Part:BBa_K5321011

nPPVp_mut

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 295
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 295
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 295
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 295
  
• 1000
  COMPATIBLE WITH RFC[1000]

Usage and Biology

To construct a molecular machine featuring a highly efficient sensor, such as an aptamer, and an upstream signal transporter, like a split protease, we have devised crosslinking via click chemistry, specifically DBCO-p-AzF click reaction, to do this task. We introduced UAG stop codon site-directed mutations into the nPPVp genes. This modification allows the pEvol-pAzFRS.2.t1 plasmids to incorporate p-AzF through an orthogonal tRNA synthetase/tRNA pair.

Figure 1 | Prediction result of PPVp structure. Cyan portion represents the N-terminal fragment (nPPVp), while green portion represents the C-terminal fragment (cPPVp).

Firstly, we need to construct the plasmids that express the required split proteases. Specifically, we will PCR the nPPVp and cPPVp fragments along with the pET28a backbones from our original plasmids to eliminate the rapamycin sensor components (FRB/FKBP). Following this, we will perform Gibson assembly to create plasmids containing the split protease genes, which can then be further modified using site-directed mutagenesis, which sites were selected by dry lab team. Finally, bacteria co-transformed with the mutated plasmids and pEvol-pAzFRS.2.t1 can be induced to express target proteins containing p-AzF.

Figure 2 | Overview of plasmid construction for click chemistry-based crosslinking. (A-B) Workflow for the construction of UAG stop codon-mutated plasmids for nPPVp and cPPVp (refer to text for details). (C) Structure prediction of the PPVp protease, highlighting the selected phenylalanine residues for mutation: PHE-6 on nPPVp and PHE-227 on cPPVp. (D) Plasmid mapping of pEvol-pAzFRS.2.t1.

Characterization

Sequencing Verification

Through Gibson assembly, we successfully constructed the pET28a_nPPVp/cPPVp plasmids, which were subsequently verified by sequencing. Following site-directed mutagenesis, sequencing was again conducted to confirm the introduction of the desired mutations.

Figure 3 | Sequencing verification of plasmids. (A-B) Sequencing results for the pET28a_nPPVp plasmid, showing the N-terminal and C-terminal regions, respectively. (C) Sequencing verification of the pET28a_nPPVp_mut, with red boxes highlighting the mutations. (D-E) Sequencing results for the pET28a_cPPVp plasmid, detailing the N-terminal and C-terminal regions, respectively. (F) Sequencing verification of the pET28a_cPPVp_mut, with red boxes indicating the mutations.

Protein Expression

We induced protein expression in co-transformed bacteria when the OD600 reached approximately 0.8 by adding IPTG and L-arabinose, along with p-AzF. SDS analysis of total proteins in the cell lysates before ultrasonication was conducted to verify the expression of the target proteins. The expression of the proteins was confirmed; however, we encountered challenges in purifying the proteins, which may have been due to low protein yield.

Figure 4 | SDS analysis of total proteins in cell lysates before ultrasonication. Lane 2-3: Total proteins from cell lysates of bacteria expressing hCG. Lane 4-5: Total proteins from cell lysates of bacteria expressing nPPV (15.2kDa) and cPPV (16.8 kDa), respectively.

Ligation Reaction

Thus, we chose to use both AU (after ultrasonication) and SU (supernatant after ultrasonication) components for the protein ligation reaction. Despite employing very gentle warming and cooling during the process, the DBCO-modified aptamer consistently exhibited large molecular weight multimerization bands. Notably, no new bands appeared in the reaction system where the protein was added, indicating that the ligation was not successful.

Figure 5 | Protein ligation reaction utilizing click chemistry. 29: DBCO_12T_thrombin_HD22_29mer; 40: thrombin_AYA1809004_40mer_8T_DBCO; 29 Click 1: 29+nPPVp* AU without heating; 29 Click 2: 29+nPPVp* AU with heating; 29 Click 3: 29+nPPVp* SU without heating; 29 Click 4: 29+nPPVp* SU with heating; 40 Click 1: 40+cPPVp* AU without heating; 40 Click 2: 40+cPPVp* AU with heating; 40 Click 3: 40+cPPVp* SU without heating; 40 Click 4: 40+cPPVp* SU with heating.

During the growth of bacteria containing two plasmids, we observed that the growth rate was significantly slower compared to bacteria containing a single plasmid. Given the complexity associated with the incorporation of unnatural amino acids, this may have contributed to a low protein yield, hindering purification efforts. Consequently, no visible shift bands were observed in the protein ligation reaction.