Part:BBa_K4294211
LuxI and mNeonGreen coding sequences seperated by the stop-start pentanucleotide "TAATG"
A chimeric gene approach by coupling the gene coding for LuxI with a gene producing a visible readout, namely a fluorescent protein.
Design
To combine LuxI’s synthesis with the production of the fluorescent protein, we deployed a phenomenon called Termination-Reinitiation (or TeRe) observed during translation [1]. TeRe occurs when two neighboring genes have overlapping start and stop codons. More specifically, once translation of the upstream gene is terminated, the 30S ribosomal subunit lingers in the area of the stop codon; if it immediately encounters a new start codon, it re-recruits the 50S subunit and reinitiates translation, this time, of the downstream gene, as shown in the figure below. This way, the downstream gene is translated at the same rate as the upstream gene.
Based on this mechanism, we enriched the TAA stop codon of the luxI gene with an extra TG, to convert its terminal A into the initial A of the fluorescent protein’s gene start codon, resulting in the coupling sequence 5’-TAATG-3’ for the characterization of senders’ RBS.
The translational coupling of the LuxI CDS with the mNeonGreen CDS via a stop-start pentanucleotide.
Measurment
This design was not successful, we did not manage to produce a visual output of mNeonGreen. Optimization is needed if used by future teams==
References
[1]Huber, M., Faure, G., Laass, S., Kolbe, E., Seitz, K., Wehrheim, C., Wolf, Y. I., Koonin, E. V., & Soppa, J. (2019). Translational coupling via termination-reinitiation in archaea and bacteria. Nature communications, 10(1), 4006. https://doi.org/10.1038/s41467-019-11999-9
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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