Difference between revisions of "Team:NEFU China/Protocols"

Overview

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Yogurt can easily get bacteria contamination when improperly stored. We generally cannot determine whether a cup of yogurt is safe for eating just through checking its appearance, so we asked this question: can we make spoiled yogurt look different? 

This year, the iGEM team of NEFU_China aims at creating a novel and handy method for the detection of pathogens in yogurt. Autoinducer 2 (AI-2), a signal molecule constantly produced by pathogens in yogurt, serves as the key in our project. We cloned genes related to the AI-2 responsive pathway in Salmonella typhimurium​ and integrated them into the genome of Lactobacillus bulgaricus. Our engineered Lactobacillus will be able to uptake AI-2 molecules from pathogens and trigger the expression of a report gene that produces a blue pigment. Since our engineered Lactobacillus can act as an auxiliary starter in yogurt fermentation, the detecting process can be greatly simplified. If you open a cup of yogurt and find it has already turned blue, you can just trash it.

BackGround

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As one type of the oldest fermented food, yogurt is very popular around the world. Different from other traditional dairy products (cottage cheese, sour cream and etc.), yogurt wins its popularity due to the health benefits it can offer. Primarily, yogurt comes from milk and is nutritionally rich in protein, calcium, riboflavin, vitamin B6 and vitamin B16. Additionally, Lactose-sensitive individuals may tolerate yogurt better than other dairy products due to the conversion of lactose to glucose and galactose, and the fermentation of lactose to lactic acid carried out by the bacteria in the yogurt. Most importantly, it generally possesses a certain amount of probiotics, which is necessary in our digestive tracts.

Fig2. Delicious yogurt

In spite of these health benefits, yogurt is easy to go bad and spoiled yogurt has more harm than good. Last semester, one of our teammates was absent for class due to eating some spoiled yogurt, although it was still within the expiration date. Generally, yogurt within expiration date is safe for eating if it is always kept cold, which was neglected by this teammate who consequently suffered from diarrhea. As you can see, yogurt may cause food poisoning even though it has not expired. This is one of the reasons why we came up with an idea of developing a yogurt guarder.

We searched the Internet for relevant information and found varieties of news about yogurt spoilage within shelf-life. Bacteria contamination in yogurt cannot be recognized by our naked eyes. Thus, many people may suffer from that.

To understand why yogurt may not be safe for eating even before the expiration date, we need to know how yogurt is produced. First, raw milk is treated with pasteurization to kill most microorganisms inside without destroying its nutritional components. Second, Lactobacillus is inoculated for fermentation. Then, yogurt is made. Unlike sterilization, pasteurization is unable to kill all microorganisms in the food. Instead, it intends to reduce the number of pathogens without significantly affecting nutrient. So yogurt must be kept in cold for a limited time period; otherwise pathogenic bacteria that survive from pasteurization can quickly grow and cause yogurt spoilage.

We did a simple experiment to test the appearance and contents of yogurt under different conditions. We bought fresh yogurt from the same batch and kept some of them in fridge and some at 35℃ for half an hour. Then, we took pictures of them. The yogurt kept at both conditions looked the same. However, our subsequent experiments revealed that yogurt kept at 35℃ had a higher number of coliform bacteria than that from the fridge and the pathogens significantly exceeded the qualify standard. This means the pathogen contents of yogurt may significantly increase when we carrying yogurt from a supermarket to home, since yogurt will be exposed to ambient temperature for a while, especially in summer. (Visit Result for more details)

Fig3. Yogurt treated with different temperature. (A) Fresh yogurt kept in 4℃ for 0.5hour (B) Fresh yogurt kept in 35℃ for half an hour (C) Yogurt kept in 4℃ until one-day past expiration

Needless to say, no one would like to eat yogurt if he or she knows it may contain such a high amount of coliform. However, the current methods to test pathogenic bacteria generally contain multiple cumbersome steps, are time consuming and require special equipment. They obviously cannot be used in our daily life. Therefore, a handy detecting approach for pathogenic bacteria in yogurt is urgently needed.

