Team:DTU-Denmark/SPL Section

Not only was the Synthetic Promoter Library (SPL) used as a method for attempting to characterize lambda’s N-antiterminator protein, it was also used as a proof of concept experiment for the BBF RFC 63 – DTU Synthetic Promoter Library Standard where we decided to ligate it in front of BBa_I13507, which contains BBa_E1010 - an RFP gene and BBa_B0015 - a double terminator that consists of parts BBa_B0010 and BBa_B0012. The results of this experiment will be outlined in this section, which will be followed by a section elucidating how the BBF RFC 63 DTU standard was developed and its subsequent advantages.

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.

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.

Strategy

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 2 illustrates how the SPL was amplified via PCR resulting in a genetic part that contains a chloramphenicol selectable resistance marker that has the prefix and suffix added to the appropriate ends and is therefore compatible with the BioBrick assembly standard.

1. SPL + BBa_I13507 + pSB3T5
2. BBa_J23101 + BBa_I13507 + pSB3T5
3. BBa_J23100 + BBa_I13507 + pSB3T5
4. BBa_J23116 + BBa_I13507 + pSB3T5

Results

As mentioned previously, two strains of E.coli were used. Constructs 1-4 were therefore transformed both into XL1-Blue and DH5α, both of these sets of transformations containing the constructs were then measured using the BioLector.

The first set of measurements, involving constructs 1-4 within strain XL1-Blue, showed a lot of variation in promoter strengths of the SPL. What was more significant was being able to map these strengths in relation to the reference promoters. The graph below illustrates the different promoter strengths obtained by plotting RFP over Biomass for the SPL and the reference promoters.

Figure 3 illustrates the variation in promoter strengths of the SPL mapped against the reference promoters. PM corresponds to BBa_J23101, PS corresponds to BBa_J23100 and PW corresponds to BBa_J23116.

Figure 4 illustrates the specific activities of the SPL promoters ranked together with the reference promoters.

Figure 5 illustrates the variation in promoter strengths of the SPL mapped against the reference promoters. PM corresponds to BBa_J23101, PS corresponds to BBa_J23100 and PW corresponds to BBa_J23116.

Figure 6 illustrates the specific activities of the SPL promoters ranked against the reference promoters.

Conclusion

We successfully designed a BioBrick compatible SPL and demonstrated its functionality. The resulting constructs contained promoters with a wide range of different promoter strengths. The strengths of the measured promoters ranged from 0 - 1.35 RPU compared to the reference standard BBa_J23101. This range is comparable to that of the existing promoter libraries already present in the parts registry. Thus we have shown that the DTU Synthetic Promoter Library Standard is a great alternative method to quickly and easily construct a promoter library, with a wide range of promoter strengths, compared to using already existing BioBrick promoters.