Help! Some Project Ideas on Ethanol Production.

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Help! Some Project Ideas on Ethanol Production.

Postby cicichen » Mon Oct 22, 2007 2:45 pm

I had posted some questions asking what topic I should focus on based on different project ideas I had. And thanks for the people leading me to ethanoal production field!

I'm interested in different cellulose degraders (bacteria, fungi, etc) and want to know how efficient they are. I actully found some interesting cellulose degraders. The JGI (Joint Genome Institute) has started sequencing the bottom mushroom in order to "identify superior enzymes and reactions that could be incorporated into fermentation organisms ".
(http://www.technologyreview.com/Energy/18958/)

I also found another article saying that: "Cytophaga hutchinsoini was originally isolated from sugarcane piles. This microorganism therefore probably produces an array of enzymes allowing it to digest cellulosic substrates. C. hutchinsonii thus represent a rich source of potentially effective cellulase ezymes that can be harnessed for consversio of biomass to simple sugars."

I think that would be interesting to test these natural cellulose degraders and maybe sequence their genes.

I thought it would be cool if I can do a project to genetically engineer a bacteria so it can "breakdown and ferment cellulose biomass directl to ethanol" like the scientists in the article:

Researchers from the Universidad Nacional Autonoma de Mexico have genetically engineered the bacterium Bacillus subtilis to ferment glucose sugar directly to ethanol with a high (86%) yield. This is the first step in their quest to develop bacteria that can breakdown and ferment cellulose biomass directly to ethanol.

In a presentation at last week’s 106th General meeting of the American Society for Microbiology, the team members described their work in engineering the bacterium to produce ethanol as a single fermentation product from the sugars resulting from the breakdown of plant cellulose into simple carbohydrates.

With its very efficient secretion systems, B. subtilis is widely used in the production of extracellular hydrolytic enzymes, but it does not produce ethanol. The researchers engineered in the ethanol production capability by using the pyruvate decarboxylase (PDC) and alcohol dehydrogenase B (ADHII) from Zymomonas mobilis, a bacterium that does produce ethanol.

Initially, the team found that the integration of the ethanol synthetic pathways allowed ethanol production with a 50% yield, but with a large amount of butanediol still produced. Further tweaking avoided the butanediol formation, and bumped the ethanol yield up to 86%.

They found, however, that while the yield was high, the rate of production from the recombinant strains was low. Further work is necessary to increase the rate.

Beyond that, the next step is to engineer the bacterium to produce the cellulase enzymes that can also initially break down the stems and leaves into the simple carbohydrates required for fermentation.
(http://www.greencarcongress.com/2006/05 ... .html#more)

The bio ethanol production field seems very interesting yet challenging too. So far I'm wondering if my ideas are applicable or any suggestion on putting them into an experiment? Please give me some feedback or anything related in this area.

Thank you! :shock:

Cici
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Re: Help! Some Project Ideas on Ethanol Production.

Postby Louise » Mon Oct 22, 2007 3:28 pm

I think you are on the correct track (and what you propose is what many researchers are doing). I cannot recall where you are planning to work, or what resources you have, so some of your ideas may be overly ambitious for someone in high school. If you could find a college mentor working in this area, I think you could have a great time. What city are you in? [Don't give me any more detail than that; I just want to search for the research institutions near you.]

I read this article in the paper and I thought you might be interested in it, as it touches on several of your themes:

http://www.washingtonpost.com/wp-dyn/content/article/2007/10/17/AR2007101702216.html

Note: Washington Post articles are only free for 2 weeks after print publication, so this link may not work forever.

The bacteria in this article is very unique, as it performes both the breakdown and ethanol production and is found in nature.

Reading what you quoted, I don't think that the bacillus subtilis can break down cellulose (unlike c. hutchinsonii), only do the fermentation on the products of breakdown.
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Postby wildfirefox » Mon Oct 22, 2007 3:29 pm

CiCi:

You've hit an awesome research that scientists have been dying to master since the ethanol-methanol vs petroleum research projects released back in the 60's for environmental friendly fuel alternatives.

