Part:BBa_K3014012

Vibrio natriegenstRNA construct

This part is an assembly of the 5 rarest tRNAs in the Vibrio natriegens genome. The tRNA genes are separated by Biobrick scars and flanked by Biobrick prefix and suffix. The implemented regulatory elements for the tRNA transcription are the rrnA P1 Promoter (from Vibrio natriegens1) and the rrnA terminator (derived from E. coliK12). With this operon, the transcription of the tRNAs should be proportional to the growth rate and adjust to the cellular levels of the remaining protein synthesis machinery. The tRNA genes contain 50 nucleotides of the upstream sequence to minimize the influence on later tRNA maturation. To prevent unwanted terminator loops, the genes contain only 8-37 nucleotides downstream. Terminator loops were identified in the sequence using the web service ARNold.^1-4 With the help of this part, it should be possible to increase the level of all rare tRNAs in Vibrio natriegens and thus to increase the translation speed and the expression. Therefore, this part is inserted into the corresponding plasmid backbone. A special tRNA plasmid (BBa_K3014006) has been designed for experiments with the tRNAs. The tRNA backbone uses a p15A ori, as it is also used in the established tRNA vector ‘pRARE’ from Merck Inc.⁵

V.natriegens rare tRNA collection

This gene is part of the V.natriegens rare tRNA collection from Team Stuttgart. The collection contains the 5 rarest tRNA genes from the genome of Vibrio natriegens. Applying the High-performance Integrated Virtual Environment-Codon Usage Tables (HIVE-CUTs) databank codon usage tables were generated for Vibrio natriegens(DSM759) using the online interface (available at hive.biochemistry.gwu.edu/review/codon).⁶ From this codon usage tables codons were selected as a 'rare' codon, if the number of codons per 1000 codons was less or equal 5 (excluding the stop-codons ‘TAA’, ‘TAG’ and ‘TGA’). Therefore, the following codons are considered by us as beeing rare in Vibrio natriegens(DSM759):

AGG (1.42 codons/1000 codons)
CGG (1.52 codons/1000 codons)
CCC (2.93 codons/1000 codons)
TGC (3.56 codons/1000 codons)
AGA (4.54 codons/1000 codons)
TCC (5.17 codons/1000 codons)

Figure 1: Graphical representation of Vibrio natrigens’ codon usage. The graphic is based on 7,530,201 codons Vibrio natriegens uses in 22.954 CDS retrieved from Genbank. Rare codons (number of codons / 1000 codons ≤ 5) are highlighted in red.

The encoded tRNA genes were identified using tRNA-Scan. For all rare tRNAs, except the CCC(Proline)-encoding tRNA, genes were successfully identified. The resulting genes encoding the rare tRNAs are listed in the following table 1.

Table 1: List of the identified rare tRNAs of Vibrio natriegens, their positions in the genome and the respective part number.

Genome ID	Location	Codon	Encoded amino acid	Part
NZ_CP009977.1	2,006,503-2,006,427	AGA	Arg	BBa_K3014000
NZ_CP009977.1	2,114,151-2,114,227	AGG	Arg	BBa_K3014001
NZ_CP009977.1	2,867,750-2,867,674	CGG	Arg	BBa_K3014002
NZ_CP009977.1	869,777-869,850	TGC	Cys	BBa_K3014003
NZ_CP009977.1	1,760,572-1,760,485	TCC	Ser	BBa_K3014004

The system is based on the famous Rosetta strain with pRARE from Merck.⁷ pRARE contains the rarest codons of E. coli to increase the translation rate. We are pursuing a similar system with the V. natriegens rare tRNA collection. However, all iGEM teams have access to this system and can fully exploit the potential of V. natriegens. In this way, we contribute towards optimizing V. natriegens with the aim of replacing E. coli as the most commonly used organism in the future.

The parts have been synthesized with Biobrick cloning sites to ensure easy cloning via BioBrick Standard Assembly.

References

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Hofacker, I. L. et al. Fast folding and comparison of RNA secondary structures. Monatsh Chem 125, 167–188; 10.1007/BF00818163 (1994).
Lesnik, E. A. et al. Prediction of rho-independent transcriptional terminators in Escherichia coli. Nucleic acids research 29, 3583–3594; 10.1093/nar/29.17.3583 (2001).
Macke, T. J. et al. RNAMotif, an RNA secondary structure definition and search algorithm. Nucleic acids research 29, 4724–4735; 10.1093/nar/29.22.4724 (2001).
Drott, D. Overcoming the codon bias of E. coli for enhanced protein expression. inNovations 12 (2001)
Athey, J. et al. A new and updated resource for codon usage tables. BMC bioinformatics 18, 391; 10.1186/s12859-017-1793-7 (2017).
Robert Novy, Don Drott KY and RM. Overcoming the codon bias of E. coli for enhanced protein expression. Newsl NOVAGEN, INC. 2001;(12):4-6.