Aflatoxin pollution is a worldwide problem. Every year, 25% of the world's grain and oil crops are contaminated by aflatoxin and other fungal mycin. In China, the annual loss of grain and edible oil caused by aflatoxin pollution is as high as 31 million tons. After understanding this problem, the project team hopes to use synthetic biology to provide a solution to the difficult problem of reducing aflatoxin pollution.

According to preliminary studies retrieved by the project team, a lassase mutant called CotAGold has the effect of hydrolyzing lactone bonds in aflatoxins to detoxify them; Nanoantibodies that can specifically bind aflatoxins have also been found. In addition, we found that the cell wall of gram-positive bacteria can physically adsorb aflatoxins to a certain extent.

Therefore, we designed an engineering bacterium using Lactobacillus rhamnosus, which can secrete CotAGold laccase in the environment with aflatoxins and degrade aflatoxins in the environment. We hope that it can alleviate the problem of aflatoxins pollution.

In this study, the target plasmids were constructed in vitro and the related plasmids were transformed by electroporation method. After the target plasmids were obtained, the strain Lactobacillus rhamnosus str. ATCC 7469 was transformed by electric shock or heat shock, and the target recombinant was verified by antibiotic positive screening bacterial liquid PCR and enzyme digestion. The strain and the reagent materials which were involved in this study are as follows:

The strain ATCC 7469, Which is usually used to research, teaching and vitamin detection. So it has good biosecurity. What’s more, Lactobacillus rhamnosus is a Gram-positive bacterium, it’s peptidoglycan cell wall can effectively catch the aflatoxin by physical absorption, this enables it to degrade the aflatoxin more effectively.

Table 1 The strain and the plasmids that were used in the study

We used four kinds of plasmids: pBS(KS+), pET-30b(+), pCDFDuetTM.1, pACYCDuet-1 as expression vectors.

For plasmid pBS(KS+), we inserted the E.coli MazF gene and regulated its expression with glucose operon. MazF is a toxin protein which is kind of RNase can cut bacteria’s mRNA and lead to suicide of the bacteria. By the way, under the control of pGlu the low glucose in environment could cause bacterial death. So the engineering bacteria won’t cause bio-safety problem when it leaks into the normal environment.

Figure 1 The expression vector pBS-MazF for suicide system

For plasmid pET-30b(+), we inserted the CotAgold gene and a GFP gene, both of them uses T7 promoter. CotAGlod laccase is an artificially engineered mutant derived from the BsCotA laccase of Bacillus subtilis. In addition, GFP gene, as a reporter gene, allows us to quickly screen recombinants that can secrete a large amount of CotAGold laccase, increasing the accuracy of screening.

Figure 2 The expression vector pCG of CotAGold laccase

For plasmid pCDFDuet-1, we built a delicate control system. It includes two VVD structural domain, each of them combine a part of T7 RNA polymerase, when it is illuminated by blue light, two VVD structural domain will combine together so the two part T7 RNA polymerase will combine into a complete T7 RNA polymerase. So the CotAGlod and GFP protein which use T7 promoter only be expressed when blue light is in the environment.

Figure 3 The expression vector pVVD of VVD

For plasmid pACYCDuet-1 is responsible for expressing a nano-antibody that can bind to aflatoxin B1(AFB1) and emit blue light. This special part consist of a nano-antibody that could bind specifically to AFB1 and a Gauss luciferase that is linked to the nano-antibody via linker. When the nano-antibody binds to AFB1, the conformation changes and the substrate oxidized by Gauss luciferase emits blue light. Blue light can restore the activity of T7 RNA polymerase, thus opening the subsequent expression of laccase.

Figure 4 The expression vector pGG of AFB1 nano-antibody

Table 2 The Culture medium and reagent used in the experiment

Reagent name	Manufacture
D-sorbitol	Psaitong
Maleic acid	Psaitong
D-KH2PO4	Psaitong
K2HPO4	Psaitong
Sucrose	Psaitong
Glycine	Psaitong
MgCl2• 6H2O	Psaitong
CaCl2	Psaitong
Glycerine	Psaitong
Lysozyme from chicken egg white	Psaitong
Spectinomycin solution	Psaitong
Kanamycin mono sulfate solution	Psaitong
Ampicillin Solution	Psaitong
Chloramphenicol Solution	Psaitong
NaOH	Psaitong
Agar	Psaitong
Peptone from Casein	Psaitong
Beef extract	Psaitong
α-D-Glucose	Psaitong
CH3COOH	Psaitong
Ammonium citrate dibasic	TWEEN 80
MgSO4•7H2O	Psaitong
MnSO4•H2O	Psaitong
CaCO3	Psaitong
Agarose	Psaitong
50×TAE	Psaitong
Restriction endonuclease	Psaitong
Restriction endonuclease	Psaitong
In-Fusion HD Cloning kit	Psaitong
Ex-Taq DNA polumersase	Psaitong
dNTP Mix	Psaitong
DNA Marker	Psaitong
PCR Mix	Psaitong
Plasmid extraction kit	Psaitong
PCR product recovery kit	Psaitong
GeneJET agarose gel recovery kit	Psaitong
MRS Broth	Psaitong
LB Broth	Psaitong
LB Agar	Psaitong

a)PEB buffer: first weigh 46.55g of sucrose, add hexahydrate and magnesium chloride 0.0506g, KH2PO4/K2HPO4 buffer pair, and adjust the pH value to 7.4, add water to 500mL.