But to further improve the processing of biomasses (such as wastes, overstocks of food/agricultural materials) directly into ethanol products, skipping all the refinement processes, the technologies haven't gotten there yet based on the current researches. You've pounded your research straight on the head when you start mentioning about the splicing of the bacteria/fungi genetic materials to enable the breakdown of the biomasses more effectively. However, the genetic researches are still in their infancy states.

If you research a bit further, and compile together the necessary data, then create a cross compare-contrast charting on the data you've collected, you will literally "WOW" everyone. Try to have these in mind as you do the researches:

1. What's the efficiency of petroleum-based fuels vs. bio-fuels (ethanol-methanol)?

2. What are the benefits of petroleum vs. bio-fuels?

3. Which fuel source sustains a longer, greater, cleaner productivity? Look into "sustainable resources".

Due to the oil crises that the modern societies been facing, with wars and political controls that the countries fight over for because of crude oils, the researches on alternative fuels are more favored by the people now more than ever.

Do your best, and we're here to help you. Ask as many questions as you need to, and we'll do our best to help you out.

Nam
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Re: Help! Some Project Ideas on Ethanol Production.

Postby cicichen » Thu Nov 01, 2007 1:33 pm

Louise wrote:I think you are on the correct track (and what you propose is what many researchers are doing). I cannot recall where you are planning to work, or what resources you have, so some of your ideas may be overly ambitious for someone in high school. If you could find a college mentor working in this area, I think you could have a great time. What city are you in? [Don't give me any more detail than that; I just want to search for the research institutions near you.]

Louise



Thank you for asking! I live in the San Francisco bay area (city of Cocord). I don't know whether you are familiar with the Joint Genome Insititute (JGI), it is actually close to where I live. And since my science teacher has a second job there she introduced me a mentor. I talked about my project idea with my mentor because I wasn't so sure if I would be able to do this project. My mentor thinks it is doable, but I need to work hard on it.

Cici
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Postby cicichen » Thu Nov 01, 2007 2:11 pm

I talked with my mentor about my experimental design.

Here is what my mentor and I came up with:

My goal is to genetically engineer a type of fermentive yeast that produces ethanol so that it is capable of coverting cellulose to ethanol in a one-step process.

First I need to find out several genes that are able to together break down the structures in biomass and produce cellulase to convert cellulose.

Second I need to run a PCR to duplicate the genes

Then I will need to ligate the genes and do a knock in to insert the genes into the genome.

I will have two sets of yeast: the control yeast (no genetically engineering) and the yeast that have been inserted the cellulase produced genes.

I will feed both types of yeast with glucose source, then after a while I will change their food source to cellulose (maybe a form of liquid).

The objective is to see whether the genetically engineered yeast will live with the cellulose source, if it does, then it proves that the yeast can breakdow the cellulose (which is something they are not supposed to do)

My second objective is to measure the ethanol produce as bioproducts from the yeast and to determine its efficiency.

My third objective is to see how the genetic mutation (using UV light) affects the yeast and its ethanol productio.

Cici
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Re: Help! Some Project Ideas on Ethanol Production.

Postby Louise » Thu Nov 01, 2007 2:18 pm

cicichen wrote:
Louise wrote:I think you are on the correct track (and what you propose is what many researchers are doing). I cannot recall where you are planning to work, or what resources you have, so some of your ideas may be overly ambitious for someone in high school. If you could find a college mentor working in this area, I think you could have a great time. What city are you in? [Don't give me any more detail than that; I just want to search for the research institutions near you.]

Louise



Thank you for asking! I live in the San Francisco bay area (city of Cocord). I don't know whether you are familiar with the Joint Genome Insititute (JGI), it is actually close to where I live. And since my science teacher has a second job there she introduced me a mentor. I talked about my project idea with my mentor because I wasn't so sure if I would be able to do this project. My mentor thinks it is doable, but I need to work hard on it.

