Team:DTU-Denmark/SPL
From 2010.igem.org
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<h1>Synthetic Promoter Library</h1> | <h1>Synthetic Promoter Library</h1> | ||
<p align="justify"> Modulation of gene expression such as of cellular enzyme activities[1] as well as regulation of transcription are amongst some of the areas where Synthetic Promoter Libraries (SPLs) are currently being used. SPL provides an alternative method for gene regulation compared to the old methods, namely those of gene knockout as well as strong over expression, these two usually executed on the basis of apparent rate limiting steps[1].<br> | <p align="justify"> Modulation of gene expression such as of cellular enzyme activities[1] as well as regulation of transcription are amongst some of the areas where Synthetic Promoter Libraries (SPLs) are currently being used. SPL provides an alternative method for gene regulation compared to the old methods, namely those of gene knockout as well as strong over expression, these two usually executed on the basis of apparent rate limiting steps[1].<br> | ||
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As previous studies indicate consensus regions outside of the -35 and -10 regions seem to contribute very little if anything at all in terms of altering promoter strengths, rather the spacer sequences surrounding the -35 and -10 regions seem to have the most significance[2]. This might be due to the three-dimensional structure that forms from the sequences that are arranged from the randomized spacer sequences[2].<br></p> | As previous studies indicate consensus regions outside of the -35 and -10 regions seem to contribute very little if anything at all in terms of altering promoter strengths, rather the spacer sequences surrounding the -35 and -10 regions seem to have the most significance[2]. This might be due to the three-dimensional structure that forms from the sequences that are arranged from the randomized spacer sequences[2].<br></p> | ||
+ | |||
+ | <table class="https://static.igem.org/mediawiki/2010/c/c8/SPL1.png" align="center"> | ||
+ | <caption align="bottom"><p align="justify"><b>Figure 1</b>: An SPL designed on the basis of randomizing both the spacer sequences surrounding the consensus regions (-35 and -10 regions) as well as randomizing two bases within each of the consensus regions is illustrated. N stands for 25% each of A, C, G and T, while S stands for 50% each of C and G, and W stands for 50% A and T.</p></caption> | ||
+ | <tr><td><img src="https://static.igem.org/mediawiki/2010/c/c8/SPL1.png" ></td></tr> | ||
+ | </table><br> | ||
+ | |||
+ | <table class="https://static.igem.org/mediawiki/2010/a/a6/SPL2.png" align="center"> | ||
+ | <caption align="bottom"><p align="justify"><b>Figure 2</b>: The linear BioBrick plasmid backbone with SPL inserted between the EcoRI and XbaI sites of the BioBrick prefix is illustrated.</p></caption> | ||
+ | <tr><td><img src="https://static.igem.org/mediawiki/2010/a/a6/SPL2.png" ></td></tr> | ||
+ | </table><br> | ||
+ | |||
+ | <table class="https://static.igem.org/mediawiki/2010/c/c7/SPL3.png" align="center"> | ||
+ | <caption align="bottom"><p align="justify"><b>Figure 3</b>: The primer binding sites on a BioBrick plasmid backbone as well as the final linear plasmid backbone that is generated by the PCR is illustrated.</p></caption> | ||
+ | <tr><td><img src="https://static.igem.org/mediawiki/2010/c/c7/SPL3.png" ></td></tr> | ||
+ | </table><br> | ||
+ | |||
+ | <table cellpadding="2" border="1px" cellspacing="0" align="center" width="300px"> | ||
+ | <caption><p align="justify"><b>Table 1</b>: illustrates the Tm of the SPL primers. IDT DNA oligo analyzer was used in order to calculate the Tm.</p></caption> | ||
+ | <thead align="center"> | ||
+ | <td><b>Primer</b></td><td><b>Tm - °C</b></td> | ||
+ | </thead></tr> | ||
+ | <font face="courier new"> | ||
+ | <tr> | ||
+ | <td align="center"><i>I) Primer SPL Suffix-F</i></td><td align="center">62.1</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td align="center"><i>II) Primer SPL Prefix-R-01 </i></td><td align="center">59.8</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td align="center"><i>III) Primer SPL Prefix-R-02</i></td><td align="center">60</td> | ||
+ | </tr> | ||
+ | </font> | ||
+ | </table> | ||
+ | |||
+ | <table cellpadding="2" border="1px" cellspacing="0" align="center"> | ||
+ | <caption><p align="justify"><b>Table 2</b></p></caption> | ||
+ | <thead> | ||
+ | <td align="left"><b>PCR substrates</b></td><td align="right"><b>Volumes - μL</b></td> | ||
+ | </thead></tr> | ||
+ | <tr> | ||
+ | <td align="left">Total volume</td><td align="right">50</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td align="left">Phusion Polymerase (0,02 U/μL)</td><td align="right">0.5</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td align="left">x5 Phusion HF buffer</td><td align="right">10</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td align="left">dNTP's (5μM)</td><td align="right">2</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td align="left">Primer SPL Suffix-F (10μM)</td><td align="right">1.25</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td align="left">Template - BioBrick plasmid backbone</td><td align="right">1</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td align="left">ddH2O</td><td align="right">33.5</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <table cellpadding="2" border="1px" cellspacing="0" align="center"> | ||
