Team:NCTU Formosa/Project/Project new

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<li><a href="https://2009.igem.org/Team:NCTU_Formosa/Notebook/Team" target="_self">Team</a></li>
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<li><a href="https://2009.igem.org/Team:NCTU_Formosa/Team" target="_self">Team</a></li>
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<li><a href="https://2009.igem.org/Team:NCTU_Formosa/WetLab/Mainworks" target="_self">Mainworks</a></li>
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<li><a href="https://2009.igem.org/Team:NCTU_Formosa/WetLab/Labworks" target="_self">Lab works</a></li>
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         <h4> Principle </h4>
         <h4> Principle </h4>
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         <p>The original observation that the regions flanking the −35 (TTGACA) and −10 (TATAAT) bacterial promoter consensus sequences affect promoter strength is the blueprint for the tuning of promoter activity. If random mutations are created at the −35 and −10 sequences, then such promoters will represent many variations in promoter strength (Fig. 1).</p>
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         <p>The original observation that the regions flanking the ?35 (TTGACA) and ?10 (TATAAT) bacterial promoter consensus sequences affect promoter strength is the blueprint for the tuning of promoter activity. If random mutations are created at the ?35 and ?10 sequences, then such promoters will represent many variations in promoter strength (Fig. 1).</p><br>
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     <a href="https://2009.igem.org/Image:Newprot1.png" ><img src="https://static.igem.org/mediawiki/2009/0/06/Newprot1.png" border="0"></a>
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        <h4>protocol</h4>
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        <p>This protocol contains four steps:
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        <ul>
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            <li>Degenerated primers design. </li>
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            <li>Using PCR to amplify the target plasmid.</li>
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            <li>PCR product transformation. </li>
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            <li>The measurement of promoter activity.</li>
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</ul>
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        <p><strong>1. Degenerated primers design</strong>
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            <li>At first, it is required to design a primer. It is no matter who has experiences about designing. Just have a check the points below when you want design your primer.</li>
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            <li>Normally, primer size is 25~45mer. Our design depends on the Tm value and GC ratio, so 30~40mer length is recommended.</li>
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            <li>The most important thing is that you have to check the Tm value, more than 78℃ or not. (At least more than 40％ of GC ratio.)</li>
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            <li>If the Tm value is under 78℃, it is necessary to change the primer length.</li>
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            <li>Design two strands, forward and reverse primers. In this step, locate the target nucleotide on the center of primer.</li>
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            <li>Avoid desalting grade. Must use over than minimum FPLC or OPC grade.</li>
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        <p>A pair of random mutation primers for the biobrick: BBa_K145279 is made as an example. In this case, we wanted to generate different GFP intensity of the promoter PtetR, so two point mutations were made in the -10 box (Table 1).</p>
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    <a href="https://2009.igem.org/Image:Newprot2.png" ><img src="https://static.igem.org/mediawiki/2009/1/16/Newprot2.png" border="0"></a>
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            <p>Table 1. The degenerated primers designed to amplify the biobrick plasmid: BBa_K145279. Yellow bars indicate the -10 boxes and N is a random mutation (A, T, C, G) in the primer.</p>
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        <p><strong>2. Using PCR to amplify the target plasmid.</strong></p>
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        <p><i>PCR Reaction Mixture Set Up</i></p>
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    <a href="https://2009.igem.org/Image:Newprot3.png" ><img src="https://static.igem.org/mediawiki/2009/7/71/Newprot3.png" border="0"></a>
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            <p>* It is important to use KOD-plus polymerase. Since DNA polymerase from Thermococcus kodakaraensis KOD is one of the most efficient thermostable PCR enzymes exhibiting higher accuracy and elongation velocity than any other commercially available DNA polymerase.</p>
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        <p><i>PCR conditions</i></p>
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    <a href="https://2009.igem.org/Image:Newprot4.png" ><img src="https://static.igem.org/mediawiki/2009/9/9b/Newprot4.png" border="0"></a>
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            <p>The linear PCR product can join by self-ligation that can increase the transformation efficiency.</p>
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        <p><i>Ligation conditions</i></p>
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    <a href="https://2009.igem.org/Image:Newprot5.png" ><img src="https://static.igem.org/mediawiki/2009/3/31/Newprot5.png" border="0"></a>
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            <p>(1) Vortex the tube and spin down in a microcentrifuge for 3-5 seconds. <br> (2) Incubate the mixture for 8 hour at 16°C. <br> (3) Use the mixture for transformation.</p>
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        <p><strong>3. PCR product transformation</strong></p>
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        <p>ECOSTM competent cells are used for transformation. </p>
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        <p>Step:
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        <ol>
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        <li>Thaw competent cells in the room temperature water bath with circulating water or hold the tube under the running tap water for ~20 seconds until 1/3~1/2 volume is thawed.</li>
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        <li>Add DNA(pre-chilled on ice, volume should be ≦ 5% of competent cells) immediately. Vortex for 1 second or tap the tube with finger to mix well.</li>
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        <li>Heat-shock the cells in the pre-warmed 42℃ water bath for 30~45 seconds.</li>
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        <li>Plate the cells using plating beads onto a pre-chilled(4℃) and dried antibiotic-selected LB agar plate.</li>
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        <li>Incubate the plates at 37℃.</li>
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Latest revision as of 19:52, 21 October 2009

New Idea - A PROTOCOL FOR ENGINEERING PROMOTERS TO FIT THE DESIGN SPECIFICATIONS


Motive

Constructing a functional genetic circuit requires assembling genetic devices and getting them to work together. The main challenge in genetic circuit design lies in selecting well-matched genetic components that when coupled, reliably produce the desired behavior. Although the parameter values are calculated by model equations, it is hard to select the biological part that reliably implements a desired cellular function with quantitative values. In general, to control gene expression is achieved by placing the gene of interest under the control of an inducible promoter. Strong and inducible promoters are widely used in recombinant protein over-expression. However, many current application in synthetic biology, particular those to metabolic optimization and control analysis, require the capacity to precisely tune the levels of gene expression. To overcome this problem, the promoters which can control the expression of downstream genes are necessary.

Our team designs a simple and rapid protocol to generate a promoter library. This promoter library with different transcriptional strength can be built to tune the specific parameter values that model equations indicated. A strategy is applied in our protocol. Degenerated primers designed for PCR are used to generate mutations in promoter regions. The promoter activity can be assayed using a reporter protein (GFP). Because the reporter protein activity has positive correlation to promoter activity, we can choose the suitable transcriptional strength of the promoter for our design scheme.




Principle

The original observation that the regions flanking the ?35 (TTGACA) and ?10 (TATAAT) bacterial promoter consensus sequences affect promoter strength is the blueprint for the tuning of promoter activity. If random mutations are created at the ?35 and ?10 sequences, then such promoters will represent many variations in promoter strength (Fig. 1).


Fig. 1. Degenerated primers designed for PCR are used to generate mutations in promoter regions. The library of promoters is created where the -35 or -10 sequences are amplified by degenerated primers with some random mutations (red star), then such a library can represent many variations in promoter strength.




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