Cici


It is great that you have a mentor! It looks like the JGI has a large biofuels program. If you did not know about it, here is the website:

http://www.jgi.doe.gov/education/bioenergy/

I think this is a perfect place for you to do your project! I couldn't come up with a better place if I wanted to! I would recommend that you study your mentor's research and the research of the JGI as carefully as possible. You have a great opportunity here, and the more effort you put in the more you will get out of this project. Good luck, and keep us posted on how your project developes!

Louise
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Postby ChrisG » Thu Nov 01, 2007 2:32 pm

Hi Cici,
It sounds like a very exciting project and a great opportunity to work with researchers at JGI. Congrats!
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Postby Louise » Thu Nov 01, 2007 2:46 pm

cicichen wrote:I talked with my mentor about my experimental design.

Here is what my mentor and I came up with:

My goal is to genetically engineer a type of fermentive yeast that produces ethanol so that it is capable of coverting cellulose to ethanol in a one-step process.

First I need to find out several genes that are able to together break down the structures in biomass and produce cellulase to convert cellulose.

Second I need to run a PCR to duplicate the genes

Then I will need to ligate the genes and do a knock in to insert the genes into the genome.

I will have two sets of yeast: the control yeast (no genetically engineering) and the yeast that have been inserted the cellulase produced genes.

I will feed both types of yeast with glucose source, then after a while I will change their food source to cellulose (maybe a form of liquid).

The objective is to see whether the genetically engineered yeast will live with the cellulose source, if it does, then it proves that the yeast can breakdow the cellulose (which is something they are not supposed to do)

My second objective is to measure the ethanol produce as bioproducts from the yeast and to determine its efficiency.

My third objective is to see how the genetic mutation (using UV light) affects the yeast and its ethanol productio.

Cici


This sounds really good (I didn't see this post when I replied earlier). I'm not entirely sure what "cellulose (maybe a form of liquid)" means, since cellulose is a polymer and usually not a liquid. Do you mean using a short polymer that would be water soluble?

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Postby cicichen » Thu Nov 01, 2007 2:52 pm

Both my mentor and I think that the hardest part is to look for the genes that work.

There are several genes that I found are related to cellulase: Cel1, Cel2, Cel3 and Cel4.

I learned that the cel1 is the endo-1,4-β-glucanase gene from Arabidopsis thaliana, a small flowering plant that is widely used as a model organism in plant biology. Cellulose chains in the cell walls of plant tissue are cross-linked to one another by the molecule xyloglucan. The CEL1 protein breaks cellulose-xyloglucan links.

http://www.cbdtech.com/Technology.html

I read about cel genes and how the genes genetically egineered to yeast Saccharomyces cerevisiae. But I don't quite understand why they are cloingt the BGL1 gene and co-expressing it with the END1, CBH1, and CEL1 gene. All these genes are not naturally occur in the yeast, so did those scientists insert the END1 gene and END1 CBH1 CEL1 genes on seperate regions? Why do they choose those genes and put they together to produce cellulase complex?

Abstract
Saccharomyces cerevisiae produces several β-1,3-glucanases, but lacks the multicomponent cellulase complexes that hydrolyse the -1,4-linked glucose polymers present in cellulose-rich biomass as well as in haze-forming glucans in certain wines and beers. We have introduced into S. cerevisiae a functional cellulase complex for efficient cellulose degradation by cloning the Endomyces fibuliger cellobiase (BGL1) gene and co-expressing it with the Butyrivibrio fibrisolvens endo--1,4-glucanase (END1), the Phanerochaete chrysosporium cellobiohydrolase (CBH1) and the Ruminococcus flavefaciens cellodextrinase (CEL1) gene constructs in this yeast. The END1, CBH1 and CEL1 genes were inserted into yeast expression/secretion cassettes. Expression of END1, CBH1 and CEL1 was directed by the promoter sequences derived from the alcohol dehydrogenase II (ADH2), the phosphoglycerate kinase I (PKG1) and the alcohol dehydrogenase I (ADH1) genes, respectively. In contrast, BGL1 was expressed under the control of its native promoter. Secretion of End1p and Cel1p was directed by the signal sequence of the yeast mating pheromone -factor (MF1), whereas Cbh1p and Bgl1p were secreted using their authentic leader peptides. The construction of a fur1 ura3 S. cerevisiae strain allowed for the autoselection of this multicopy URA3-based plasmid in rich medium. S. cerevisiae transformants secreting biologically active endo--1,4-glucanase, cellobiohydrolase, cellodextrinase and cellobiase were able to degrade various substrates including carboxymethylcellulose, hydroxyethylcellulose, laminarin, barley glucan, cellobiose, polypectate, birchwood xylan and methyl--d-glucopyranoside. This study could lead to the development of industrial strains of S. cerevisiae capable of converting cellulose in a one-step process into commercially important commodities.