+ | <caption><p align="justify"><b>Table 3</b></p></caption> | ||
+ | <thead> | ||
+ | <td align="left"><b>Cycle step</b></td><td align="right"><b>Temperature - ºC</b></td><td align="right"><b>Time</b></td><td align="right"><b>Cycles</b></td> | ||
+ | </thead></tr> | ||
+ | <tr> | ||
+ | <td align="left">Initial denaturation</td><td align="right">98</td><td align="right">30 sec</td><td align="right">1</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td align="left">Denaturation</td><td align="right">98</td><td align="right">10 sec</td><td align="right">-</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td align="left">Annealing</td><td align="right">63*</td><td align="right">30 sec</td><td align="right">20-25</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td align="left">Extension</td><td align="right">72</td><td align="right">30 sec / kb</td><td align="right">-</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td align="left">Final extension</td><td align="right">72</td><td align="right">10 min</td><td align="right">1</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td align="left">Hold</td><td align="right">4</td><td align="right">forever</td><td align="right">1</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | |||
+ | <table cellpadding="2" border="1px" cellspacing="0" align="center"> | ||
+ | <caption><p align="justify"><b>Table 4</b>: Prefix SPL primers that SHOULD be used depending on which BioBrick plamid backbone is selected for amplification is illustrated.</p></caption> | ||
+ | <tr><thead> | ||
+ | <td align="left"><b>BioBrick Plasmid Backbone</b></td><td align="right"><b>Primer II</b></td><td align="right"><b>Primer III</b></td><td align="right"><b>Sizes - bps</b></td> | ||
+ | </thead></tr> | ||
+ | <tr> | ||
+ | <td align="left">pSB1A3</td><td align="center">+</td><td align="center">-</td><td align="right">2157</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td align="left">pSB1AC3</td><td align="center">+</td><td align="center">-</td><td align="right">3055</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td align="left">pSB1AK3</td><td align="center">+</td><td align="center">-</td><td align="right">3189</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td align="left">pSB1AT3</td><td align="center">+</td><td align="center">-</td><td align="right">3446</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td align="left">pSB1C3</td><td align="center">+</td><td align="center">-</td><td align="right">2072</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td align="left">pSB1K3</td><td align="center">+</td><td align="center">-</td><td align="right">2206</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td align="left">pSB1T3</td><td align="center">+</td><td align="center">-</td><td align="right">2463</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td align="left">pSB2K3</td><td align="center">+</td><td align="center">-</td><td align="right">4425</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td align="left">pSB3C5</td><td align="center">-</td><td align="center">+</td><td align="right">2738</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td align="left">pSB3K5</td><td align="center">-</td><td align="center">+</td><td align="right">2936</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td align="left">pSB3T5</td><td align="center">-</td><td align="center">+</td><td align="right">3252</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td align="left">pSB4A5</td><td align="center">-</td><td align="center">+</td><td align="right">3395</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td align="left">pSB4C5</td><td align="center">-</td><td align="center">+</td><td align="right">3221</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td align="left">pSB4K5</td><td align="center">-</td><td align="center">+</td><td align="right">3419</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td align="left">pSB4T5</td><td align="center">-</td><td align="center">+</td><td align="right">3735</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
</td> | </td> | ||
<td width="163px" height="100%" valign="top"> | <td width="163px" height="100%" valign="top"> |
Revision as of 23:27, 26 October 2010
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Synthetic Promoter Library Modulation of gene expression such as of cellular enzyme activities[1] as well as regulation of transcription are amongst some of the areas where Synthetic Promoter Libraries (SPLs) are currently being used. SPL provides an alternative method for gene regulation compared to the old methods, namely those of gene knockout as well as strong over expression, these two usually executed on the basis of apparent rate limiting steps[1].
The spacer sequences that surround the consensus regions contribute significantly to the strengths of promoters[1]. In our design, we decided to both randomize the spacer sequences as well as randomize 2 bases in both of the consensus regions as seen in the provided diagram. N stands for 25% each of A, C, G and T, while R stands for 50% each of A and G, and W stands for 50% A and T. The point of randomizing both would be to obtain a promoter library that is not biased towards being all strong, by giving 2 bases within each of the consensus regions a 50% chance of being their original bases only 1/16 of all promoters will be strong, this being without taking into consideration the fraction of strong promoters obtainable from the randomized spacer sequences.
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