Part:BBa_K3699007

Tail protein

Tail, make the recombinant cyanophage safer

As mentioned in the design, we will use recombinant cyanophage as the chassis to metabolize the microcystins. In any case, cyanophage is a virus, and safety issues always come first. After the artificial "upgrade" of the cyanophage, we also expect to add "handcuffs" to ensure that the cyanophage would always be implemented in the right place and would not spread to the environment or break up the original ecological balance. The handcuff mentioned above is the “tail” protein with UAG inserted.

Design

The “tail” protein encodes the putative phage tail sheath protein of Microcystis virus Ma-LMM01. (Protein ID: BAF36182.1)

We have made a base substitution at position 747 (T747G), which converted the Tyrosine codon to a stop codon and lead to the early termination of protein translation. In order to facilitate plasmid conctruction, we added BamHI and SacI cutting sites at both ends of the gene.

Figure 1. sequence of the tail protein. We made a base substitution at position 747 (T747G), converting TAT to TAG.

We speculate that the mutated phage tail sheath protein will not be fully expressed, but a truncated protein.

Construction

Plasmid pKMV-Tail was synthesized by BGI Tech. On this basis, we constructed the plasmid pET28a-Tail by digesting pKMV-tail and pET28a with BamHI/SacI enzyme digestion and ligation. Plasmid pET28a, which contains T7 promotor, is an excellent expression vector in E. coli BL21 (DE3) with IPTG addition. The tail protein gene will be highly expressed.

Figure 2. Skeleton map of pET28a-Tail.

Figure 3. Construction of pET28a-Tail. BamHI-SacI double enzyme digestion of pKMV-tail and pET28a to construct plasmid pET28a-Tail. T7 promoter was introduced to facilitate expression in E. coli.

Testing

We demonstrated the success of plasmid construction by enzymatic digestion.

Figure 4. DNA gel electrophoresis for pET28a-Tail digensted with BamHI-SacI (2.3kb + 5.3kb).

Expression of the truncated Tail protein

We transferred pET28a-Tail into E. coli BL21 (DE3). As expected, it should express a truncated protein of 29.7 kDa.

Figure 5. The expected truncated tail protein sequence.

Result

Figure 6. SDS page of BL21 (DE3) harboring plasmid pET28a-Tail.

From left to right:

1. Bacterial lysis, no IPTG; 2. Bacterial lysis, IPTG induction;

3. Bacteria cell pellets, no IPTG; 4. Bacterial cell pellets, IPTG induction; M protein marker

The introduction of stop codon caused the early termination of protein translation. The recombinant E. coli expressed a truncated phase tail shear protein as expected. In other words, if the stop codon was introduced into the cyanophage genome, it will become a virus that can not release or infect cyanobacteria, thus preventing the escape of recombinant phage.

Reference

[1] 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-.

[2] 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.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 2449
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 53
  Illegal BglII site found at 1289
  Illegal BamHI site found at 105
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 218
  Illegal NgoMIV site found at 1845
  
• 1000
  COMPATIBLE WITH RFC[1000]