http://www3.interscience.wiley.com/cgi- ... 1&SRETRY=0

I would really like to know what those genes: BGL1, END1, CBH1, and CEL1 do and how do they work together?
And what's a β-1 4-glucose?

I don't really know a lot about genetics, so I really appreciate if some experts could help me on that. Thank you!

CIci
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Postby wildfirefox » Thu Nov 01, 2007 4:15 pm

CiCi, welcome to biotechnology 101. You're in a field where genetic recombination plays a critical roles in making the final product. In other words, we're playing a mixing-and-matching game. Here's a simpler explanation of the abstract.

cicichen wrote:Saccharomyces cerevisiae produces several β-1,3-glucanases, but lacks the multicomponent cellulase complexes that hydrolyse the -1,4-linked glucose polymers present in cellulose-rich biomass as well as in haze-forming glucans in certain wines and beers.


In other words, S. cerevisiae produces the necessary glucose-deconstruction enzymes to turn glucose into usable fuel source.

cicichen wrote:We have introduced into S. cerevisiae a functional cellulase complex for efficient cellulose degradation by cloning the Endomyces fibuliger cellobiase (BGL1) gene and co-expressing it with the Butyrivibrio fibrisolvens endo--1,4-glucanase (END1), the Phanerochaete chrysosporium cellobiohydrolase (CBH1) and the Ruminococcus flavefaciens cellodextrinase (CEL1) gene constructs in this yeast.


By using yeast, since yeast is a bacteria that easily adapt to genetic materials without dying within the first generation, they strategically place the needed genetic markers on a certain region of the yeast to enable it to break down the available materials in the biomasses easily. Think of the yeast as a ball, and you want the surround LEGO pieces to attach to the round ball. To do that, you have to glue some opposite pieces of LEGO onto the ball, so the ball can have the rest of the LEGO pieces attach to the ones that have been glued onto the ball. The glued LEGO pieces that are attached to the ball are BGL1 and CBH1. By having these two pieces attached to the yeast, the genes END1 and CEL1 can then be attached to the yeast with minimal complication (or so the scientists and geneticists hope.

cicichen wrote:The END1, CBH1 and CEL1 genes were inserted into yeast expression/secretion cassettes. Expression of END1, CBH1 and CEL1 was directed by the promoter sequences derived from the alcohol dehydrogenase II (ADH2), the phosphoglycerate kinase I (PKG1) and the alcohol dehydrogenase I (ADH1) genes, respectively.


When the yeast finally have the genes inserted into it, it an then (hypothetically) be instructed to break down the biomasses material into the final products with ADH2 and ADH1. ADH2 and ADH1 are instruction manuals for the 4 genes being inserted into the yeast, but the three genes (END1 CBH1 CEL1) are the primary genes that will do all the works in breaking things down.

cicichen wrote:In contrast, BGL1 was expressed under the control of its native promoter. Secretion of End1p and Cel1p was directed by the signal sequence of the yeast mating pheromone -factor (MF1), whereas Cbh1p and Bgl1p were secreted using their authentic leader peptides. The construction of a fur1 ura3 S. cerevisiae strain allowed for the autoselection of this multicopy URA3-based plasmid in rich medium. S. cerevisiae transformants secreting biologically active endo--1,4-glucanase, cellobiohydrolase, cellodextrinase and cellobiase were able to degrade various substrates including carboxymethylcellulose, hydroxyethylcellulose, laminarin, barley glucan, cellobiose, polypectate, birchwood xylan and methyl--d-glucopyranoside. This study could lead to the development of industrial strains of S. cerevisiae capable of converting cellulose in a one-step process into commercially important commodities.