 

Design

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The major pathogens in spoiled yogurt include E. coli, Salmonella and Bacillus. If we can find a common feature among these bacteria, we may develop a method to detect them simultaneously. We searched literatures and discovered that a signal molecule, autoinducer 2 that is a signaling molecule in quorum sensing, is common among these pathogenic bacteria.

Quorum sensing is a process of bacterial cell-to-cell communication involving the production and detection of extracellular signaling molecules called autoinducers. As the density of the bacterial population increases, so does the amount of secreted autoinducer molecules. When the concentration of the autoinducer reaches a critical threshold, it wa  transported back into the cell and activates or represses certain target genes. While most autoinducers are species specific, autoinducer 2(AI-2) is generated by many Gram-positive and Gram-negative bacteria and serves as a 'universal signal' for interspecies communication.

AI-2 is a byproduct of the Activated Methyl Cycle, which recycles S-Adenosyl-L-Methionine (SAM). As a main methyl donor in eubacteria, archeabacteria and eukaryotes, SAM is converted to S-Adenosyl-L-Homocysteine (SAH), which is subsequently detoxified by the Pfs enzyme (also called S-Adenosylhomocysteine Nucleosidase) to generate Adenine and S-Ribosyl-Homocysteine(SRH), the sole intracellular source of the substrate of LuxS. LuxS then produces the precursor of AI-2, 4,5-Dihydroxy-2,3-Pentanedione(DPD), during the conversion of SRH to Homocysteine (HCY).

Fig3.  Activated Methyl Cycle 

DPD can be converted from SRH by LuxS in the cytoplasm and then exported to culture medium, where DPD undergoes spontaneous cyclization to form AI-2. Depending on the bacteria, response to AI-2 can follow one of the two currently identified routes. In pathogens exemplified by Salmonella, AI-2 response involves ATP binding cassette transporter encoded by four LuxS-regulated (lsr) genes. lsrB encodes the periplasmic AI-2 binding protein, lsrC and lsrD encode two membrane channel proteins, and lsrA encodes the ATPase that provides energy for AI-2 transport. The extracellular AI-2 can bind LsrB to re-enter the cytoplasm and be phosphorylated by LsrK. Then the phosphorylated AI-2 can activate the lsr operon through binding the repressor protein LsrR and release it from the promoter. This will lead to the synthesis of LsrA,C,B, D and increase AI-2 entry.

Fig 4. AI-2 response in Salmonella typhimurium

AI-2 response pathway in Lactobacillus is different from that in pathogenic bacteria. So we will take the advantage of this difference and use the mechanism of AI-2 pathway in these pathogenic bacteria to build our detecting system. We choose Lactobacillus as our chassis. As beneficial bacteria, they are in food-grade and widely used in food fermentation. Lactobacillus bulgaricus can improve nutrient absorption and human gastrointestinal function, and inhibit the reproduction of pathogenic bacteria in guts. If fully developed into real products, our engineered Lactobacillus can be directly used in yogurt fermentation, which will make our detecting process even more convenient.

We cloned genes related to the AI-2 response in Salmonella and integrated these genes in Lactobacillus and Lactococcus genomes. In our engineered bacteria, the lsrA, C, B, D genes will constitutively express to form the membrane transporter. We will clone the promoter sequence of the lsr operon and use it to drive the expression of the report gene. According to the previous studies, we have chosen an identified pigment in the Registry: the biobrick of amilCP (BBa_K592009). It encodes a blue pigment that can be recognized by naked eyes. Thus, when pathogenic bacteria express AI-2 molecules and secrete them extracellularly, these molecules can be transported into our engineered bacteria and trigger the expression of the report gene to produce the blue pigment. 

Fig5. Working mechanism of our engineered bacteria

In our design, the PnisA promoters, which can be activated by nisin, are used to drive the expression of lsrA, C, B, D, R and K genes. Nisin is a anti-microbial peptide consisting of 34 amino acids. Because of its broad host spectrum, it is widely used as a food preservative.

The nisin-controlled gene expression (NICE) system is one of the most commonly used regulatory gene expression system of Gram-positive bacteria. In the natural situation, nisin binds to the receptor NisK, which activates NisR through phosphorylation. The activated NisR drives the nisA promoter. Sub-toxic amounts of nisin in the ng/mL range are sufficient to fully activate the otherwise tightly repressed promoter.

