# Team:HFLS H2Z Hangzhou/Applied Design

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Jianan Li
Qingrui Sun
Yiming Rong
Jiayue Guo
Zhiyuan Lu
Meiqi Yuan
Zhengyao Lin
Caiyi Feng
Shuyun Zhang
Tenghao Huang
Yining Huang
Yanyue Zhu

Design

# Industrial Specifics

After visiting Qiumei Food Corporation, we compiled out information about pickle production process to give a general idea of the industry. The fellowing is written in a worker's perspective for rhetorical purposes.

As a worker in Qiumei Food Corporation, I recount on every process of pickle production to give you a more holistic view of how the pickles in our everyday life is made step by step. The first step to do is to harvest mustard—the fundamental ingredient of pickles. We have our own vast field grown solely Chinese mustard( it is not the US style mustard; it's much more like lettuce). Click the picture for further information.

# Parameters

To solve this industrial specific problem, we listed several parameters to meet.

## Goal one

Our final product must be environmental friendly, any toxic residue that remained in the food is unacceptable.

## Goal two

The product must work faster than the nitrite's natural degradation rate in pickles. We set the maximum days for the enzyme to turn nitrite content under Chinese FDA's standard to be Three Days.

## Goal three

Our chassis selection must be wise, or it will be impractical to applied in industry (unable to convince consumers and FDA officers that our product is safe).

## Goal four

The device must be regulated: because we expecting our device to express several proteins(or one fusion protein) the constitutive promoter will cause too much pressure on the bacteria, and may even cause the bacteria not functional at all.

## Goal five

The product should work under pickle's storage temperature, because it will not be worth it if the Qiumei Food changed the storage temperature leading changed taste just to use our product.

# Our designs

## Design concerning goal one

See our Enzyme page, we have selected three target enzymes to work with: NiR(catalyzing reaction of Nitrite ions to Nitrogen Monoxide), NOR(catalyzing reaction of Nitrogen Monoxide to Nitrous Oxide), nosZ(catalyzing reaction of Nitrous Oxide to Nitrogen and Oxygen).

We believed the enzyme selection will form a reaction pathway: from NO2- to completely harmless nitrogen and oxygen. This is also know as substrate channeling.

Substrate channeling is the passing of the reaction intermediate of one enzyme directly to the active site of another enzyme without release into the bulk solvent. The use of molecular tunnels for the passage of reaction products provides a mechanism for the protection of reactive intermediates from the external medium, and the transit time for the movement is reduced. Moreover, it could prevent the release of unstable intermediates.
The substrate channeling of our project is as shown in the figure: Furthermore, as can be seen in Enzyme page, the reaction rate of our first enzyme NiR(Nitrite Reductase) in the reaction pathway. The first enzyme NiR has much slower reaction rate compared with other two enzymes, so NiR becomes a limiting factor in the system which is perfect: its following enzymes can spontaneously process its product.

## Design concerning goal two

As can be seen in our Performance page, our fusion protein NzFE(Nitrite Reduction Fusion Protein) when expressed in chassis $E.coli$ BL21 DE3, is ultimately able to eliminate 41%(in 16 °C for 39h) and 46%(in 25 °C for 39h) of nitrite in pickle extract.

## Design concerning goal three

Although to fully analyze our three target enzymes reactivities, we first use $E.coli$ as our chassis. Fully aware of the fact this is not a good choice for final product, we researched possible chassises for final product, and decided on using $B.substilis$. See Chassis Selection.

## Design concerning goal four

Since our trip to Qiumei Food suggested that the company keeps their storage in open air to dry their vegetables after fermentation, it is best we choose promoter responsive in aerobic conditions. We inquired the CEO of Qiumei Food whether it is technically feasible to place the vegetable after fermentation in anaerobic without affecting the quality of the product, and while she said it is completely possible, we realized the metabolic product of our chassis under anaerobic condition (eg. alcohol for yeast, lactic acid for $L.bacillus$) will change the favor of Daodie Chai(Qiumei's pickle product).

For all these reasons, we chose promoter Pyear, see our Genetic Circuit page. Pyear is the only $E.Coli$ promoters responsive to nitrate and nitrite under aerobic conditions, and to test its characteristics, we constructed several sensors.

The promoter is constitutively repressed by protein NsrR, and will open for transcription if any of nitrite, nitrate, nitric oxide is present. This is perfect for our design: though nitrite is not sufficient to activate the transcription, there is always enough nitrate in the pickles.

## Design concerning goal five

Qiumei Food's storage temperature is 25 °C, and our fusion enzyme working under this condition performed the best compare to in 16 °C and 30 °C.

# Reference

 1 Huang X, Holden H M, Raushel F M. Channeling of Substrates and Intermediates in Enzyme-Catalyzed Reactions[J]. Annual Review of Biochemistry, 2001, 70(1):149. 2 Lin H, Bledsoe P V. Activation of yeaR-yoaG operon transcription by the nitrate-responsive regulator NarL is independent of oxygen- responsive regulator Fnr in $E. coli$ K-12.[J]. Journal of Bacteriology, 2007, 189(21):7539.

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