The remaining is merely an explanation about how the gene breaks down the contents within the biomass to convert the biomass into a final product for usage.

cicichen wrote:I would really like to know what those genes: BGL1, END1, CBH1, and CEL1 do and how do they work together?
And what's a β-1 4-glucose?


BGL1, END1, CBH1, and CEL1 (as indicated earlier) are genes that being used to make the yeast have the abilities to eat up the biomasses without any problem. The biomasses are the other LEGO pieces laying around. Your yeast is the ball. You want your yeast to grab onto the LEGO pieces laying around, and clump all those miscellaneous LEGO pieces into larger LEGO pieces that you can use. The instruction manuals are the ADH1 and ADH2, telling the genes what to do with the biomassive, whether to convert them into alcohol, or some form of product. The β-1 4-glucanase is the complex enzyme (like your saliva in your mouth) to break the biomasses down to the products that the instruction manuals telling the yeast, with the genes, to do. Think of the β-1 4-glucanase as the glue that holds all the miscellaneous LEGO pieces together permanently, after the LEGO pieces attached to ball's LEGO pieces.

Does that help a bit? Bio-genetic recombinant is a wonderful thing to learn and study. Fascinating subject that not only reveal the hidden secrets to life, but the complexities that make us who we are. You're more than welcome to ask us more questions when ever you're confused about something.
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Postby cicichen » Sun Dec 09, 2007 11:21 pm

Thank you for everyone's help!
I've been trying to keep my project on track.
That's how I'm doing so far: I collected wild samples of white and brown rot fungus and hope to PCR CBH1, CelF, BGL1 genes (they are supposed to produce cellulase and do all the magic) from the fungi DNA. I then will ligate the three genes and transform them into the S. cerevisiae.
I will have a control of S. cerevisiae growing on regular sugar rich media, and ones without. We will try to use psyllium husk to feed the genetically modified yeast and see whether they survive or not. If they do, then it means the genes work and they can digest cellulose.

I'm doing research on yeast growing media. Wikipedia says that "Common media used for the cultivation of yeasts include; potato dextrose agar (PDA) or potato dextrose broth, Wallerstien Laboratories Nutrient agar (WLN), Yeast Peptone Dextrose agar (YPD), and Yeast Mould agar or broth (YM). The antibiotic cycloheximide is sometimes added to yeast growth media to inhibit the growth of Saccharomyces yeasts and select for wild/indigenous yeast species."

But what's best for S. cerevisiae? And I wonder if I have a sugar rich media for the yeast, when I add the psyllium husk to the media, how should I know if the yeast live from cellulose or the sugar from the media?
But if I start with the plain agar plate, the yeast wouldn't grow on the media right?

So I was thinking about culturing S. cerevisiae in the the broth. The control would be in some kind of sugar rich broth, and the transform yeast would be in the psyllium husk broth. I will incubate the broth for a period of time, and then transfer them on plain agar plate. The control would show growth, and if the other plate show growth also, that means the yeast can digest cellulose an live with it. Does this sound right? And would anyone please give me some suggestion on which media is best for
S. cerevisiae ?

Thank you!

Cici
12/9/07
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Postby Lise Byrd » Sat Dec 15, 2007 3:05 pm

Hi Cici,

Your project sounds very interesting! Here is a page I found that lists several types of yeast media and how to make them (although they should also be available commercially):
http://www.phys.ksu.edu/gene/g1.html
It's part of an entire website devoted to yeast experiments, so if you click on the "Click here to return" link at the bottom of the page, you can browse around the rest of the website and see if there's anything else that might help you. Even though you won't be working with the same genes the site discusses, you may be able to use some of their methods.

I believe the general method for a transformation (if you are going to use a broth) is to incubate the yeast in a standard broth and then plate aliquots of the broth+yeast on sugar-rich or psyllium-husk plates, for example. You want to grow as much yeast as you can before selection, since you may have relatively low proportion of transformants, so you start with less specific media and move to more specific ones.

