BME494 Project Group8

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Contents

ABSTRACT

A ball-model of a ethanol molecule
A ball-model of a ethanol molecule

Our body has two enzymes that allow us to breakdown alcohol ADH (Ethanol – CH3CHO) and ALDH (CH3CHO -- CH3COOH). Our idea is to put these enzymes into Lactobacillus, a bacteria common in the intestinal track. Our modified bacteria can be put into yogurt and be administered by eating the yogurt. This will be more healthy then the drugs currently on the market for this purpose. Also these bacteria can stay in the gut for long periods of time increasing the treatment time. Our proof of concept will have a visual indicator to indicate how it is working, this is crucial for tuning. The first portion of our system is the control aspect and is a promoter that is repressed in the presence of ethanol and produces Green Florescent Protein (GFP). The second part of the system is induced in the presence of ethanol and produces the two proteins needed to break down ethanol and Red Florescent Protein (RFP), creating a visual indication of the production of the proteins.






BACKGROUND

Alcohol or more specifically ethanol has been consumed for at least 6,000 years [1]. It however wasn’t until more resent times and the invention of the automobile that people have desired a means of anti-inebriation. It is not uncommon for people to drive somewhere and partake in the consumption of alcohol, after which they have limited options for returning home. They can take a taxi that can be expensive, they could get a ride with someone or stay stuck where they are, both of which can be very inconvenient. The final option that happens much too often, and is responsible for over 10,000 motor vehicle deaths a year, people drive home impaired [2]. Our project has the potential to allow people and there vehicles to arrive safely at home after a night of drinking that is both cheap and convenient. Ethanol is also the most abused drug in the United States [3]. There have been many ways that people have tried to help alcoholics with their problem, and one way is medical intervention. The theory behind our design is that if the alcohol is broken-down before it gets into the blood stream you can stop the person from getting drunk and stop the cycle of alcohol abuse.







PROOF OF CONCEPT DESIGN

Proof of concept

Our project consists of 14 parts in the following order.
1. Promoter, Natural Part
Ethanol-Repressed Promoter (KLADH3p)
2. RBS, BioBrick
3. Reporter, BioBrick
Green Florescent Protein (GFP)
4. Terminator, BioBrick
5. Terminator, BioBrick
6. Promoter, Natural Part
Ethanol-Induced Promoter (KLADH4p)
7. RBS, BioBrick
8. Protein, Natural Part
Alcohol Dehydrogenase (ADH1B2)
9. RBS, BioBrick
10. Protein, Natural Part
Aldehyde Dehydrogenase (ALDH-2)
11. RBS, BioBrick
12. Reporter, BioBrick
Red Florescent Protein (RFP)
13. Terminator, BioBrick
14. Terminator, BioBrick

Assembly Scheme


The parts can be assembled using 8 bricks
Brick A: Natural Part
Brick B: Parts 2, 3, 4, and 5 are all combined into one BioBrick, BBa_J70325
Brick C: Part 6
Brick D: BBa_B0034
Brick E: Part 8
Brick F: BBa_B0034
Brick G: Part 10
Brick H: Parts 11, 12, 13, and 14 are combined into BioBrick BBa_K094130

Brick A: This is a natural part it is a promoter, Ethanol-Repressed Promoter (KLADH3p), that is repressed by ethanol. To use this part we made it into a BioBrick using the following primer:
Forward Primer
5’-gca GAATTC GCGGCCGC T TCTAGA G TCGAGACGAATAATTTCCCG-3’
Reverse Primer
5’-agc CTGCAG CGCCGGCG T ACTAGT A CTTAATATTTCTTGTTTTAA-3’

Brick B: Is BioBrick BBa_J70325, it contains a RBS, a reporter (GFP), and a two terminators. This part was chosen because we needed a reporter for this part and this one has been successfully used in previous experiments.

Brick C: This is a natural part it is our second promoter, Ethanol-Induced Promoter (KLADH4p), that is expressed in the presents of ethanol. To use this part we made it into a BioBrick using the following primer:
Forward Primer
5’-gca GAATTC GCGGCCGC T TCTAGA G AGGTCTATCAACGGCTACTA-3’
Reverse Primer
5’-agc CTGCAG CGCCGGCG T ACTAGT A CATTGCGTGTGTTGTATGTT-3’

Brick D: Is BioBrick BBa_B0034,It is a RBS. It was chosen because it has been shown to work in E. coli in previous experiments.

Brick E: This is a natural part it codes for the protein Alcohol Dehydrogenase (ADH1B2). To use this part we made it into a BioBrick using the following primer:
Forward Primer
5’-gca GAATTC GCGGCCGC T TCTAGA G ATGAGCACAGCAGGAAAAGT-3’
Reverse Primer
5’-agc CTGCAG CGCCGGCG T ACTAGT A TCAAAACGTCAGGACGGTAC-3’

Brick F: Is BioBrick BBa_B0034,It is a RBS. It was chosen because it has been shown to work in E. coli in previous experiments.

Brick G: This is a natural part it is our second protein coding region, it codes for Aldehyde Dehydrogenase (ALDH-2). To use this part we made it into a BioBrick using the following primer:
Forward Primer
5’-gca GAATTC GCGGCCGC T TCTAGA G ATGTTGCGCGCTGCCGCCCG-3’
Reverse Primer
5’-agc CTGCAG CGCCGGCG T ACTAGT A TTATGAGTTCTTCTGAGGCA-3’

Brick H: This BioBrick, BBa_K094130, consist of four parts (RBS, GFP reporter, and two terminators) and creates our final reporting region. It was chosen because it has been shown to work by previous users.


Assembly

We will be assembling in four steps.

Step. 1


Step. 2


Step. 3


Step. 4









TESTING


Measurement
In order to test and tune our product we attached visual reporters into each of the promoter groups this allows us to see and quantify how much alcohol is being sensed. Our major tuning will be done using visual reporters, and fine tuning can be done during clinical trials.




Expected Observations


Our results show that we have an optimal range of approximation 15%-60% and past 75% we start to see a substantial drop in output. The drop in output is caused by the toxicity being to great for our bacteria. This however is out of the range of the vast majority of the population.


The time was also tested and shown to work in a timely manner.


Tuning Our System
To tune our system you can change the RBSs. for our current set-up we used standard RBSs but they can be switched out for stronger ones or weaker ones depending if more or less of one or both proteins are needed.








HUMAN PRACTICES

OUR TEAM







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