Parts characterization

Cellulose degradation

For this part, we want our chassis organism degrade lignocellulose into cellobiose firstly, so we designed a plasmid which can express C. fimi endoglucanase CenA and C. fimi β-1,4-exoglucanase Cex and can secrete these two enzymes out of E.coli using hemolysin secretion system, which is the best-characterized and most widely used TISS is the ahemolysin (HlyA) secretion pathway from uropathogenic E. coli. The secretory machinery of this pathway consists of three components: HlyB, an ATP-binding cassette (ABC transporter); HlyD, a membrane fusion protein; and TolC, an outer membrane protein.

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

In order to use E. coli DH5α to express recombinant protein into the growth medium through the a-hemolysin secretion pathway, the α-hemolysin specific signal peptide HlyAs, which corresponds to residues 965-1024 of α-hemolysin, was fused to the C-terminus of cenA and cex by overlapping PCR. In addition, the genes encoding the components of translocator, hlyB and hlyD, were amplified together by PCR using part:BBa_K1166002 as template.

Enzyme activity of Cex-HlyA/CenA-HlyA

To make sure our final construction would work, we first analyzed the enzyme activities of the fusion protein cenA-HlyA and cex-HlyA. HlyA was amplified from part:BBa_K1166002 and cenA was amplified from part:BBa_K523015, while cex was amplified from the gene synthesized by Genewiz, and the sequence was obtained from ncbi. The PCR products were cloned into a modified pET28b (+) plasmid pIN2 and enzyme activity within cells harboring cloned genes were tested qualitatively using carboxymethyl cellulose (CMC) substrates and FPA substrates.

Figure 2. Plasmids for Cex-HlyA and CenA-HlyA expression
cex: Cellulase exoglucanase gene, cenA: Cellulase endoglucanase gene,hlyA, hlyB, hlyD: α-hemolysin system gene

Due to mRFP had been inserted into our backbone pIN2 for the construction of the invertor system, the negative colonies would show red for expressing mRFP, and white colonies were mostly positive. We conducted colony PCR and Sanger sequencing for further confirmation.

After successfully constructed both clones, we followed Crude enzyme extraction protocol and cellulase enzyme activity assay protocol to obtained the enzyme activity of Cex-HlyA and CenA-HlyA, and the results are shown below. We also measured the enzyme activity curve of CenA alone for the characterization of Part: BBa_K523015. for more information)

To fully verify our cellulase hydrolysis products were mostly cellobiose for the activation of cellobiose promoter and as a result turning off cellulase expression and turning on bacterial cellulose synthase, we performed HPLC/TLC for our FPA assay products and CMCNa assay products and results are as follows:

Secretion efficiency of Cex-HlyA/CenA-HlyA

After validating the secretion ability of a-hemolysin system by HlyA fused with RFP, we also want to make sure that Cex and CenA can be secreted and to analyze the secretion efficiency separately, we planned to construct pCDFDuet-MCS1-cenA-HlyA-MCS2-HlyBD and pCDFDuet-MCS1-cex-HlyA-MCS2-HlyBD.

Firstly, the hlyBD was cloned into MCS2 by overlapping PCR. After obtaining pCDFDuet-MCS1-MCS2-HlyBD, we tried multiple methods to insert cenA-hlyA into MCS1, such as seamless cloning, double enzymes digestion and ligation, and overlapping PCR, but all these attempts failed for a mysterious missing of a part of hlyBD in the final construction while the sequence of pCDFDuet-MCS1-MCS2-HlyBD was proved to be correct by Sanger sequencing of the whole sequence.

Interestingly, we inserted cenA-hlyA, cex-hlyA, HlyBHlyD into the plasmid pIN2 of our invertor system just before deadline, and we performed western blot for both pellet and supernatant to evaluate secretion efficiency.

Supernatant enzyme activity of Cex-HlyA/CenA-HlyA

Also, we used the above two plasmids-- cex-hlyA/pIN2 and cenA-hlyA/pIN2 to measure to secreted enzyme activity for cex and cenA, and the results are as below.

Collaboration with Hubei University

Due to the fact that all of these proteins were fused with flag-tag, we were able to detect their presence by Western Blot using a mouse-derived anti-flag primary antibody and detection antibody probed with fluorescence.

From the Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and Western blot, we can conclude that the Secretion Efficiency of PhoD-CelA outshines other two types, while ZM4 showed higher expression efficiency of GAP promoter than E.coli T1.

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.

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.