Do you know that the yeast will grow on the sugar-rich medium, or is that something you need to find out? And were you planning on adding the psyllium husk to the sugar-rich plates, or transferring colonies from the sugar-rich plates to psyllium-husk plates?

Especially if you're not sure if the yeast will grow on the sugar-rich medium, you might consider having two controls, one of yeast grown on a non-specific medium such as YED and one of yeast grown on the sugar-rich medium. The experimental colonies could be picked from the sugar-rich plates and transferred to the psyllium husk medium. In a two-step process like this, you would know whether the yeast were surviving in the sugar-rich or psyllium-husk medium by the difference between the number of colonies on the two plates.

I hope this helps. I apologize if I've misunderstood something in your post!
Sonia
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Postby cicichen » Sat Dec 15, 2007 5:19 pm

Thank you for your reply Sonia!

Sorry that I didn't explain my experimental procedure clearly.

I want to do a yeast transformation so S. cerevisiae would have the foreign genes from white/brown rot fungus and be able to digest cellulose.

BGL1,CelF,CBH1(cellulase gene) ---transformation--->S.cerevisiae (ethnaol produce yeast)--------> celllulose digestible yeast + ethanol!


I don't really know what kind of media is best to grow yeast, and in this experiment particularly, to be the control plate for yeast transformation. (my supervisor told me yeast can grow on glucose, so I thought the control would be sugar rich plate, but I guess I confuse you by that!)

Do you think YED plate can be the media for growing the yeast?
My supervisor was thinking to grow yeast colonies on media first, and then spit the mixture. This way all the yeast come from the orginal stock yeast. We'll transform half of the yeast and once they are transformed, we will grow half on the media as control, and half on psyillum husk media. So if no colonies grow on control we'll know the transformation process (which can kill yeast) is causing failure.

After reading the material on yeast, I think I'll use YED as growing media and controll media.

Does this procedure sound clear to you? I'm only using control to compare to psyillum husk media, because yeast can live on sugar base mdeia and generally cannot live on cellulose. If yeast colonies show up on
psyillum husk media, that means the transformation is successul and the genes from fungus work.

We want to use psyillum husk as cellulose source in our experiment because it's high in fiber and doesnot contain any sugar, does that sound good to you or do anyone have better suggestion on which kind of (pure)cellulose source can be put on agar plate?

Thank you for your help!

Cici
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Postby Lise Byrd » Sat Dec 15, 2007 6:36 pm

Thanks for the clarifications, Cici. The procedure your adviser was thinking of doing sounds fine. It shouldn't take too much time to perform (provided, of course, it works smoothly!), and taking all the yeast from the same stock will cut down on a lot of independent variables. You might also consider plating some non-transformed yeast on a psyllium plate as a negative control, just to double-check that only the transformed yeast will grow there.

From what I understand, YED is the standard yeast medium, so any kind of yeast (wild type, enhanced, knockout, etc) can grow on it. It is probably your best bet for the growing and control medium.

Cellulose itself is a sugar (a string of glucose molecules), so I presume that when you say psyllium has no sugar, you mean no simple sugars.

Sounds like you're well on your way! Keep it up, and feel free to post if you have more questions.

Sonia
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Postby cicichen » Sun Dec 16, 2007 7:26 pm

Thanks Sonia!
Your suggestion on a double negative control sounds very helpful, I will certainly add that to my experiment!

I have some other questions that have been bothering me. I wasn't sure how many trial I should do for my experiment. My supervisor thinks once would be fine, and if the experiment fail, we will have many explaination to it. Do you think it's ok for me to do just once? If more trial is not neccessary, I would be happy to just finish my experiment and start doing my papers since my school science fair is about three weeks from now.

Second is that I want to use Fleischmann's yeast for S. cerevisiae, that's the only yeast will be tested on. Should I go and find more yeast with different brands? I'm thinking that it would all be S. cerevisiae so maybe I just stick with the Fleischmann's yeast.

Thank you!

Cici
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