Team:DTU-Denmark/SPL

From 2010.igem.org

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<h1>Synthetic Promoter Library</h1>
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<h1>Introduction to Synthetic Promoter Libraries</h1>
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<p align="justify">Modulation of gene expression of i.e. cellular enzyme activities (Solem and Jensen 2002), as well as regulation of transcription are amongst some of the areas where SPLs are currently being used. SPL provides an alternative method for gene regulation compared to older methods, namely those of gene knockouts and strong over expression. These two methods are usually based upon apparent rate limiting steps within metabolic pathways (Jensen and Hammer 1998). <br>
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When working with gene regulation, it is important to elucidate where expression levels are optimal for the given gene being worked on. Under these specifications it is essential to be able to have slight increments in expressional strength when attempting to optimize gene expression. This can be achieved by the usage of an SPL, where the variability in strengths can be achieved by either randomizing the spacer sequences, namely the 17 bases that reside between the -35 and -10 consensus regions, and/or some of the bases within the consensus regions, being the -35 and -10 regions. <br>
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The spacer sequences that surround the consensus regions contribute significantly to the strengths of promoters (Hammer et al. 2006). In our design, we decided to both randomize the spacer sequences as well as two bases in both consensus regions as seen in Figure 1 below. 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.<br></p>
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Revision as of 12:43, 27 October 2010

Welcome to the DTU iGEM wiki!

Introduction to Synthetic Promoter Libraries

Modulation of gene expression of i.e. cellular enzyme activities (Solem and Jensen 2002), as well as regulation of transcription are amongst some of the areas where SPLs are currently being used. SPL provides an alternative method for gene regulation compared to older methods, namely those of gene knockouts and strong over expression. These two methods are usually based upon apparent rate limiting steps within metabolic pathways (Jensen and Hammer 1998).

When working with gene regulation, it is important to elucidate where expression levels are optimal for the given gene being worked on. Under these specifications it is essential to be able to have slight increments in expressional strength when attempting to optimize gene expression. This can be achieved by the usage of an SPL, where the variability in strengths can be achieved by either randomizing the spacer sequences, namely the 17 bases that reside between the -35 and -10 consensus regions, and/or some of the bases within the consensus regions, being the -35 and -10 regions.

The spacer sequences that surround the consensus regions contribute significantly to the strengths of promoters (Hammer et al. 2006). In our design, we decided to both randomize the spacer sequences as well as two bases in both consensus regions as seen in Figure 1 below. 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.

Figure 1: 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.


Figure 2: The linear BioBrick plasmid backbone with SPL inserted between the EcoRI and XbaI sites of the BioBrick prefix is illustrated.


Figure 3: 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.


Table 1: illustrates the Tm of the SPL primers. IDT DNA oligo analyzer was used in order to calculate the Tm.

PrimerTm - °C
I) Primer SPL Suffix-F62.1
II) Primer SPL Prefix-R-01 59.8
III) Primer SPL Prefix-R-0260

Table 2

PCR substratesVolumes - μL
Total volume50
Phusion Polymerase (0,02 U/μL)0.5
x5 Phusion HF buffer10
dNTP's (5μM)2
Primer SPL Suffix-F (10μM)1.25
Template - BioBrick plasmid backbone1
ddH2O33.5

Table 3

Cycle stepTemperature - ºCTimeCycles
Initial denaturation9830 sec1
Denaturation9810 sec-
Annealing63*30 sec20-25
Extension7230 sec / kb-
Final extension7210 min1
Hold4forever1

Table 4: Prefix SPL primers that SHOULD be used depending on which BioBrick plamid backbone is selected for amplification is illustrated.

BioBrick Plasmid BackbonePrimer IIPrimer IIISizes - bps
pSB1A3+-2157
pSB1AC3+-3055
pSB1AK3+-3189
pSB1AT3+-3446
pSB1C3+-2072
pSB1K3+-2206
pSB1T3+-2463
pSB2K3+-4425
pSB3C5-+2738
pSB3K5-+2936
pSB3T5-+3252
pSB4A5-+3395
pSB4C5-+3221
pSB4K5-+3419
pSB4T5-+3735