Overview

The project

I have a dream
I am happy to join with you today in what will go down in history as the greatest demonstration for freedom in the history of our nation.

Five score years ago, a great American, in whose symbolic shadow we stand today, signed the Emancipation Proclamation. This Momentous decree came as a great beacon light of hope to millions of Negro slaves who had been seared in the flames of withering injustice. It came as a joyous daybreak to end the long night of their captivity.

But one hundred years later, the Negro still is not free. One hundred years later, the life of the Negro is still sadly crippled by the manacles of segregation and the chains of discrimination. One hundred years later, the Negro lives on a lonely island of poverty in the midst of a vast ocean of material prosperity. One hundred years later, the Negro is still languishing in the corners of American society and finds himself an exile in his own land. So we have come here today to dramatize the shameful condition.

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Background

Yogurt is manufactured using a culture of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus salivarius subsp. thermophilus bacteria. This fermented product is nutritionally rich in protein, calcium, riboflavin, vitamin B₆ and vitamin B₁₂^[1]. In addition, consumers who are moderately lactose-intolerant can consume yoghurt without ill symptoms, because much of the lactose in the milk precursor is converted to lactic acid by the bacterial culture^[2]. Yoghurt containing live cultures is sometimes used in an attempt to prevent antibiotic-associated diarrhea. Yoghurt contains varying amounts of fat. There is non-fat (<0.5% fat), low-fat (usually 2% fat) and plain or whole milk yoghurt (4% fat). A study published in the International Journal of Obesity also found that the consumption of low-fat yoghurt can promote weight loss, mainly due to the abundance of calcium in the yogurt^[3].

About post-acidification

Although yogurt possesses many health benefits there exists a major problem in yogurt manufacturing and during storage prior to consumption, i.e., post-acidification. As bioactive ingredients, Lactobacillus bulgaricus and Streptococcus thermophilus continue to produce lactic acid after production fermentation, making the yogurt too sour. This phenomenon is not desirable. Post-acidification shortens yogurt’s shelf life results in an unacceptable taste by consumers. Therefore, the objective of this project is to minimize post-acidification in yogurt achieving a consistent acidity (pH) and a prolonged shelf-life.

About collagen

Collagen is well known to consumers, especially to female consumers. Collagen is one of the long, fibrous structural proteins, whose functions are quite different from those of globular proteins such as enzymes. Tough bundles of collagen called collagen fibers are a major component of the extracellular matrix that supports most tissues and gives cells structure from the outside. Collagen is also found inside certain cells. Collagen has great tensile strength and is the main component of fascia, cartilage, ligaments, tendons, bone and skin^[4]. Along with soft keratin, it is responsible for skin strength and elasticity, and its degradation leads to wrinkles with aging. It strengthens blood vessels and plays an important role in tissue development. It is present in the cornea and lens of the eye in crystalline form. Hydrolyzed collagen can also play an important role in weight management, as a protein, it can be advantageously used for its satiating power. Thus it is of great interest in producing a yogurt rich in collagen.

Outline

In order to achieve our goals, we have designed four new biobricks. With these biobricks, three steps are needed to minimize the process of post-acidification and produce collagen in yogurt. First, a food-grade vector is being designed. Secondly, biobricks for minimizing postacidification are being reconstructed with a food-grade vector and the recombinant plasmid will be transduced into Lactobacillus bulgaricus. Thirdly, the food-grade vector reconstructed with the genes coding a section of human collagen will be transduced into Streptococcus thermophilus.

Food-grade vector

The proposed food-grade vector is based on the plasmid pMG36e (FIG 1.1), which has the erythromycin resistance gene from Staphylococcus aureuis. The plasmid, pMG36e, functions in a wide range of bacteria [5]. These include E. coli, B. subtilis and especially Lactococcus lactis. But for a food-grade vector, we need to change the antibiotic gene to a safer resistance gene to avoid potential risks. A Nisin resistance gene nisI becomes our interest. Nisin is an antimicrobial peptide produced by Lactococcus lactis. It has a long history of safe use in food production [6]. So we insert the nisI gene between XbaI and PstI. And then, we cut off the erythromycin resistance gene using primers with EcoRI in both ends [7] (FIG 1.2 and FIG 1.3). For a better function, the coding of nisI gene has being optimized separately in L. bulgaricus and S. thermophilus.

Devices for postponing the process of postacidification

Yogurt will become more and more acid for the two strains, L. bulgaricus and S. thermophilus, keep producing lactic acid all the time, especially L. bulgaricus. To reduce the production of lactic acid, L. bulgaricus needs to perform two functions. First, it should have a pH sensor to sense the external pH value and release a signal when the pH value declines to 5.5 or lower. Then, with the signal, L. bulgaricus should cut off the pathway for lactic acid.

pH sensor

Although L. bulgaricus is one of the most extensively studied lactic acid bacteria, little information is available on the acid induced gene expression. The rcfB promoter is the most recently reported gene which is highly induced by acidity. When the external pH value declines to 5.5, rcfB promoter will be highly upregulated [8]. Although, the rcfB gene, encoding RcfB protein which has unknown function, is found in Lactococcus lactis IL1403, we still try to figure out whether it will work in L. bulgaricus.

Repressor

Two important enzymes are needed for the lactic acid bacteria to convert lactose to glucose and then lactic acid. They are lactose permease and β-galactosidase which are located in the lac operon (FIG 2.1). In L. delbrueckii subsp. lactis, the lactose permease (lacS ), the β-galactosidase (lacZ ) and the repressor (lacR ) constitute the lac operon per se. The three genes are linked together as a lacSZR operon without any promoter in between. The repressor is able to bind to the promoter region which is located upstream the lacS gene. Thus the transport of the lactose is being blocked. However, in L. delbrueckii subsp. bulgaricus, the lacR gene was inactivated by small nucleotides insertions and deletions in the sequence resulting in the constitutive phenotype of this subspecies (FIG 2.2)_[9]. So, the normal express of lacR would cut off the pathway for lactic acid production. The overview of the pH sensor and repressor is shown in FIG 2.3.

Diagram for producing collagen

Our collagen gene contains a coding region encoding 101 amino acids, which comes from the α1 chain of human collagen type Ⅲ (ColⅢ), a signal peptide called blpC and a His tag (FIG 3.1). The gene blpC, which is found in Streptococcus thermophilus CNRZ1066, will transport the collagen out of the bacteria _[10]. And the His tag is for the purification of the collagen.

Team:HIT-Harbin/Project

From 2011.igem.org

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