Parts characterization

Linker optimization

Since the cellulose fiber cannot cross the cell membrane, we expected the cellulase, including endoglucanase(CenA) and exoglucanase(Cex), to be secreted through α-hemolysin system.

The hemolysin (type I) secretion system belongs to the ABC transporter family, which recognizes the C-terminal amino acids of hemolysin toxin HlyA for protein secretion without requiring an N-terminal signal peptide.

So we planed to fuse the cex and cenA gene with hlyA tag at the C-terminus. Besides, we also co-expressed the HlyB and HlyD, two inner membrane proteins necessary to recognize and transport fusion protein across the inner and outer membrane directly.

Figure 1. Gene circuit of cellulase secretion system
cex: Cellulase exonuclease gene, cenA: Cellulase endonuclease gene,hlyA, hlyB, hlyD: α-hemolysin system gene

But before we constructed our final plasmid, which is load with cex-hlyA, cenA-hlyA, hlyB, and hlyD(Figure above), we firstly tried to replace the function gene, namely cex and cenA, with mRFP.

Figure 2. Gene circuit of mRFP secretion system

We used BBa_K1166002 as the template to acquire the hlyA-tag, hlyB ,and hlyD as a whole by PCR. And in the similar way, we cloned the mRFP-containing pIN2 backbone form one of our former constructed plasmid. In order to diminish the conformation effects of the hlyA on the target gene(in this case, mRFP), we constructed 3 types of linker— flexible, rigid, and OmpT-cutting linker—form the original linker which was reported to interfere with the GFP folding when fused together.

Figure 3.3 types of linker optimization

We modified the linker by introducing desired 5’ primer sequence.Through touch-up PCR using Phanta high fidelity polymerase from Vazyme, we got all the fragments and tested their length through electrophoresis.

We purified the PCR products and detected the concentration of DNA using NanoDrop2000.

Figure 4.Touch-up PCR results
O:original linker, F:flexible linker, R:rigid linker, C:OmpT-cut; line1-4: pET+mRFP plasmid backbone(～3500bp); line5-8: hlyA+secretion system(～5200bp); line9: marker III

Diagram 1.Touch-up PCR of hlyA+secretion system

Using Gibson-Assembly method, we ligated the backbone and target gene. Ligated plasmid were transferred into E.coli DH5α competent cell, then applied onto the Kan-containing LB plate and incubated at 37℃ for 12hr.

Diagram 2.Concentration of purified DNA measured by NanoDrop2000

Diagram 3.Gibson Assembly Reaction System

We selected 8 visible(purple) colonies of each four plates and used 3 pairs of primer to test whether these 2 fragments are connected.

Figure 5.plates of DH5α transformants (pIN2 + mRFP + linker + hlyA + araC + pBAD + hlyB + hlyD) of 4 types of linker on LB-kan plates and in liquid M9 media.

Three different pairs of primer has been used to test the connection link. and the colony PCR results showed that positive clones were: flexible-linker F12357, rigid-linker R12367, OmpT-cleavable-linker C12357, and original-linker O4568.

So we chose F3, R2, C3, and O8 to be amplified and further tested by sequencing(BGI company). The link sequences were all proved to be in accordance with our designed linker sequence, indicating we’ve successfully constructed 4 types of plasmid with original, flexible, rigid and cleavable linker respectively. These four clones were then use to prepare protein sample to run SDS-PAGE and Western Blot.

Figure 6.colony PCR results shown on 1% agarose gel.

All the colonies proved to be positive by colony PCR and first generation sequencing could be easily spotted red on plates and liquid media, directly indicating that the all the linker+hlyA tags were not interfering the emission of fluorescence of mRFP. Compared with using GFP as the secreted target gene, mRFP is apparently more suitable for the fusion of hlyA to testify the secretion efficiency of hemolysin system.

Figure 7.liquid media of DH5α transformants (pIN2 + mRFP + linker + hlyA + araC + pBAD + hlyB + hlyD) of 4 types of linker on LB-kan plates and in liquid M9 media.

Positive transformants with different linker were cultured and induced by arabinose. We collected the same weight of E.coli by restricting the value(OD multiple volume) equal to 16.32. And then separate the media and cell by centrifugation. The supernate were concentrated 100 fold and the pellet were resuspended with PBS. Supernate and pellet were both pretreated to prepare protein sample and run SDS-PAGE.

Figure 8.liquid LB media containing positive colonies with(Y) or without(N) arabinose induction after 16 hours. Left three media are pIN2+mRFP, and pIN2(empty vector) as negative control.

The SDS-PAGE shows that, the fusion protein—mRFP-hlyA(in the red frame, about 32.6kD)—were all expressed inside the cell and successfully secreted to the media.And in the OmpT-cleavable linker case, the mRFP and hlyA-tag were also successfully cleaved, since the cleaved mRFP(in the rgreen frame, about 26.0kD) clearly surpassed the fusion protein on the gel. Although the mRFP-hlyA fusion protein and mRFP were both slightly smaller than expected size, we highly suspected that’s because, the pI of the mRFP-hlyA and mRFP are estimated to be 5.12 and 5.07, both were lower than pH7, which might dramatically affect the mobility ratio of these protein in the SDS-PAGE buffer and shows at a lower molecular weight on the gel.

Figure 9.SDS-PAGE of supernatant and pellet of types of linker-transformants.

So we’ve demonstrated the OmpT-cleavable linker to be functional, and the flexible and rigid linker were also proved to be not hindering the secretion of fusion protein while offering several alternative options for perspective users who want to express their protein in an secretory form.