Part:BBa_K3699006

Pyrrolysyl-tRNA pair

Orthogonal translation system including PylRS/tRNA(CUA)

Introduction

From virus to human, the same set of genetic code is employed, which contains the start codon, three-stop codons (UAA, UAG, UGA), and the remaining 61 codons encoding 20 kinds of amino acids. However, some exceptions have been found, such as pyrrolysyl tRNA synthetase/tRNA (pylrs/tRNA) from Methanosarcina mazei, which can recognize the amber codon UAG and translate it into pyrrolysine. Moreover, in E. coli, yeasts, and mammalian cells, this PylRS/tRNA pair is orthogonal, which means it does not interfere with the translation system of the chassis[1]. The orthogonal translation system has been used in vaccine safety design [2]. Now, we will use it to achieve the anti-escape design of cyanophages.

Design

We finally decided to use the orthogonal translation system from Methanosarcina mazei. It has been used in E. coli. The Pyrrolysyl-tRNA pair was designed according to the paper published by Wang et al, 2012 [3]. In addition, Tatsuo Yanagisawa et al found that the Y384F mutant of MmbocRS had higher aminoacylation activity [4]. Based on the previous reports, the MmBocRS (GenBank NZ_ Cp009514.1 3998898-4000262) was designed to convert Tyr (306) and Tyr (384) to Ala and Phe, respectively. The J23119 promoter was employed to drive MmBocRS expression. PyltRNA (GenBank: NZ_ Cp009514.1 3998599-3998670) was initiated by proK promoter. BamHI restriction site at 5 'end and HindIII restriction site at 3' end were introduced to facilate plasmid construction.

Figure 1. Schematic map of pMCS3-RS-tRNA. The part integrates orthogonal translation systems together, including PylRS gene BBa_3699003 & tRNA gene BBa_3699004.

The BBa_3699006 part integrates orthogonal translation systems together, which facilitates the introduction of the system in E. coli, which can specifically recognize UAG and introduce Boc-lysine. We firstly tested whether the system works well in E. coli. When the system is finally introduced into the recombinant cyanophage genome, the reproduction of cyanophage is controlled by addition of unnatural amino acids.

Process

1. Plasmid pMV-RS-tRNA was synthesized.

2. Plasmid pMCS3-RS-tRNA was constructed by digesting pMV-RS-tRNA and pMCS3 with EcoRI-XhoI double enzyme digestion and ligation.

Figure 2. Construction of pMCS3-RS-tRNA. RS: the pyrrolysyl-tRNA synthetase gene of Methanosarcina mazei (gene, MSMAC_3241, protein id, AKB73131.1); tRNA, the pylT gene (tRNA) of Methanosarcina mazei.

Testing

We tried to verify whether pMCS3-RS-tRNA was successfully constructed. It demonstrated the success of plasmid construction.

Figure 3. DNA gel electrophoresis for pMCS3-RS-tRNA digested with EcoRI-XhoI (4.7kb + 1.7kb).

Expression of the full-length Tail protein

To test whether the orthogonal translation system works well, the previously mentioned plasmids pET28a-Tail and pMCS3-RS-tRNA were co-transformed into E. coli BL21 (DE3). If the full-length tail protein is expressed, the system works normally.

At first, we couldn't transfer the two plasmids pET28a-Tail and pMV-RS-tRNA, which contains the Tail protein and the pyrrolysyl-tRNA synthetase respectively, into E. coli BL21 (DE3). They had different antibiotic resistance gene but share the same replicon protein. Therefore, pMCS3-RS-tRNA was constructed, and it can be co-transfomed with pET28a-Tail into E. coli BL21 (DE3).

Result

Figure 4. SDS page of BL21 (DE3) / pET28a-Tail (Lane 1-4) and BL21 (DE3) / pET28a-Tail / pMCS3-RS-tRNA (Lane 5-8).

From left to right:

1. Bacterial lysis, no IPTG, BL21 (DE3) / pET28a-Tail;

2. Bacterial lysis, IPTG induction, BL21 (DE3) / pET28a-Tail;

3. Bacteria cell pellets, no IPTG, BL21 (DE3) / pET28a-Tail;

4. Bacterial cell pellets, IPTG induction, BL21 (DE3) / pET28a-Tail;

M protein marker;

5. Bacterial lysis, no IPTG, BL21 (DE3) / pET28a-Tail / pMCS3-RS-tRNA;

6. Bacterial lysis, IPTG induction, BL21 (DE3) / pET28a-Tail / pMCS3-RS-tRNA;

7. Bacteria cell pellets, no IPTG, BL21 (DE3) / pET28a-Tail / pMCS3-RS-tRNA;

8. Bacterial cell pellets, IPTG induction, BL21 (DE3) / pET28a-Tail / pMCS3-RS-tRNA.

Figure 5. The expected full-length tail protein sequence.

Bacteria with double plasmids was expected to express the full-length tail protein of 87.1 kDa. However, experimental results indicated the expression of truncated tail protein (Lane 8). The protein expression level is lower than that of the strain harboring a single plasmid (Lane 4).

Possible Reasons:

Codon expansion technology needs further optimization, such as the concentration of unnatural amino acids added, the expression level of aminoacyl-tRNAase, etc.

There may also be weak expression of the full-length proteins, but the expression level is not high enough to be clearly visible by SDS-Page electrophoresis.

Subsequently, the fluorescent protein will be employed as indicators to validate codon expansion technology, and the expression of related genes would be optimized. These results would be probably presented in our next year iGEM projects.

Reference

[1] Mukai T , Kobayashi T , Hino N , et al. Adding l-lysine derivatives to the genetic code of mammalian cells with engineered pyrrolysyl-tRNA synthetases[J]. Biochemical & Biophysical Research Communications, 2008, 371(4):818-822.

[2] Si L , Xu H , Zhou X , et al. Generation of influenza A viruses as live but replication-incompetent virus vaccines.[J]. Science, 2016.

[3] Yane-Shih Wang, Xinqiang Fang, Ashley L. Wallace, et al. A Rationally Designed Pyrrolysyl-tRNA Synthetase Mutant with a Broad Substrate Spectrum[J]. Journal of the American Chemical Society, 2012.

[4] Tatsuo, Yanagisawa, and, et al. Multistep Engineering of Pyrrolysyl-tRNA Synthetase to Genetically Encode Nɛ-(o-Azidobenzyloxycarbonyl) lysine for Site-Specific Protein Modification[J]. Chemistry & Biology, 2008.

[5] Daichi M , Shigeko K , Yoshihiko S , et al. Transcriptome Analysis of a Bloom-Forming Cyanobacterium Microcystis aeruginosa during Ma-LMM01 Phage Infection[J]. Frontiers in Microbiology, 2018, 9:2-.