Fig6. Nisin induced regulation system

Previous studies indicate that the nisR and K are the only nis genes required for nisin-mediated signal transduction and PnisA promoter activation. However, our chassis, Lactobacillus bulgaricus Lb14, does not have nisR or nisK genes in its genome. In order to implement this strictly controlled expression system in such lactic acid bacteria, various nisR and K expression constructs were generated, and we picked pNZ9530 among them. Thus, apart from the plasmids for the expression of the essential parts in charge of AI-2 response, we also need to transform pNZ9530 into our engineered Lactobacillus.

This system for regulated gene expression shows many desirable characteristics: (1) nisin is an ideal molecule to be used as an inducer since it is already widely used in the food industry and can therefore be regarded as a food-grade inducer; (2) the protein expression levels are very high in this system; (3) the expression of the intergrated genes appears to be very tightly controlled, leading to undetectable protein expression in the uninduced state. So once our engineered bacteria are consumed as auxiliary starters in the yogurt fermentation, they will not express these genes modulated by PnisA, since the inducer nisin has been destroyed during digestion.

Plasmid construction

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We have carefully considered the functions of these genes involved in AI-2 response, antibiotic resistance of expression vectors and plasmid incompatibility before transformation. In order to achieve the final goal of constructing the AI-2 response pathway of Salmonella in our engineered bacteria, we chose 3 types of plasmids with different replication origins and different antibiotics so that they can replicate in one host cell and provide convenience for screening post transformation. To visualize AI-2 existence, we chose a reporter gene, amilCP (BBa_K592009), from previous registered parts. This adds up to 7 plasmids for essential parts of AI-2 response system: (1) pNZ8148 is used to express lsrB, R and K; (2) pBBR1MCS-5 is used to express lsrA, C and D; (3) pHY300PLK is used to express the blue pigment, which is under the control of Plsr. We linearized these expression vectors and proceeded to stably integrating them into the genome of the host bacteria.

pNZ8148-lsrB, R, K

We chose pNZ8148 for the expression of those three genes. The replicon of the vector pNZ8148 is originally from the Lactococcus lactis plasmid pSH71. However, this replicon has a broad host range. Apart from Gram-positive bacteria, pNZ8148 can also replicate in E. coli, but require a recA+ strain like MC1061. It is chlorampenicol resistant.

pBBR1MCS-5 - lsrA, C, D

In Salmonella, AI-2 response involves an ATP binding cassette transporter. lsrC and lsrD encode the membrane channel proteins, and lsrA encodes the ATPase that provides energy for AI-2 transport.

Those three coding sequences were first inserted in pNZ8148. Afterwards, we used PCR to isolate them together with the upstream nisA promoters. And then we used double digestion and ligation to pBBR1MCS-5 to construct those three vectors.

pHY300PLK-plsr- amilCP

First, we isolated the promoter sequence of the plsr operon from the genomic DNA of Salmonella. Second, we isolated the coding sequence of amilCP together with the terminator from pET-14b, which was constructed by our iGEM team last year. After that, we spliced these two parts using SOE-PCR. Finally, the product was inserted to pHY300PLK using double digestion and ligation.

 

Our parts are designed for the construction of the AI-2 response pathway in the engineered bacteria. The following Biobrick parts have been submitted to the registry:

 

BBa_K1666000: This is a coding region part for lsrA. LsrA is a part of the ABC transporter complex involved in autoinducer 2 (AI-2) import and responsible for energy coupling to the transport system.

BBa_K1666001: This is a coding region part for lsrB. LsrB is a solute-binding protein that can bind AI-2 specifically and help to transport the AI-2 into the cytoplasm.

BBa_K1666002 and BBa_K1666003: LsrC and LsrD are parts of the ABC transporter complex LsrABCD, probably responsible for the translocation of the substrate across the membrane.

 

BBa_K1666004: This is a coding region part for lsrK. LsrK catalyzes the phosphorylation of autoinducer 2(AI-2) to phospho-AI-2, which subsequently inactivates the transcriptional regulator LsrR and leads to the transcription of the lsr operon.

