Part:BBa_K5121012

GGG-mNeon

Background

mNeonGreen is a monomeric green-yellow fluorescent protein engineered from a tetrameric fluorescent protein from Branchiostoma lanceolatum (Shaner et al., 2013). Since its design, mNeonGreen has been used as a fluorescent reporter in numerous fields. We were supplied with a pCDFDuet plasmid containing mNeonGreen with an N-terminal polyhistidine tag. We engineered this plasmid to add a TEV protease cleavage site (ENLYFQ/G) and tri-glycine sequence downstream of the his-tag — see more details in the design section.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 124
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Characterisation

mNeonGreen was expressed using a pCDFDuet-1_His_mNeon_TmTP plasmid. To make mNeonGreen compatible with sortase A-catalysed conjugation, an N-terminal triglycine motif was added, preceded by a TEV protease recognition sequence to cleave off the N-terminal methionine residue. The sequences were added using two-fragment PCR. Each set of primers has overlapping regions that anneal to the existing sequence of mNeonGreen and regions containing the new sequences to be added to mNeonGreen (Figure 1). The products of the two PCR reactions were designed to have overlapping regions on each end, allowing Gibson assembly to be used to re-assemble the plasmid with the new sequences. These assembled plasmids were immediately transformed into Bl21 E. coli for expression and DH5-alpha cells for plasmid propagation. Successful modification and transformation was indicated by growth on plates with the selective marker spectinomycin (Figure 2). Plasmid purification was performed on DH5-alpha samples and were sequenced to further verify that mNeonGreen had the correct sequences inserted.

Figure 1. Plasmid map of the mNeonGreen plasmid along with the primers binding sites used to at modifications. A TEV protease site was added before the triglycine motif to facilitate cleavage of the N-terminal methionine.

Figure 2. Generation of modified mNeonGreen for compatibility with sortase A conjugations. A) PCR of mNeonGreen coding region, with tri-glycine and TEV cleavage site introduced on primer tails. PCR was performed with six replicates, loaded in each lane of the agarose gel. B) PCR of mNeonGreen backbone with complementary regions to the insert amplicons. The designed product should be 3.6kb, corresponding to the topmost band. The presence of smaller bands indicates off-target amplification, necessitating gel-purification of the desired PCR product for downstream applications. C) DH5-alpha cells transformed with the Gibson-assembled GGG-mNeonGreen plasmid on plates containing spectinomycin.

Our modified mNeonGreen was overexpressed in BL21 cells. Overnight expression was induced with 1mM IPTG when the optical density at 600 nm of the cell culture reached 0.6. The cells were pelleted and lysed by sonication (Figure 3A). The cleared lysates were incubated with a Ni-NTA affinity resin (Figure 3B). After washing the resin, rather than eluting mNeonGreen with imidazole, we performed on bead cleavage overnight with the TEV protease. While a considerable yield was achieved with on-bead cleavage, the beads were still coloured green, indicating there was mNeonGreen still bound. We eluted these off the beads with imidazole (50mM Tris pH 7.5, 300mM NaCl, 500mM imidazole), (Figure 3C). The TEV-cleaved mNeonGreen samples now had an N-terminal tri-glycine motif. We further purified this sample with size-exclusion chromatography (Figure 4).

Figure 3. His-Tag Purification of GGG-mNeonGreen. A) Pellets of cells expressing mNeonGreen (left) and wild-type R-bodies (right). B) Cell lysates bound to the Ni-NTA matrix during His-Tag purification. C) mNeonGreen samples eluted from the Ni-NTA column. The tube on the right contains mNeonGreen that was cleaved from the beads via overnight incubation with the TEV protease. This sample went on to further purification with size exclusion chromatography. The tube on the left was eluted with a high-imidazole concentration buffer. In this sample, mNeonGreen still has an N-terminal his-tag and TEV cleavage site.

Figure 4. Size Exclusion Chromatography of GGG-mNeonGreen. A) His-Tag-purified mNeonGreen entering the size-exclusion column. B) SDS-PAGE of SEC input and the fractions selected for mNeonGreen. Some contaminants are visible in the input column, which have been removed during the purification process. C) The fractions were compiled and concentrated into a GGG-mNeonGreen stock solution. This was used in subsequent sortase-A reactions.

References

Shaner, N. C., Lambert, G. G., Chammas, A., Ni, Y., Cranfill, P. J., Baird, M. A., Sell, B. R., Allen, J. R., Day, R. N., Israelsson, M., Davidson, M. W., & Wang, J. (2013). A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum. Nature Methods, 10(5), 407–409. https://doi.org/10.1038/nmeth.2413