b)MRS hypertonic: 119.8g of sucrose, 2.504g of magnesium chloride hexahydrate, 1.385g of calcium chloride solid, add MRS to 500mL. SMRS: take 85.575g of sucrose, then add 10g of glycine, add MRS to 500mL.

c)SMM Buffer: 85.575g of sucrose, then 11.6g of maleic acid, 40.46g of magnesium chloride hexahydrate solid, sodium hydroxide was used for neutralization, and adjusted pH to 7.8.

d)Resuscitation: 85.575g of sucrose, 2.023g magnesium chloride hexahydrate, 1.11 g of calcium chloride solids, and add MRS to 500 mL.

e)LB medium: Weigh 40g of LB Agar or 25g of LB Broth, stir with 800ml distilled water, dissolve the medium, hold the medium to 1 L, and sterilize, 121℃, 20 min.

the medium, hold the medium to 1L, and sterilize, 121℃, 20 min. g)Antibiotics:

Table 3 The antibiotic used in the experiment

The sterilize 50% glycerin was divided into 2 mL frozen storage tubes after sterilization in a super-clean bench, each tube was 1 mL, loosen the cap and sterilized again,then refrigerate at 4℃ for use. The target strains were inoculated into 5 mL MRS liquid medium containing corresponding antibiotics at 200 rpm, cultured overnight at 37℃, at 5000 rpm, centrifuged for 5 min, abandoned the supernatant, and then added to 1mL fresh antibiotic-free MRS medium to reinsert the bacteria at 5000 rpm. Centrifuge for 5 min, wash the bacteria and discard the supernatant, then add 1 mL fresh MRS medium, reinsert the bacteria, add all of them into the frozen storage tube, mix them upside down, label the strain name, resistance, production time and producer, and freeze them in the refrigerator at -80℃. Prepare the corresponding resistance MRS solid plate, dry the condensation of water on the surface of the plate to prevent the formation of bacterial moss after the water flow through the colony during inoculation. A small amount of bacterial solution was obtained from the glycerol tube with a sterilized inoculation ring, and then activated on a plate with a line. After the bacterial colonies grew out, a single colony was selected and transferred to MRS medium containing corresponding antibiotics at 200 rpm and cultured overnight at 37℃.

The specific steps to prepared the competent cell are as follow: A)The activated bacterial solution was inoculated into the SolⅠ medium, and the ratio of bacterial solution to SolⅠ medium was 1:50. Culture at 37℃ until the OD600 reached 0.3~0.6.

B)Culture solution ice bath for 30min, centrifuge at 4℃ and 6000g for 5min, discard the supernatant liquid.

C)Add appropriate amount of SM buffer, The suspended bacteria solution was centrifuged at 6000g and 4℃ for 5min, and the bacteria were collected and repeated twice. Add 200µL pre-cooled SM buffer, re-suspension bacterial solution, divided into 50µL per tube.

We use two method to transform the Lactobacillus rhamnosus: heat shock method and electric shock method. The experimental result shows that the efficiency of the latter is higher than the former.

A)The heat shock method:

The receptive cell suspension was taken at -80°C and placed on ice for 10min. Add plasmids DNA into 100~200µL receptive cell suspension, gently shake well, and leave on ice for 30 minutes.

Heat shock in water bath at 42℃ for 30-90 seconds, and quickly put on ice to cool for 3-5 minutes.

1mL liquid medium without antibiotic MRS Was added into the tube, mixed and oscillated for 1 hour at 37℃, so that the bacteria returned to normal growth state and expressed four antibiotic resistance genes encoded by the plasmids. The bacterial solution was shaken and coated with 100μL on a screening plate containing antibiotics (75 mg/mL), inverted petri dish, and cultured at 37℃ for 16-24h.

B)The electric shock method:

After mixing 1μL plasmid DNA with 100μL receptive cell suspension, the plasmid was placed in a 0.2cm electroshock cup and placed on ice for 10min. Click on the sample: electric voltage 1.75kV, resistance 200Ω, capacitance 25μF; Immediately after the shock, 1mL of MRS Hypertonic medium was added (for Lactobacillus rhamnosus receptive state resuscitation culture). After mixing, all the products were transferred into 1.5 mL centrifuge tube, incubated at 37 ℃ for 3h, coated with 100μL on resistant MRS Plate, and incubated for 48-72 h.

Prepare PCR tubes, primers, another new plate, primers, pipette tips and PCR Mix, etc., and prepare the appropriate system on the lab bench. Scraping a small amount of single colony with a white pipette tip in the super-clean bench, stir gently in the PCR system for 5~10 times, and then put the tip on the prepared plate with the same antibiotic added, and mark it accordingly, and cultivate it under the corresponding conditions.

Determine the correct transformants according to the agarose gel electrophoresis results of PCR products, and then line purification. Inoculate the purified transformants into 5 mL of MRS medium with appropriate antibiotics, incubate overnight and extract the plasmids, quantify by agarose gel electrophoresis, and then perform enzyme digestion for verification. Expand the culture of the correctly verified transformants and preserve them in glycerol tubes.

The digestion reaction is used to verify whether the target plasmid is connected correctly, generally use single or double digestion; digestion template needs to be determined according to the size of the target bands, generally 10 μL system digestion 200~300 ng.

The amount of conventional PCR template is 100 ng, and the system is 20 mL; in the case of colony PCR, the enzyme, buffer and dNTP in the system are included in the PCR Mix.

The temperature and time of the PCR program were determined based on the enzyme used and the length of the PCR and the annealing temperature of the primers.

Table 7 The Reaction Procedures of PCR