by real time PCR.
RNA extraction → Reverse transcription → Real time PCR
RNA extraction: (Link to the protocol)
To extract the total RNA from the engineered E. coli
Reverse transcription: (Link to the protocol)
To convert the total RNA into cDNA for the subsequent real time PCR. Real time PCR can only quantify the levels of cDNA but not RNA.
Real time PCR: (Link to the protocol)
To amplify our desired cDNA (lacZ and lacY) if the RNA extract contains the lacZ and lacY transcript. It will be difficult to detect the small amount of RNA in the cell, if the RNA is not amplified.
RNA extraction → Reverse transcription → Real time PCR
RNA extraction: (Link to the protocol)
To extract the total RNA from the engineered E. coli
Reverse transcription: (Link to the protocol)
To convert the total RNA into cDNA for the subsequent real time PCR. Real time PCR can only quantify the levels of cDNA but not RNA.
Real time PCR: (Link to the protocol)
To amplify our desired cDNA (lacZ and lacY) if the RNA extract contains the lacZ and lacY transcript. It will be difficult to detect the small amount of RNA in the cell, if the RNA is not amplified.
Characterization on LacZ
To determine the level of β-galactosidase encoded by lacZ, ONPG assay was performed.
ONPG assay: (Link to the protocol)
Besides lactose, ONPG (ortho-Nitrophenyl-β-galactoside) is also a substrate of β-galactosidase. ONPG is digested into galactose and ortho-nitrophenol (ONP) which is yellow in colour. Chloroform was used to lyse the cells and the enzymes were released into the surrounding. By measuring the O.D. at 420 nm after lysing the cells, the level of the β-galatosidase was determined.
ONPG assay: (Link to the protocol)
Besides lactose, ONPG (ortho-Nitrophenyl-β-galactoside) is also a substrate of β-galactosidase. ONPG is digested into galactose and ortho-nitrophenol (ONP) which is yellow in colour. Chloroform was used to lyse the cells and the enzymes were released into the surrounding. By measuring the O.D. at 420 nm after lysing the cells, the level of the β-galatosidase was determined.
PL8-UV5 characterization
L8-UV5 is a regulated promoter which can be induced by either lactose or the analog IPTG. To characterize the inducible property of the L8-UV5 promoter, PL8-UV5 was engineered upstream of the GFP gene. The GFP will be transcribed and translated after the promoter is induced. GFP fluorescent signal will be given by GFP. By measuring the GFP fluorescent signal after adding different concentration of IPTG to the bacteria, the inducible property of L8-UV5 promoter can be determined.
Lysis cassette characterization
To characterize the efficiency of the lysis cassette, the lysis cassette was put under the control of the T7 promoter in the pSNAP plasmid. By inducing the T7 promoter with lactose, the genes in the lysis cassette was transcribed and translated into the components of the lysis proteins, which cause cell lysis.
The O.D. and the colony forming units (CFU) were measured to determine the efficiency of the lysis cassette.
Inducing the promoter with lactose → Measure O.D. → Serial dilution → Spread plate → Incubate → Count the number of colonies
The lower the CFU/O.D., the higher the efficiency of the lysis cassette after induction.
The O.D. and the colony forming units (CFU) were measured to determine the efficiency of the lysis cassette.
Inducing the promoter with lactose → Measure O.D. → Serial dilution → Spread plate → Incubate → Count the number of colonies
The lower the CFU/O.D., the higher the efficiency of the lysis cassette after induction.
Characterization of the lacY lacZ operon BBa_S04055 (from 2008 Caltech: Curing lactose intolerance)
Western blotting analysis (Link to the protocol) was used to detect the expression level of β-galactosidase inside E. coli harboring BBa_S04055.