Team:XJTU-China/Engineering

<!DOCTYPE html> Design

XJTU-China

Engineering Success

This year we were committed to finding an effective solution to the global problem of desertification. And our project aimed to achieve efficient production of extracellular polysaccharide (EPS) , the key component of biological soil crusts, by engineered bacteria with an anti-escape system. A genetic circuit containing the key enzymes was constructed for the production of EPS, and controlled production of suicide protein under the regulation of arabinose operon was also attempted to realize the suicide of engineered bacteria once they leave the desert environment for biosafety. Meanwhile, a co-culture system of Bacillus subtilis and cyanobacteria Nostoc sp. was investigated primarily, to achieve more efficient desert soil fixation of engineered strain under tough growth condition of desert.

1. Engineered bacteria with GalU and PGM expression were able to produce EPS efficiently

Research

For the production of EPS, we found the synthesis pathway of bacterial EPS[1] and two key enzymes in EPS production, UDP-glucose pyrophosphorylas（GalU）and a-phosphate Glucose Mutase（PGM）, after extensive literature research. UDP glucose can be used as a raw material for the synthesis of EPS. It has been reported that by overexpressing genes for both enzymes in Streptococcus thermophilus, the production of EPS in bacteria can be significantly increased. Fredrik Levander et al. achieved EPS yield increase from 0.17 g/mol to 0.31g/mol when both pgmA gene and galU gene were overexpressed in Streptococcus thermophiles[2]. Therefore, we hypothesized that increased expression of pgmA and galU genes in Bacillus subtilis could also lead to increased EPS production.

Fig．1 EPS precursor synthesis pathway

Note: FK: fructokinase; GalE: UDP galactose-4-epimerase; GalK: galactose kinase; GAIT: galactose-1-phosphate uridine yltransferase; GalU: UDP-Glucose pyrophosphate; GK: glucokinase; GalM: N-acetylglucosamine-1-phosphate uridylyltransferase; GPI: glucose-6-phosphate isomerase; LacZ: Igalactosidase; PGM: a-phosphate Glucose Mutase; PM: Phosphomutase; RmlB: dTDP-Glucose-4, 6. Dehydratase; SPH: sucrose-6-Phosphohydrolase; TGP: dTDP-Glucose pyrophosphorylase; TRS: dTDP-4 dehydrogenation

Rhamnose-3,5-epimerase; UDP-GalNAc: UDP-N-acetylgalactosamine; UDP-GIcNAc: UDP-N. Acetyl glucosamine; UGDH: UDP-glucose. 6-Dehydrogenase; UGM:UDP-Galactopyranose mutase.[1]

Design

Based on the above assumptions, we searched for three isoenzymes with higher enzyme activity of PGM in three different species, from Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa. We linked these three genes to the galU gene derived from Bacillus subtilis, and regulated their transcription by the Bacillus subtilis constitutive promoter. We constructed the following three gene circuits to realize the expression of galU and PGM enzymes in engineered bacteria, and efficiently produce EPS.

FIG 2. Three expression plasmids galU from Bacillus subtilis and pgmA genes from different strains

Build

Based on our design, three pgmA genes from Escherichia coli, Bacillus subtilis and Pseudomonas aeruginosa respectively were synthesized, amplified by PCR and connected to the galU gene from Bacillus subtilis under the P43 promoter of Bacillus subtilis, and three plasmids would be constructed containing different combinations of galU and pgmA gene (See Figure…), using the backbone vector PHT43, a the shuttle vector of in E. coli and B. subtilis.

FIG. 3 PCR amplification of linare vector, galU gene and pgmA genes

  FIG 4. Three expression plasmids：galU from Bacillus subtilis and pgmA genes from different strains under P43 promoter  

Test

1. We have characterized gene expressions in these plasmids at the transcriptional level using RT-qPCR.

（BB：E.coli DH5α with galU gene from Bacillus subtilis, and pgmA gene from Bacillus subtilis）

（BE：E.coli DH5α with galU gene from Bacillus subtilis, and pgmA gene from Escherichia coli）

（BP：E.coli DH5α with galU gene from Bacillus subtilis, and pgmA gene from Pseudomonas aeruginosa）

FIG 5-1 galU gene verification in mRNA levels of three DH5a strains transferred with above 3 plasmids

FIG 5-2（a） pgmA gene from Bacillus subtilis verification in mRNA levels of DH5a strains transferred with plasmid B.S.galU+B.S.pgmA

FIG 5-2（b） pgmA gene from Escherichia coli verification in mRNA levels of DH5a strains transferred with plasmid B.S.galU+E.coli-pgmA

FIG 5-2（c） pgmA gene from Pseudomonas aeruginosa verification in mRNA levels of DH5a strains transferred with plasmid B.S.galU+P.A.pgmA

At the mRNA level, the transcription level of the corresponding gene was significantly improved after the gene was transferred, indicating the expression of these genes in our engineered strain.

