RECONNECT NERVES
Achievements:
- Successfully cloned a part coding for secretion of NGF in pET43.1a and iGEM plasmid backbone pSB1C3, creating a new composite part BBa_K2616000
- Successfully sequenced BBa_K2616000 in pSB1C3 and sent to iGEM registry
- Successfully co-transform E. coli with plasmid secreting NGF and plasmid expressing the secretion system, creating bacteria capable of secreting NGF in the medium
- Successfully characterized production of NGF thanks to mass spectrometry
- Successfully observe axon growth in microfluidic chip in presence of commercial NGF
Next steps:
- Purify secreted NGF, and characterize its effects on neuron growth thanks to our microfluidic device
- Global proof of concept in a microfluidic device containing neurons in one of the chamber, and our engineered bacteria in the other
DNA assembly
The sequence we designed codes for two different proteins: proNGF (Nerve Growth Factor) and TEV protease (from Tobacco Etch Virus). These two proteins are fused in C-terminal with a signal peptide for E. coli Type I Secretion System which consists in the last 60 amino-acids of HaemolysinA (HlyA). Each coding sequence is separated from the signal peptide by the cleavage sequence for TEV, in order to get the protein without its signal peptide (Figure 3).
Figure 1: proNGF and TEV production cassette
This DNA construct was ordered in two parts, named Seq1 (1096 bp) and Seq2 (1153 bp) in commercial plasmids pEX-A258 from gene synthesis. Seq1 and Seq2 were amplified in competent E. coli DH5-α. After bacteria culture and plasmid DNA extraction, we digested commercial vectors with restriction enzymes (NheI and BamHI for Seq1, MscI and HindIII for Seq2). We extracted the inserts from the gel and performed a ligation by using specific overlaps into linearized pET43.1a for proNGF expression and into pSB1C3 for iGEM sample submission.
We repeated the procedure (transformation in E. coli Stellar competent cells, bacteria culture, plasmid DNA extraction, digestion) and we proved that our vector pet43.1a contained Seq1 and Seq2 (Figure 2) and that pSB1C3 contained Seq1 and Seq2 (Figure 3) after digestion and DNA electrophoresis. Plasmid DNA of pSB1C3 construction was purified and sent for sequencing (Figure 4).
Figure 2: Agarose 1% gel after electrophoresis of digested pET43.1 containing Seq1 and Seq2 (Bba_K2616000) with NdeI. Colonies 6, 9, 10 ,11, 15 have the correct construction.
Figure 3: Agarose 1% gel after electrophoresis of digested pSB1C3 containing Seq1 and Seq2 (Bba_K2616000) with EcoRI/PstI. Colonies 3, 7 and 8 have the correct construction.
Alignment of Sequencing Results then confirmed that pSB1C3 contained Seq1 and Seq2, BBa_K2616000 .
Figure 4: Alignment of sequencing results for BBa_K2616000. Sequencing perform in pSB1C3 and three primers were designed (FOR1, FOR2, FOR3) to cover the whole sequence. Image from Geneious.
The construction was successfully assembled. On Figure 4, mismatches are visible which correspond to the reduced precision of sequencing after 600 bp. To avoid this lack of precision, we used three different primers, allowing us to cover the whole sequence without mistakes. As visible, the mismatches are only present at the extremities of each primer sequencing.
proNGF characterization and purification
Our chassis is Escherichia coli BL21(DE3) pLysS, a specific strain dedicated to producing high amounts of desired proteins under a T7 promoter. Thus, we co-transformed our bacteria with BBa_K2616000 and pVDL 9.3, generously provided by Dr. Victor de Lorenzo, from Centro Nacional de Biotecnologia of Madrid, bearing HlyB and HlyD (Type I secretion system) sequences, in order to get a chance to secrete NGF out of the cell.
Bacteria were grown at a large scale (800 mL), and proNGF expression was induced with 0.1 mM IPTG for 2 hours at 37°C.
We tried to achieve His-tagged proNGF purification using Ni-NTA affinity purification column. We eluted our protein using a gradient of imidazole-containing buffer and one peak was detected.
Figure 5: FPLC proNGF purification with ÄKTA pure (General Electric) Ni-NTA column was equilibrated with buffer A (50 mM Tris, pH 7.4, 200 mM NaCl). Supernatant of lyzed bacteria was introduced through the column. Washing with 5% of buffer B. Elution by buffer B gradient (buffer A + imidazole 250 mM). UV absorbance at 280nm is shown in blue, conductivity in red, and concentration of buffer B in green.
We analyzed bacterial lysate and purification fractions using SDS-PAGE electrophoresis and Mass spectrometry.
Figure 6: SDS-PAGE gel Bis-Tris 4-12% of bacterial lysate and proNGF purification fraction by SDS-PAGE.
The proNGF purification using NiNTA column is not conclusive. Many proteins are found on elution fractions. His-tagged proNGF fused to HlyA export signal should be found at 33 kDa while the proNGF cleaved by TEV protease should be found at 27 kDa. We finally analyzed five gel bands of the FPLC flow-through (lane 2, Figure 6) by mass spectrometry, by LC/MS/MS, to verify the presence of proNGF.
According to Figure 7, proNGF pattern are found on each lane sent to mass spectrometry. The major amount is found on fraction 5, corresponding to 33 kDa, at this molecular weight, the proNGF is still fused to the signal export. The TEV protease, 34 kDa fused to signal export and 28 kDa cleaved from the signal export are found.
Figure 7: Distribution of proNGF and TEV protease by gel fractions after mass spectrometry analysis.
Analysis of Fraction 5 of the gel shows our protein proNGF is present but is still bound to its signal peptide HlyA. (Figure 8) Mass spectrometry spectrum of Peptide A, IDTACVCVLSR, from proNGF sequence is shown in Figure 9. Mass spectrometry spectrum of Peptide B, IISAAGSFDVKEER from fused HlyA signal export is shown in Figure 9. The presence of mass spectrometry identified peptides corresponding to the fusion of proNGF and HlyA indicate some proNGF uncleaved from the signal export
Figure 8: Alignment sequences of proNGF fused to HlyA export signal and peptides identified by mass spectrometry. In light blue peptides that match proNGF amino acids sequence. In light yellow, peptides that match HlyA signal export. Sequence has been annotated to match corresponding protein amino acid sequences : In orange His tagged proNGF, in red TEV protease cleaving site, in rose HlyA signal export.
Figure 9: Mass spectrometry spectrum. A) Peptide identified corresponding to proNGF. B) Peptide identified corresponding to the fusion of proNGF and HlyA export signal.
The proNGF did not seem to be retained on the affinity column. We performed batch purification using NiNTA beads under native and partial denaturing conditions (Urea 2 M) followed by Western Blot analysis with immunodetection through Anti-His Antibodies Alexa Fluor 647. (Figure 10) Detection of His-tag in the pellet supernatant of induced BL21 with 1 mM IPTG and flow through when partially denatured.
His-tagged proNGF was not retained on NiNTA beads. N-terminal His tag may be hidden in the protein fold. Consequently, we did not manage to purify the proNGF.
Figure 10: Western Blot analysis of batch purification proNGF under native and partial denaturing conditions.
