Team:NEFU China/fit
Team:NEFU China/Description
Background
Yogurt, as one of the oldest and most ordinary fermented foods, is known all of the world. However, the yogurt section practically taken over the dairy aisle of most grocery stores. Differ from other traditional dairy products(cottage cheese, sour cream and etc.), yogurt wins itself the most popularity due to the many health benefits that it can gives.
And, what kinds of health benefits of yogurt?
Primarily, everyone needs to have a healthy amount of ''good'' bacteria in the digestive tract, and many yogurts are made using active, good bacteria. And then, let us notice that yogurt comes from milk and is as well nutritionally rich in protein, calcium, riboflavin, vitamin B6 and vitamin B16, but it has nutritional benefits beyond those of milk, namely due to its probiotics. In fact, the health benefits of yogurt are so impressive that many health-conscious people make it a daily habit. Here are five possible health benefits of having a yogurt a day:
(1) Yogurt may help prevent Osteoporosis,
(2) Yogurt may reduce the risk of high blood pressure.
(3) Yogurt with active cultures helps the gut
(4) Yogurt with active cultures may discourage vaginal infections
(5) Yogurt may help you feel fuller
In spite of this health benefits, spoiled yogurt is more harm than good if you carelessly eat it. Firstly, let us talk about the shelf-life of yogurt. Yogurt provides two significant barriers to pathogen growth (heat and acidity) to ensure a safe product. Discard batches that fail to set properly, especially those due to culture errors. Yogurt generally has a 10-21 day self-life when made and stored properly in the refrigerator below 40¡ãF. Then let we look into yogurt manufacturing process:
(1) pasteurization
(2) combine ingredients
(3) inoculate
(3) incubate
(4) refrigerate
The first step pasteurization is used for sterilization, but the pathogenic bacteria like E.coli, salmonella and bacillus still possibly survive from the first step that can spoil the yogurt during prolonged storage.
However, from the view of the premise of self-life is numerous and strict, is self-life really reliable? On the one hand, there are too many reasons like long-time transport can not make sure yogurt is stored in a 40¡ãF environment. On the other hand, when you do yogurt yourself, you should also need to make sure the use of clean equipment and containers. To make matter worse, if yogurt spoiled within expiration date you can not tell them apart, for their appearances were almost the same.(illustrated by fig.1)
fig.1(those nine photos of yogurt with a same production date were taken maintain a two days interval. Those yogurt were kept in room temperature, and their packing were open only when took photos )
This is for sure, the pathogenic bacteria can be detected. We then did an experiment based on our national food safety standards. (protocols click here)
Needless to say, no one would willing to eat yogurt with such amount of coliform if he or she had see our results. However, considering the test method containing cumbersome steps, costing much time and requiring certain experimental apparatus, it on no condition can be use in daily life. That is to say, if we don¡¯t do something we could still eat yogurt that maybe contain much bacteria we don¡¯t want.
Now, a handy detector of pathogenic bacteria is really needed.
Design:
1.Introduction of AI-2
2.Why we choose AI-2
3.Chassis(why we choose them)
4.The system we will construct
5.NICE system
6.Plasmid construction
7.Quantification
Quorum sensing (QS) is a chemical communication system among bacterium,and QS requires the production and detection of specific signaling molecules,which are commonly referred to as autoinducers.Autoinducer-2 (AI-2) molecules are one class of signaling molecules involved in QS.AI-2 is a byproduct of the Activated Methyl Cycle,which recycles S-adenosyl-l-methionine (SAM).As a main methyl donor,SAM is converted to S-adenosyl-l-homocysteine (SAH).Then SAH is subsequently hydrolyzed to adenine and 4,5-dihydroxy-2,3-pentanedione (DPD) by the nucleosidase Pfs and the Luxs enzyme.Upon formation,DPD spontaneously cyclizes to form at least two different interspecies communication molecules described as AI-2.
Why we chose AI-2 as signaling molecule,that¡¯s because the production of AI-2 is the only quorum-sensing mechanism shared by both Gram-positive and Gram-negative bacteria.There are many pathogens in spoiled yogurt, Ecoli, Salmonella and Bacillus are the main part.Then AI-2 can be used for a universal language among pathogens.Besides,AI-2 response pathway of our chassis, lactobacillus and Lactococcus, is different from that kinds of pathogens.
We choose lactobacillus and Lactococcus as our chassis.As beneficial bacteria, they are food grade and are widely used in food production for thousands of years.Lactobacillus can improve nutrient absorption and human gastrointestinal function.They can also inhibit the reproduction of pathogenic bacteria in guts. Lactococcus lactis is a Gram-positive lactic acid bacterium that is widely used in food fermentations, such as in cheese and butter production.
LuxS converts SRH to the precursor of AI-2,DPD,in the cytoplasm.DPD then undergoes spontaneous cyclization and expert to the culture supernatant.Depending on the bacteria,response to AI-2 can follow one of the two currently identified routes.In pathogens exemplified by Salmonella, AI-2 is bound to a homologue of the periplasmic binding protein LsrB.LsrB is part of an ABC transporter(encoded by the lsrACDBRK operon).This operon is regulated by the repressor LsrR,the gene of which is located upstream. Immediately downstream of lsrR is a gene (lsrK). LsrK phosphorylates AI-2, Phosphorylated AI-2 then causes LsrR to relieve its repression of the lsr operon, allowing further AI-2 import.
The nisin-controlled gene expression system NICE of Lactococcus lactis is one of the most widely used expression systems in Gram-positive bacteria.Biosynthesis of nisin is encoded by a cluster of 11 genes, NisR and NisK belong to the family of bacterial two-component signal transduction systems.NisK is a histidine–protein kinase that resides in the cytoplasmic membrane and is proposed to act as a receptor for the mature nisin molecule. Upon binding of nisin to NisK, it autophosphorylates and transfers the phosphate group to NisR, which is a response regulator that becomes activated upon phosphorylation by NisK. Activated NisR* induces transcription from two of the three promoters in the nisin gene cluster: PnisA and PnisF. In our parts,we use inducible promoter PnisA to express gene A¡¢B¡¢C¡¢D¡¢R¡¢K,the inducer nisin¡¢gene nisR¡¢nisK are necessary for PnisA.
Plasmid construction
¢ÙGene A¡¢B¡¢C¡¢D¡¢R¡¢K ¡¢plsr was cloned from Salmonella typhimurium
¢ÚPlasmid pNZ8148 served for amplifying gene B¡¢R¡¢K respectively
Plasmid pBBR1MCS-5 served for amplifying gene A¡¢C¡¢D respectively
We replace promoter of pET-14b with plsr,The final construct called pHY300PLK contains a blue pigment gene.
¢ÛPlasmid pNZ8148(¡Á3)¡¢pBBR1MCS-5(¡Á3) and pHY300PLK were linearized and introduced by electroporation into our chassis.
¢ÜAs for eletroporation,we have two different routes.One of which is that seven linearized palsmid and pNZ9530(Low copy plasmid with pAM¦Â1 origin of replication, which carries the nisR and nisK genes.) were introduced into lactobacillus.Or seven linearized palsmid containing gene A¡¢B¡¢C¡¢D¡¢R¡¢K¡¢Blue pigment were introduced into Lactococcus NZ9000(containing nisR and nisK)by eletroporation.
The molecular structure of AI-2 is somewhat similar to ascorbic acid. It was expected that AI-2 would also act as a reducing agent and reduce Fe(III) ions in the presence of 1,10-phenanthroline to form the colored [(o-phen)3 Fe(II)]SO4 ferroin complex that could be quantified colorimetrically.