 

2. In order to further verify whether our engineered bacteria can truly and efficiently produce EPS, we conducted the extraction and content detection of polysaccharides in the 24h culture medium.

The experimental methods were as follows

(1) Draw the standard curve of glucose Colorimetry (OD620)

① 0.0ml, 0.2ml, 0.4ml, 0.6ml, 0.8ml, 1.0ml of glucose solution were added into 6 test tubes, and 1.0ml, 0.8ml, 0.6ml, 0.4ml, 0.2ml, 0.0ml of distilled water were added in turn.

② 0 ml of anthrone sulfuric acid solution was added to each tube, and the mixture was homogenous.

③ The test tube was immersed in 95 ℃ water bath for 10 minutes, and the solution was kept in a uniform state at all times.

④ After the color of the solution is stable, colorimetry is carried out at 620nm with the enzyme labeled instrument.

⑤ Three parallel experiments were conducted

(2) Determination of polysaccharide solution concentration

① 1.0ml polysaccharide solution of the bacteria to be tested is added into the test tube, and then 3.0ml of anthrone sulfuric acid solution is added, and the mixture is homogeneous.

② The test tube was immersed in 95 ℃ water bath for 10 minutes, and the solution was kept in a uniform state at all times.

③ After the color of the solution is stable, colorimetry is carried out at 620nm with the enzyme labeled instrument.

④ The concentration of polysaccharide was calculated by colorimetric standard curve.

FIG 6. EPS content in the culture medium after 24h DH5a culture with 3 combination plasmids and control

From the above data it can be concluded that the EPS yield of engineering bacteria has been increased compared to negative control, demonstrating the successful production of EPS.

Improve

However, the yield of EPS of expression strains were not satisfying. According to our survey results in Yulin and the feedback we got in the interview with Doctor Lide Wang, such polysaccharide production may not significantly improve the efficiency of soil crusting. Doctor Wang pointed out that according to our data, the production of polysaccharides is not very high. That would make our system difficult to spread, and not conducive to the formation of large-scale and efficient soil crusts. So we further searched other alternative galU genes with high enzyme activity from the database. Finally we chosen the galU gene from E.coli, and constructed another 3 expression vector and comprehensively compared 6 combinations of engineered bacteria.

FIG 7.  PCR amplification of galU gene from Bacillus subtilis and Escherichia coli

We repeated the above detection operation and got the following results.

As shown in Figure 8, the relative mRNA levels of the six strains were much higher than those of the blank strain, among which E.coli-galU and E.coli-pgmA were more active in transcription. Figure 9 showed the engineered strains with co-expression of galU and pgmA were able to produce EPS compared to the negative control, with varied yield. And the combination of E.coli-galU and E.coli-pgmA was the highest, corresponding with the mRNA results.

FIG 8-1. Transcriptional levels of galU and pgmA in DH5a transferred from 6 combinations of plasmids and controls

FIG 8-2. Polysaccharide content in the culture medium after 24h DH5a culture with 6 combination plasmids and control

The above results of gene expression at mRNA level and the yield of EPS in engineered strains have demonstrated our engineering success as expected.  

As a result, we found the three gene combinations for high-yield EPS and submitted 6 composite parts : BBa_K3331009  BBa_K3331010  BBa_K3331011  BBa_K3331012  BBa_K3331013  BBa_K3331014

Due to various reasons, we have not succeeded in introducing the plasmid into Bacillus subtilis for further verification, but it can be foreseeable that one of the six plasmids we constructed would achieve efficient transcription in Bacillus subtilis and significantly increase the level of EPS.

http://parts.igem.org/wiki/index.php?title=Part:BBa_K3331009

http://parts.igem.org/wiki/index.php?title=Part:BBa_K3331010

http://parts.igem.org/wiki/index.php?title=Part:BBa_K3331012

http://parts.igem.org/wiki/index.php?title=Part:BBa_K3331013

http://parts.igem.org/wiki/index.php?title=Part:BBa_K3331014

2. A primary investigation on the symbiotic system of Bacillus subtilis and cyanobacteria Nostoc sp. demonstrates the coordinated growth of two microorganisms.

