Part:BBa_K5121026

RebB TAG Amber

Biology

RebA encodes an 18 kDa protein which forms one of the main structural components of type 51 R bodies. This part is based off of the wildtype rebA part — BBa_K2912000 — uploaded by SZU-China in 2019, however has been modified to be compatible with amber stop codon suppression to facilitate the incorporation of unnatural amino acids. This is to allow for conjugation with entire R bodies. Amino acids can be incorporated by co-expressing a corresponding aminoacyl tRNA synthetase/tRNA pair in the presence of the target mRNA and the non-canonical amino acid.

Our group used the modified rebA monomer to incorporate p-azido-l-phenylalanine (AzF) into the N-terminus of rebA. This amino acid contains an azide functional group, which can be reacted to an alkyne via a Cu(I)-catalyzed azide–alkyne cycloaddition (CuAAC) click reaction. This method facilitates conjugation of compounds such as fluorophores and drugs onto R bodies assuming they have an alkyne functional group.

Part overview

In order to maximise the chance of successful conjugation, the N-terminal AzF was to be added onto the end of a glycine-serine linker, such that the full amino acid sequence added onto the N-terminus was Azf-GGGGS. This was achieved with a PCR of the original reb1 plasmid (BBa_K5121011) using overlapping primers to add in 5’-TAGGGCGGGGGTGGAAGC-3’ between the start codon and the rest of the rebA open reading frame. Rather than amplifying the whole plasmid in one reaction, the plasmid was amplified in two sections — each containing one of the two overlapping primers and a primer within the kanamycin resistance gene — followed by gibson assembly of the two fragments to reconstruct the full plasmid. This avoided annealing of the overlapping primers. The kanamycin resistance gene was chosen as the site of the second set of primers to ensure only cells with correctly assembled fragments at this site were viable — reducing the possible set of incorrectly assembled fragments. To incorporate Azf into R bodies, bacteria were co-transformed into BL21 cells with the modified plasmid, as well as pEVOL-AzF encoding the amino acyl tRNA synthetase/tRNA pair, and supplemented with AzF.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 806
  Illegal NheI site found at 868
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 748
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Characterisation

Successful Gibson assembly was confirmed by sequencing plasmid minipreps from transformants. Additionally, a positive control using E. coli cotransformed with pEVOL_AzF and pCDF RFP amber plasmid was performed. The former encodes the p-azido-L-phenylalanylyl-tRNA synthetase and tRNACUA for the amber codon. The latter encodes a modified RFP where the first amino acid is p-azido-L-phenylalanine (AzF). Taking from Ma et al. (2014), we performed a qualitative test to see whether RFP expression was enhanced by the addition of organic solvents, which helps overcome resistance to AzF uptake through the cell membrane (Figure 2).

The presence of RFP indicates that amber codon suppression is successfully incorporating AzF. However, organic solvent appeared to make little difference to observed changes. This may be due to the qualitative nature of our analysis and due to the small culture volumes (2 mL), as Ma et al. (2014) report significant effects on protein yield. Optimising amber codon suppression using organic solvents could be of interest in scaling-up operations, but presently we were hesitant to introduce an untested compound into R body expression medium. We therefore chose to use AzF alone.

The amber codon is positioned close to the N-terminus of RebA, and unsuccessful incorporation would result in a highly truncated protein and affect R body formation. Purification from TAG-RebA expression cultures yielded sizable pellets and could be imaged under microscopy (Figure 3), albeit contaminated, suggesting that amber codon suppression was achieved. This alone cannot verify successful AzF incorporation, as amino acid misincorporation may produce false positives.

However, the success of CuAAC conjugation (Figure 4) implies that AzF had been incorporated into RebA or RebB monomers. Amber codon suppression can be a challenging technique to implement. Although powerful and easy to execute, its implementation in the lab is commonly hampered by unpredictable variations in incorporation efficiencies at different amber codon positions. Flanking sequences, cell lines, ribosomal fidelity and even permeability of the non-canonical amino acid (ncAA) all influence rates of misincorporation or translation termination. Bartoschek et al. (2021) have developed a predictive tool to identify permissive amber codon sites, which we could use in the future to identify which contextual factors enabled our success with ncAA incorporation.

References

Bartoschek, M. D., Ugur, E., Nguyen, T.-A., Rodschinka, G., Wierer, M., Lang, K., & Bultmann, S. (2021). Identification of permissive amber suppression sites for efficient non-canonical amino acid incorporation in mammalian cells. Nucleic Acids Research, 49(11), e62–e62. https://doi.org/10.1093/nar/gkab132

Ma, X., Wei, B., & Wang, E. 2022. Efficient incorporation of p-azido-l-phenylalanine into the protein using organic solvents. Protein Expression and Purification. 200: 106158. https://doi.org/10.1016/j.pep.2022.106158.