Team:DTU-Denmark/SPL
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<li>The BioBrick plasmid backbone should now contain the SPL and is ready to be used as a vector in a BioBrick assembly.</li> | <li>The BioBrick plasmid backbone should now contain the SPL and is ready to be used as a vector in a BioBrick assembly.</li> | ||
</ol> | </ol> | ||
+ | <h1>Advantages of Using the DTU SPL Standard</h1> | ||
+ | <ul> | ||
+ | <li>An SPL allows the fine tuning of gene expression by creating a promoter library with a wide variety of strengths. For data from our SPL proof of concept experiments, please visit our <a href="https://2010.igem.org/Team:DTU-Denmark/SPL_Section" target="_blank">SPL Results page</a> | ||
+ | <li>The SPL will necessarily give you the allowed upper and lower bounds of gene expression for cell viability. | ||
+ | <li>The method for creating the BioBrick compatible SPL is quick and easy to perform. The method is practically identical to the procedure for generating linear BioBrick plasmid backbones, which many teams will most likely have to do anyway. | ||
+ | <li>Once the SPL is in the linear BioBrick plasmid backbone, it can be used in a variety of experiments. A BioBrick part or device can be ligated into the plasmid backbone and the resulting ligation can be transformed and screened for colonies containing the promoter strength needed. | ||
+ | </ul> | ||
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Revision as of 21:52, 27 October 2010
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Introduction to Synthetic Promoter LibrariesModulation 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).
The point of randomizing both areas is to obtain a promoter library that is not biased towards being strong. This is achieved by giving two bases within each of the consensus regions a 50% chance of being their original bases, ensuring that only 1/16 of all promoters will be strong. This is without taking into consideration the fraction of strong promoters obtainable from the randomized spacer sequences. The DTU Synthetic Promoter Library StandardWe utilized the SPL technology to create a new BioBrick Standard in the form of a BioBrick Foundation Request For Comments (BBF RFC). The DTU Synthetic Promoter Library Standard was submitted as BBF RFC 63.Strategy for Integrating SPL into the BioBrick Assembly StandardThere are many different ways to integrate an SPL into the BioBrick Standard, and a lot of ideas were considered when creating this RFC. However, in the end a method was chosen based on the fact that it would be least time consuming for teams looking to use SPL, and at the same time, be easy to do. Instead of relying on ligations to successfully insert the SPL onto the BioBrick plasmid backbone, a Polymerase Chain Reaction (PCR) method was designed to not only amplify the backbone but also add the SPL onto the linear BioBrick plasmid backbone at a specific chosen site (see Figure 2). Since most teams will probably have to amplify their backbones during the course of a project, this method will only require a small amount of extra work.
The design of the SPL leads to the possibility of illegal restriction sites being present within the randomized spacer sequence. If a given promoter is to be used in further ligations it is vital that the promoter is sequenced first to ensure that it does not contain any recognition sites for EcoRI, XbaI, SpeI or PstI. The presence of these recognition sites could lead to the promoter being cut in a future restriction digest Primer DesignA PCR MUST be used in order to add the SPL onto the BioBrick plasmid backbone. The following primers for amplification of BioBrick plasmid backbones were used as a starting point for the design of our SPL primers:
The primers were taken from Parts Registry. The restriction enzyme recognition sites are marked with the following colors:
In order to amplify and add the SPL successfully, the following modifications were made to both of the annealing primers:
For this primer, a tail with the recommended extra bases has been added. For more information click here. Depending on which backbone needs to be amplified, one of the following SPL primers should be used:
These primers have the SPL sequence inserted between the EcoRI and XbaI sites. Furthermore, 14-18 nt have been added to the 3’ end of the primer to ensure that the primers’ annealing sequences are long enough. Table 1 contains a list showing which primer to use with regard to which backbone is chosen.
Protocol for Adding SPL to a BioBrick Backbone PlasmidIn terms of primer annealing specificity, a touch down ramp PCR (Don et al. 1991)may be used, but as Table 2 illustrates, the melting temperatures (Tm) are relatively high and a standard PCR can therefore be run instead of a touch down ramp PCR.
Advantages of Using the DTU SPL Standard
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