After a large number of literature searches, the symbiotic growth of the two microorganisms is supported by some literatures. The experiment of wild-type cyanobacteria and Bacillus subtilis by Hossein Kheirfam et al. proved that the co-culture of Bacillus subtilis and cyanobacteria can produce a larger thickness of soil crusts on a sandy river bed, compared to cyanobacteria alone or Bacillus alone inoculation[3]. Our primary co-culture test of these two strains as well as the modeling simulation on growth (See Growth model) also demonstrated the feasibility of symbiotic system for our further optimizations.

3. Controllable regulations of two arabinose operons from B.subtilis and E.coli using GFP as a proof of concept can be achieved, demonstrating the controllable anti-escape system was feasible with suicide MazF protein under regulation of arabinose operon.

Design

Considering biosafety issues, we also designed a suicide switch based on the arabinose operon. The elements of the arabinose operon here are all derived from the genome of Bacillus subtilis 168. Although the effect is similar to the commonly used E.coli arabinose operon, the principle of action is different. When there is no arabinose in the environment, the araR protein binds to the araE promoter and acts as a repressor to inhibit the expression of downstream genes. When there is enough arabinose in the external environment, the combination of arabinose and araR protein cancels the inhibitory effect of araR, and downstream gene expression produces MazF protein. And Bacillus subtilis MazF-bs (EndoA) is a UACAU-specific mRNA interferase, which is a toxic protein to Bacillus subtilis.
Through literature search, we found that the content of arabinose in desert soil is scarce, but there is more arabinose in normal soil environment, which confirms the feasibility of suicide switch conceptually.

FIG 9. Suicide switch based on the arabinose operon of Bacillus subtilis

Before constructing the suicide circuit, we also constructed a verification plasmid for the efficiency of the arabinose operon of Bacillus subtilis, replaced the toxic protein MazF gene with the green fluorescent protein gene GFP, and characterized the efficiency of the arabinose operon by fluorescence.

FIG 10. Validation circuit of suicide switch based on Bacillus subtilis arabinose operon

Build

Based on the above gene circuit design, we constructed the following two plasmids. and verified the function of the arabinose operon in Bacillus subtilis.

FIG 11. Validation plasmid of suicide switch based on Bacillus subtilis arabinose operon

FIG 12. Plasmid of suicide switch based on Bacillus subtilis arabinose operon

In order to further verify the arabinose operon of Bacillus subtilis, we constructed a verification plasmid with green fluorescent protein and arabinose operon in E. coli.

FIG 13. Validation plasmid of suicide switch based on E.coli arabinose operon

Test

After constructing a verification plasmid for the arabinose operon, we characterize the translation level of the transformed strain. We take the bacterial liquid after a certain induction to detect the fluorescence, and the results are as follows

FIG 14. Fluorescence intensity in Bacillus subtilis and E.coli  at different concentrations and induction times

Because of COVID-19 and limited time, our project was delayed and we failed to obtain the anti-escape system with suicide MazF protein under regulation of ara operon, which will be further optimized in the future.
But we are able to quantitatively determine the controllable regulations of two ara operons from B.subtilis and E.coli using GFP as a proof of concept, and results showed these two ara operons are both functional under induction of different concentrations of arabinose, exhibiting low leakage and wide dynamic range. From Figure14 we can see, the optimal induction concentration of brabinose is 1.5 Mm/L and 1.0 mM/L for ara BS and ara E.coli respectively. E.coli has wider dynamic range indicating it response faster after inducing, and lower leakage expression, while BS ara showed higher fluorescence intensity ,demonstrating some improvement, and provide more alternatives for arabinose regulation promoter.

References：

[1] Kong L H, Zhao L S, XIA Y J, et al. Progress in the biosynthesis of exopolysaccharides from Streptococcus thermophilus [J].Journal of Food Safety and Quality Inspection, 2019, 10(02):14-20.

[2] Pang Ning, Zhang Jiaqi, QI Jin, et al. Research Status of Extracellular polysaccharides and their biosynthesis pathways in microorganisms [J].Frontiers of Microbiology, 2017, 006(002):P.27-34.

[3] Hossein Kheirfam, Farrokh Asadzadeh, et al. Stabilizing sand from dried-up lakebeds against wind erosion by accelerating biological soil crust development [J] European Journal of Soil Biology,Volume 98,2020,103189,ISSN 1164-5563