Team:SZU-China/Procedure

Procedure

Construction of Plasmid vector

We used the Shuttle plasmid pP43NMK (donated from ZIU-iGem) for our project, which can propagate in both Bacillus subtilis and Escherichia coli. The vector contains the following parts:

Fig 1. Gene expression vector

Gene	Decription
repB	The origin of replication of pP43NMK vector in host Bacillus subtilis.
PsspB	This is the promoter (PsspB) of the sspB gene, which is expressed at high level during sporulation in a forespore-specific manner, that is to say, the promoter PsspB only switch on the transcription after the initiation of sporulation. Synthesized by biological company.
gerAa	This part is a 300-bp upstream region of gerAA, which is one of the structural genes in the operon gerA.It concerns with the triggering of spore germination by L-alanine and its analogues. Synthesized by biological company.
P43	It is a constitutive promoter and a strong promoter in Bacillus subtilis, which initiates transcription in the logarithmic phase and stable phase.
OF4-nhaC	This part is the coding sequence (CDS) of the Na⁺/H⁺ antiporter from the Bacillus pseudofirmus OF4 (GenBank Acc.No. CP001878), which regulates cytoplasmic pH value by coupling net H⁺ uptake with Na⁺ extrusion. Synthesized by biological company.
C125-tupA	The primary translation product of this gene, TupA, is likely a cytoplasmic protein involved in the synthesis of TUP, a copolymer of polyglutamic acid (PGlu) and polyglucuronic acid (PGlcU), which is one of major structural components in the cell wall of the Bacillus lentus C-125 and can neutralize the extracellular hydroxyl. Synthesized by biological company.
CA	This part is the coding sequence (CDS) of Carbonic anhydrase(CA). CA is a metalloenzyme with zinc, which is highly efficient and one of the fastest enzymes catalyzes the reversible hydration of CO₂ forming bicarbonate and protons rapidly. Synthesized by biological company.
RBS	Ribosome binding site of Bacillus subtilis.
rrnBT1	Transcriptional terminator consisting of a 64 bp stem-loop from E. coli rrnB. Synthesized by biological company.
Kanr	Kanamycin Resistance Gene in host Bacillus subtilis.

Inside the vectors，the first promoter P43 is the original component and initiates the transcription of downstream coding sequence, including the OF4-nhaC , C125-tupA and CA.These parts have respective RBS and the common terminator rrnBT1.The second promoter PsspB is the 5' sequence located before the gene coding region of gerAa. Through homologous recombination, a single cross-over event between the gerAa region in our plasmid and the endogenous gerAa sequence on chromosome inserts the sspB promoter upstream of the gerA operon, therefore gerA will overexpress during sporation, All of these parts above were combined through Restriction site. Kanr is responsible for the resistance gene of Kanamycin, so we can use Kanamycin B to verify positive transformants.

Transformation and Expression

The Bacillus subtilis we use in our project is WB800 (bought from Youbio Technology Company, China). This kind of Bacillus subtilis is a strain deficient in eight extracellular proteases, which can prevent the proteolytic cleavage of the enzymatic subunit between the catalytic and dockerin domains, to reduce the degradation of expression product by the extracellular proteases.

First, we transformed the expression vectors into Bacillus subtilis WB800 by optimization of Spizizen method, which increase the efficiency of transformation obviously. Second, the positive clones were obtained by Kanamycin resistance screening. Plasmid extraction and Digestion of restriction endonuclease and nucleic acid electrophoresis confirm real Positive clones.

Fig 2. Gene expression test procedure

Next, we detected the expression of our parts and test their function by methods as follow:

CA:
The transformed strain WB800 was grown in optimized CA production medium, and obtain crude enzyme solution by centrifugation and ultrasonic crushing. And then we detect the native molecular mass by SDS-PAGE and coomassie blue staining. In addition, For determining the activity of CA, hydration of CO2 was measured using electrometric Wilbur-Anderson assay according to Khalifah et al. with certain modifications, and crude cell-free extract were also checked for esterase activity according to the Verpoorte et al.
OF4-nhaC & C125-tupA:
In order to test the alkali resistance of the gene-modified strain WB800, we used the alkaline complex medium containing Na₂CO₃ and other necessary nutrients to set a serial pH value gradient culture from 7.5 to 9.0. Strain transformed and original WB800 were plated at 37°C in the alkaline complex medium simultaneously, then we recorded the OD600 of them at the same time to compare their growth rates, which indicates the function of these two parts to increase the alkali tolerance of B.subtilis.
PsspB-gerAa:
This part is used for increasing the germination rate of spore of B.subtilis, so we need to plate WB800 at 37°C on 2XSG medium agar plates without antibiotics and harvest spores after 72h, cleaned by repeated rounds of centrifugation and washing with water. Spores were resuspended and optical densities at 600nm(A600) of 0.8 to 1 in PBS. Germination was by monitored by reading the drop in absorbance (A600) in a 96-well microplate reader (Tecan Infinite M200) after add the L-alanine-NaCl mixed solution.The drop rate of absorbance (A600) is proportional to the germination rate so that we can compare the difference between original WB800 and recombinant WB800.

Application and Realization

In order to certificate the ability of our chassis strain WB800 to produce calcium carbonate and application in self-healing of concrete, we take a step forward, establishing a concrete-model to understand the incredible interaction between concrete and the recombinant B. subtilis.

Fig 3. micro-capsule granule we made

Using a special material-microcrystalline cellulose (Fig.3), we add around 10^-8/ml endospores with clay and of course , L-alanine(in powdery) together, making a tiny but powerful microcapsule. Next, pouring some newly-mixed cement, which is still in viscous liquid state into the mold until the cement reach its 2/3, add a microcapsule into it, and fill the mold with cement as shown in Fig.2 and Fig.3

Fig 4. Mould for concrete experiment.

Fig 5. The cement block we made

After some days for the drying of concrete mold. Take out the column-like concrete from the mold. Then spilt the concrete right through the side ditches, exposing the microcapsule. Now if we put this two part of concrete column together by sticking them with After some days for the drying of concrete mold. Take out the column-like concrete from the mold. Then spilt the concrete right through the side ditches, exposing the microcapsule. Now if we put this two part of concrete column together by sticking them with a tape, we can build a microcrack on the upper surface. So we mark these concrete columns. And put them into the artificial seawaters for 28 days. And you find the microcrack on the upper surface be repaired like they never ever existed.(As shown in Fig.4,5,6,7)

Fig.6 The comparison of repairing between WB800 (shown in upper two figures) and recombinant WB800 (shown in under two figures)

Reference

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[2] Løvdal I S, From C, Madslien E H, et al. Role of the gerA operon in L-alanine germination of Bacillus licheniformis spores[J]. Bmc Microbiology, 2012, 12(1):1-12.

[3] Tjalsma H, Bolhuis A, Jongbloed J D, et al. Signal peptide-dependent protein transport in Bacillus subtilis: a genome-based survey of the secretome[J]. Microbiology & Molecular Biology Reviews Mmbr, 2000, 64(3):515.

[4] Li W, Yu L J, Yuan D X, et al. A study of the activity and ecological significance of carbonic anhydrase from soil and its microbes from different karst ecosystems of Southwest China[J]. Plant & Soil, 2005, 272(1-2):133-141.

[5] Prágai Z, Eschevins C, Bron S, et al. Bacillus subtilis NhaC, an Na+/H+ antiporter, influences expression of the phoPR operon and production of alkaline phosphatases.[J]. Journal of Bacteriology, 2001, 183(8):2505.