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Team:DTU-Denmark/SPL Section
From 2010.igem.org
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Introduction 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. 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. 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.
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 a selectable marker that can be screened for as long as the plasmid backbone inserted into contains a different resistance marker than chloramphenicol.
The fragment was now large enough for digestion and subsequent ligation with BBa_I13507 into pSB3T5. Being able to judge how much variation between promoter strengths were obtained was accomplished by following guidelines of BBF RFC 19 - "Measuring the Activity of BioBrick Promoters Using an In Vivo Reference Standard" written by Endy et al. The backbone pSB3T5 was chosen on the basis of having the same origin of replication p15A as the backbones pSB3K3 and pSB3C5 that were used in BBF RFC 19. Reference promoters were thus chosen from the Anderson promoter library. BBa_J23101 was chosen first and foremost due to serving as an in vivo reference standard for promoter activity as described in BBF RFC 19, it was thereafter selected as the medium reference promoter which had a relative strength of 0.7, J23100 was chosen as the strong reference promoter as it had a relative strength of 1 and J23116 was chosen as the weak reference promoter which had a relative promoter strength 0.16. Strains were constructed containing:
These were transformed into two different E.coli strains, namely XL1-Blue and DH5α. Once colonies were visible on the plates, 15 random colonies were picked from the SPL containing constructs, 2 of the BBa_J23101 containing constructs, 2 of the BBa_J23100 containing constructs and 3 of the BBa_J23116 containing constructs. References were also needed in order to make sure we were indeed measuring RFP and to see how the growth curve of the strains the constructs were residing inside were behaving, therefore pSB2K3 which contains an RFP coding device, namely BBa_J04450 was taken from the DNA distribution kit, transformed and plated, and reference XL1-Blue and DH5α were plated as well. Overnight pre-cultures were started of these 24 colonies (15 SPL, 7 reference promoters, and 2 references) in order to run subsequent measurements with BioLector. Once BioLector measurements were made for all of the above mentioned constructs within both of the strains, specific activity of the SPL promoters could be calculated and more importantly the Relative Promoter Unit (RPUs) in relation to the standard reference BBa_J23101 as per BBF RFC 19. 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 SPL and reference promoters can then be plotted, ranking them according to their specific activities. The graph below displays the rankings.
Table 1 shows the calculated specific activities and RPU relative to the in vivo reference standard BBa_J23101 for the first set of measurements. This can be represented as the ratio of the specific activity of the SPL promoters to the specific activity of the reference standard BBa_J23101. The second set of measurements involving constructs 1-4 within strain DH5α showed a greater variation in promoter strengths of the SPL compared to the first set. Again these strengths were mapped 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.
The SPL and reference promoters can then be plotted, ranking them according to their specific activities. The graph below displays the rankings.
Table 2 shows the calculated specific activities and RPU relative to the in vivo reference standard BBa_J23101 for the second set of measurements. This can be represented as the ratio of the specific activity of the SPL promoters to the specific activity of the reference standard BBa_J23101. ConclusionWe 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. |
