Construction of BioBricks

The SPL + I13507 construct was ligated into the BioBrick plasmid backbone pSB3T5, this due to pSB3T5 being a low to medium copy number plasmid which contains the p15A replication of origin and a tetracycline resistance marker. The expression of RFP via an SPL would best be controlled and measured through a low copy number plasmid in case too high expression of RFP proved to be detrimental to the cell’s viability, therefore pSB3T5 was chosen as the backbone as it had the best range of copy number amongst the other backbones within the parts-registry. The pSB2K3 backbone could have been used as well, though pSB3T5 gave us more flexibility with the construct as additional regulatory elements were not required such as the case with pSB2K3.

Characterization

The SPL technology was used in order to create a BioBrick compatible standard for fine tuning the expression of BioBrick parts and devices. The methodology used in this proof of concept and what was documented in the standard has slight differences, although the main technology was unchanged, namely the SPL. The differences will be outlined later on.

As mentioned previously, a construct was made where the SPL was ligated with BBa_I13507 into the pSB3T5 backbone. In order to verify and illustrate the variation of the promoter strengths from the SPL, promoters with set known strengths, namely the Anderson promoter library were used in order to benchmark the different promoters generated from the SPL. To further illustrate the flexibility of the usage of SPL, two different strains of E.coli were used, namely XL1-blue and DH5α.

The SPL per se can be illustrated as the following:

**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 1 illustrates how the SPL was designed, where the spacer sequences in-between the -35 and -10 regions, namely N17 have been randomized, where N stands for 25% each of A, C, G and T. Two bases within each of the consensus regions, have been randomized as well although to the degree that they are given a 50% chance of being their original base, this contributing towards having an unbiased promoter library instead of only strong promoters. Within the consensus regions S stands for 50% each of C and G, and W stands for 50% each A and T.

The difference between this proof of concept and what the DTU SPL standard contains is the placement of the SPL and the addition of a selectable marker, namely chloramphenicol. Primers were designed in the way that the SPL would be amplified along side the chloramphenicol resistance marker having the prefix and suffix upstream and downstream of the amplicon, respectively. Having the SPL on its own would be too short in terms of handling viability, i.e. visualizing from gel electrophoresis on a UV image, digesting and retrieving such a small genetic fragment and obtaining a high enough yield, therefore it was more viable amplifying the chloramphenicol resistance marker with it’s own promoter together with the SPL taking care of issues in relation to handling, such as visualizing the fragment on a UV image after running the amplicon through gel electrophoresis, digesting and retrieving the digested product as well as the yield. Another advantage to this was that now the SPL contains can be screened for as long as the plasmid backbone inserted into contains a different resistance marker than chloramphenicol.

Team:DTU-Denmark/SPL Section

From 2010.igem.org

Introduction

Construction of BioBricks

Characterization

Strategy

Results