Team:Stuttgart/Design

Project

Design

Summary

By the introduction of the newly developed ptRNA plasmid for Vibrio natriegens we want to increase the cellular concentration of the rare tRNAs (TCC, TGC, AGA, AGG and CGG), in order to reduce the codon bias, acting as a bottleneck in heterologous gene expression using Vibrio natriegens.

Strain development

Applying the High-performance Integrated Virtual Environment-Codon Usage Tables (HIVE-CUTs) databank codon usage tables (CUTs) were generated for Vibrio natriegens (DSM759) using the online interface (available at hive.biochemistry.gwu.edu/review/codon)1. Here the coding sequences (CDS) from Genbank (GenBank Release July 24, 2018) and NCBI's RefSeq were separately used. The CUTs take 36.399 CDS (RefSeq) or 22.954 CDS (Genbank) into account. From this CUT, 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 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.

In order to increase the cellular concentrations of the previously identified rare tRNAs in Vibrio natriegens, the genes encoding the corresponding tRNAs had to be identified. With the whole genome of Vibrio natriegens (NCBI Reference Sequence: NZ_CP009977.1 and NZ_CP009978.1), the encoded tRNA genes were identified using tRNA-Scan2 implemented at the Galaxy server of the University of Stuttgart (access was kindly provided by the group for Computational Biology). 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.

In advance to the design of the plasmid, Dr. Josef Altenbuchner was consulted as an external expert, due to his long-year experience in plasmid design and the work with Vibrio natriegens at the Institute for Industrial Genetics (University of Stuttgart). Besides that, we also consulted the iGEM team Marburg 2018, as winners of last year’s grand prize with their exceptional foundational progress with Vibrio natriegens. As proposed by Dr. Altenbuchner we chose a two-plasmid system, one plasmid to raise the cellular rare tRNA concentrations and one plasmid for protein expression. By using a two-plasmid system, we want to maintain the possibility for the experimenter to use an expression plasmid of choice [individual adaptation of transcript level by the plasmid origin of replication (ori)]. This approach also keeps the respective plasmid size small to allow a simple experimental workflow. The tRNA plasmid uses a p15A ori, as it also used in the established tRNA vector ‘pRARE’ from Merck Inc., which is used in the famous E. coli strain Rosetta3. As selection markers, we chose tetracycline and chloramphenicol, as Vibrio natriegens is not majorly susceptible to beta-lactamase inhibiting antibiotics (e.g. ampicillin, kanamycin, ...), as advised by the iGEM team Marburg 2018 (experimental validation as shown here: https://2018.igem.org/Team:Marburg/Results). The mentioned parts were combined, using the iGEM's BioBrick cloning vector pSB3T5-I52001 as a template., to create our ptRNA vector backbone (Figure 2).


For each identified rare tRNA gene 50 nucleotides of the upstream sequence and varying lengths of the downstream sequence were copied to minimize the influence on later tRNA maturation. To prevent the inclusion of unwanted regulatory elements. The length of the downstream sequence was varied from 8 to 37 nucleotides, because terminator loops were identified in these sequences using the web service ARNold4–7. The implemented regulatory elements for the tRNA transcription are the rrnA P1 Promoter (from Vibrio natriegens8) 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 remain protein synthesis machinery9. The resulting plasmid is called ptRNA and is roughly 2.9 kb long. The plasmid design was conducted with the SnapGene software (from GSL Biotech; available at snapgene.com). Figure 3 shows the resulting plasmid map.

Table 1 – Listing of the identified rare tRNAs of Vibrio natriegens and their positions in the genome.

Genome ID

Location

Codon

Encoded amino acid

NZ_CP009977.1

1,760,572-1,760,485

TCC

Ser

NZ_CP009977.1

869,777-869,850

TGC

Cys

NZ_CP009977.1

2,006,503-2,006,427

AGA

Arg

NZ_CP009977.1

2,114,151-2,114,227

AGG

Arg

NZ_CP009977.1

2,867,750-2,867,674

CGG

Arg

Figure 2 – Plasmid map of the ptRNA plasmid backbone. The features are individually annotated, with ‘TcR’ being the tetracycline efflux protein.
Figure 3 –Map of the ptRNA plasmid. The five rare tRNAs and their respective up-and downstream elements of Vibrio natriegens are inserted between the Biobrick prefix and – suffix into the ptRNA plasmid backbone (Figure 2). The features are individually annotated, with ‘TcR’ being the tetracycline efflux protein.

Experimental plan for the testing

To create a new strain of Vibrio natriegens with reduced codon bias in heterologous gene expression, two main factors need to be validated experimentally:

  • The functionality of our plasmid ptRNA. This plasmid should raise the cellular concentrations of the rare tRNAs in Vibrio natriegens After the successful construction of the plasmid system, we want to determine selected tRNA levels in cell samples using a specialized qPCR method.
  • The compatibility of the ptRNA plasmid with a second expression plasmid (based on the yeast collection from Dueber et al.10) and the improvement of heterologous protein expression in Vibrio natriegens. First, we want to compare the expression of sfGFP (Part:BBa_I746916) and a codon-optimized sfGFP for wild-type Vibrio natriegens and our new Vibrio natriegens strain additionally containing the ptRNA plasmid. Second, we want to compare the expression of sfGFP and special sfGFP mutants (with 5 rare codons added as tandem repeats at the N-terminus, this should reduce the translation rate) in Vibrio natriegens and our new natriegens strain. This will show if the use of our ptRNA plasmid is a valid strategy for expression optimization, like the established codon optimization.

Graphical Abstract/Summary

Figure 4 – Graphical summary of the experimental validation process. A Expression of sfGFP (Part:BBa_I746916) in a wild-type Vibrio natriegens strain. B Expression of R_sfGFP (sfGFP with 5 rare codons added as tandem repeats at the N-terminus) in a wild-type Vibrio natriegens strain. The amount of expressed sfGFP is reduced in comparison to the setting shown in A, due to the reduced translation rate. C Expression of R_sfGFP in a Vibrio natriegens strain possessing the ptRNA plasmid, which increases the cellular rare tRNA level. Therefore, the expressed R_sfGFP is significantly increased. D Expression of O_sfGFP (codon optimized sfGFP (rare codons removed)) in a wild-type Vibrio natriegens strain. The amount of expressed sfGFP is comparable to the setting shown in C.


References

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