BBa_K1666005: This is a coding region part for lsrR. In the absence of autoinducer 2 (AI-2), LsrR represses transcription of the lsr operon and itself. Phospho-AI-2 can bind LsrR and inactivate it through releasing it from the repressed promoters, leading to the transcription of the lsr genes.

BBa_K1666006: This is an inducible promoter. Plsr is the promoter of the lsr operon. It is under the repressive regulation of LsrR. In our project, we use it to regulate the expression of the reporter gene, amilCP (BBa_K592009).

Protocols

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The microbiological test of Lactobacillus in yogurt (GB/T16347-1996)

1. Put 25 ml full-shaked sample into sterilized wide mouth bottle containing 225 ml sterile saline aseptically to make uniform dilution of 1:10.Samples are selected for the same brand of yogurt which date 4, 10, 20 days,and expired one day.

2. Suck up 1ml 1:10 dilution with 1ml sterile pipette and inject it slowly into a test tube containing 9 ml sterile saline along the tube wall (note not to touch the tip of the pipette tube dilution).

3. Increase by 10-fold dilution increments every time, that is replaced with a 1 ml sterile pipette according to the above steps. So it is total diluted 10^-15.

4. Choose dilutions from 10^-6 to 10^-15 and suck up 1 ml dilution into sterile plates respectively while doing the 10-fold dilution increments. Make two plates each dilution.

5. Inject 15 ml Lactobacillus count medium (modified MC) which was cooled to 50℃ into the plate as soon as the dilutions was shifted into the plate. Rotate it to mix them. Meanwhile, to make a blank comparison, pour the count medium of Lactobacillus into a sterile plate containing sterile saline which is used to test 1 ml dilution. The whole process including adding the culture to the plate to finishing pouring should be done within 20 minutes.

6. Invert the plate and put it into a 36 ± 1℃ incubator for 72±3 hours after the agar has set. Observe the lactobacillus in the plate, select colonies between 30 to 300 and count them. After the calculation, the colonies are randomly taken the Gram stain: (1) fix the smear.(2) stain for 1 min with ammonium oxalate crystal violet. (3) wash with running water.(4) add iodine to cover approximately 1 minute. (5) wash with water and absorb the water with absorbent paper. (6) add a few drops of 95% alcohol and gently shake to decolorize it. Wash with water after 20 seconds and absorb the water. (7) stain with fan red for 1 minute,wash it with running water, dry it and then take microscopic examination. Gram-positive bacteria are blue-purple and gram-negative bacteria are red.

7. Do the catalase test: pick up a colony from the solid media into a clean tube, drop 2 ml 3% hydrogen peroxide solution and observe. Those who has bubbles in 30 s are positive, the others are negative.

8. Results identification: The Lactobacillius can be identified according to the following fades: gram-positive, catalase-negative, non-spore sphaerita or bacillus. Calculate the number of Lactobacillus in one plate and multiply the dilution and then we get the number of Lactobacillus of per milliliter of the sample.

 

Table1  The colony morphology of lactobacillus in the modified MC medium

The Most Probable Number(MPN) Method of Coliform bacteria in yogurt(GB/T 4789.3-2008)

1. Sample Preparation:

    (1)Put 25 ml full-shaked sample into sterilized wide mouth bottle containing 225 ml sterile saline aseptically to make uniform dilution of 1:10.Adjust pH to 6.5~7.5 with 1M NaOH or 1M Hcl. Samples are selected for the same brand of yogurt which expiration date, expiration date 35℃ 0.5h,and expired one day.

    (2)Suck up 1ml 1:10 dilution with 1 ml sterile pipette and inject it slowly into a test tube containing 9 ml sterile saline along the tube wall (note not to touch the tip of the pipette tube dilution).

    (3)Increase by 10-fold dilution increments every time, that is replaced with a 1 ml sterile pipette according to the above steps. The whole process including adding the culture to the plate to finishing pouring should be done within 20 minutes.

2. Fermentation Test:

    Sample Dilution: 10^-1, 10^-2, 10^-3, respectively. Inoculate each 1 ml Sample to 3 tubes of LST. Put them into a 36 ± 1℃ incubator for 24±2 hours. Observe gas producing situation in tubes. If no bubble produced, continue incubate to 48 h±2 h.Record the number of producing bubble tubes in 24h and 48h. (No gas producing tubes are Coliform bacteria Negative. Gas producing tubes carry out next test.)

