Because we want to mimic
the process of melatonin production in human, we need to elongate the period cycle of
repressilatorto 24hr. According to the modeling results of repressilator, faster
translation is helpful to the period elongation; thus, a strong ribosome binding site
may be required. Then, we scanned the iGEM registry, finding that we only have a small
number of ribosome binding sites whose strengths have been determined. Therefore, we
decided to modify an existing RBS in iGEM registry by ourselves to obtain a series of
stronger ribosome binding sites.
Random mutation of RBS will generate a huge library, which will increase our workload grandly with numbers of useless mutations. Thus, we turned to a bioinformatics method to predict the sequence of a synthetic RBS with a target translation initiation rate on a proportional scale. We designed our RBS via the modeling in the website (https://salislab.net/software/doForwardRBS.)1, which was based on the theory that build on previous work that characterized the free energies of key molecular interactions involved in translation initiation and on measurements of the sequence-dependent energetic changes that occur during RNA folding and hybridization.
Firstly, we inputted the mRNA sequence of the original device BBa_I13521, and predicted the translation initiation rate of the original device BBa_I13521 with RBS BBa_B0034 by the online software tool (https://salislab.net/software/reverse). According to the position of the start codon of coding sequence in the transcript, we found the translation rate prediction result of BBa_I13521 was 2297.59 au. (Fig.1)
Then, in order to test the consistency of the
predicted results with the experimental results, we predicted 3 RBS (BBa_B0029,
BBa_B0032, BBa_B0035) collected in the Community RBS Collection of Part Registry (
). According to the document in the Part Registry, only BBa_B0035 is stronger than
BBa_B0034, and others are weaker. Their respective measured strength documented in Part
Registry and predicted strength by us via the tool listed in the table 1. (Table. 1)
This table showed that there were a consistent trend between the predicted results and
the measured results.
|RBS||Relative Measured Strength||Relative Predicted Strength||Predicted Strength|
Thus, it convinced us that the RBS calculator could guide our RBS modification. And, we inputted a nucleotide sequence (5 to 20 bp) that appears before the ribosome binding site and the sequence of mRFP (CDS of the original device BBa_I13521) in the online RBS calculator. Next, we entered the target translation initiation rate, estimated by the prediction result of BBa_B0034 within BBa_I13521. Sequentially, the RBS Calculator performed accurate calculation of the final state's free energy, a modified ribosome footprint length and additional interactions between the ribosomal platform and mRNA at upstream standby sites, and outputted a series of synthetic RBS with target translation initiation rate. (Fig.2)
We choose three designed RBS，which was predicted to be 5、10、50 times stronger than B0034 respectively, and was separately named as P1, P2,P3. (Table. 2)
|Synthetic RBS||Sequence||Translation Initiation Rate||Relative Prediction Strength|
|P1 (BBa_K2276007)||TATAAGGAGTAAATACC||11153.91 au||4.86|
|P2 (BBa_K2276008)||AAATAAGGAGGTATAATA||25075.0 au||10.91|
|P3 (BBa_K2276010)||AAATAATAAGGGGTTTAC||41213.03 au||17.94|
In order to characterize properties of such three designed RBS, we did some experiments to substituted the BBa_B0034 in the BBa_I13521 to these synthetic RBS. (More details about experiments)
1. Salis, H. M., Mirsky, E. A. & Voigt, C. A. Automated design of synthetic ribosome binding sites to control protein expression. Nat. Biotechnol.27, 946–50 (2009).