3. Refermentation Test:

Inoculate all Gas producing tubes in 48h ± 2h to BGLB tubes. Put them into a 36 ± 1℃ incubator for 48 ± 2h.Observe gas producing situation in tubes. (Gas producing tubes are Coliform bacteria Positive.)

4. The MPN of Coliform bacteria report:

    According to the number of Coliform bacteria Positive tubes, search MPN Key. Report the MPN of Coliform bacteria in per ml of sample.

 

Table2 The MPN Key of Coliform bacteria in per ml (g) of sample

Media Component (g/L) , 25℃ 

1. Modified Chalmers Agar (MC):

 

2. Lauryl Sulfate Tryptose Broth (LST) :

 

3. Brilliant Green Lactose Bile Broth (BGLB):  

Genome Extraction
For bacterial genome extraction we used TIANamp Genomic DNA Kit according to manufacturer's instructions.

Plasmids extraction
For bacterial plasmid extraction we used EndoFree Maxi Plasmid Kit and TIANprep Midi Plasmid Kit. 

Gel Extraction 
We used TIANgel Midi Purification Kit according to manufacturer's instructions.

AI-2 Quantification

1. E.coli and Bacillus were inoculated into LB media (2%, v/v), respectively, and shaken overnight at 37°C.

2. The bacteria were centrifuged at 6000 g for 3 min. The supernatant was collected and filtered through a 0.22μm membrane

3. Preparing the working solution: A working solution of 10 mM 1, 10-phenanthroline/3.32 mM Fe (III) was prepared by dissolving 0.198 g of 1,10-phenanthroline in 50 ml of deionized distilled water. The solution was adjusted to pH 2 using 1M HCl. Ferric ammonium sulphate (0.16g) was added and the solution was brought to 100 ml using deionized distilled water.

4. For the Fe (III) ion reduction test, 1ml of the cell free supernatant was mixed with 1 ml working solution to develop the full color. The solution was then diluted to 5ml and filtered through a 0.22 μm membrane，followed by scanning for the absorption spectrum against a blank solution within 3 min using a Lambda 25 UV/VIS spectrometer. 

 

Transformation by electroporation

1. Inoculate 100 μl bacterial culture into 50 ml of MRS and incubate the bacteria at 37°C overnight. 

2. Harvest the cells by centrifugation.

3. Washed the bacteria three times with cold electroporation buffer (PB).

4. Resuspend the cells in PB to an OD₆₀₀ of about 50.

5. Mix 100 μl electrocompetent cells with 10 μl plasmid DNA.

6. Incubate the cuvettes for electroporation on ice and the above mix as well.

7. Subject the sample  to a 2.4 kV, 200 Ω, 25 μF electric pulse.

8. Add 950 μl SMRS as quick as possible.

9. Incubate for 2 h at 37°C.

10. Plate on MRS supplemented with the appropriate antibiotic. 

11. Incubate the plates at 37°C for 2 to 3 days under anaerobic conditions.

12. Use isolated colonies to check the correct insertion.

 

Functional identification of the engineered bacteria
1. Inoculate E. Coli CD-2 and E. Coli DH5alpha into 100 ml medium, 180 rpm, incubate for 8 hours.

2. Centrifuge the culture to harvest the supernatant.

3. Pass the liquid through a 0.22 μm filtering membrane.

4. Add the supernatant to the culture medium of Lactobacillus.

5. Add nisin to the final concentration of 50 ng/ml.

6. Incubate the engineered Lactobacillus overnight.

7. Centrifuge at 6000 rpm for 5 min. 
 
1. Incubate E. Coli CD-2 and E. Coli DH5alpha on LB agar plate.

2. Wash the colonies with fresh MRS and centrifuge the colony wash to harvest the supernatant.

3. Pass the liquid through a 0.22 μm filtering membrane.

4. Add the supernatant to the culture medium of engineered Lactobacillus.

5. Add nisin to the final concentration of 50 ng/ml.

6. Incubate the engineered Lactobacillus overnight.

7. Centrifuge at 6000 rpm for 5 min. 

 

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