Team:Acton-BoxboroughRHS/Research

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             <p style="font-family:arial">For the active mock Wiki, go <a href="https://c9.io/ab_igem/igem2014/workspace/iGEM_Main_Page.html">here</a>.</p>
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             <li><a href="https://2014hs.igem.org/Team:Acton-BoxboroughRHS/Research">Research</a></li>
             <li><a href="https://2014hs.igem.org/Team:Acton-BoxboroughRHS/Research">Research</a></li>
             <li><a href="https://2014hs.igem.org/Team:Acton-BoxboroughRHS/Labs">Labs</a></li>
             <li><a href="https://2014hs.igem.org/Team:Acton-BoxboroughRHS/Labs">Labs</a></li>
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         <h style="font-size:50px;font-family:Arial">Research On Kopi Luwak</h>
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         <h style="font-size:50px;font-family:Arial">Research</h>
         <br>
         <br>
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             <h style="border:1px solid black;padding:5px;background-color:yellow"> E. Coli vs. Yeast</h>
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            <h style="border:3px solid black;padding:5px;background-color:green;border-radius:5px"> Pre-Research</h>
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            <br></br>
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             <h style="border:1px solid black;padding:5px;background-color:yellow;border-radius:5px"> E. Coli vs. Yeast</h>
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 +
            <p>
 +
E.coli can live in a higher range of pH and should be easier to engineer, but may be less productive.
 +
Yeast will most likely be more be more productive in protein production in the long run but is otherwise inferior to the use of E. coli, so our decision is to use the latter. Also, we used a k12 strain of E. coli from <a href="http://www.carolina.com/bacteria/escherichia-coli-living-k-12-strain-tube/155068.pr">Carolina Labs</a> and a highly-competent strain of E. coli from <a href="https://www.neb.com/products/c2987-neb-5-alpha-competent-e-coli-high-efficiency">New England BioLabs</a>.
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            </p>
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<br>
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<br>
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            <h style="border:1px solid black;padding:5px;background-color:yellow;border-radius:5px">DNA Sequences</h>
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            <p>
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We need an open reading frame (orf) a ribosome binding site (rbs), a promoter, at least 3-5 enzymes:(pepsin, salivary amylase, trypsin, chymotripsin, and pancreatic amylase), and a terminator, to create a fully functional plasmid to digest the coffee beans.
 +
<br>
 +
<br>
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We planned on using the amylase orf that already existed in our ordered tray, in part to treat the coffee beans for breaking down complex carbohydrates to simple sugars. This will make the resulting coffee less bitter and more sweet.  The part <a href="http://parts.igem.org/Part:BBa_K523006">BBa_K523006</a> codes for amylase and also includes it's own secretion mechanism, which enables the displaying of amylase on the cell’s membrane. 
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E.coli has a higher pH range and may be easier to assemble, but may be less productive.
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        <br></br>
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Yeast will probably be more productive in the long run but is otherwise inferior
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        <br></br>
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        <p style="font-family:Arial">This image shows the template for a typical plasmid requirement</p>
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        <img src="https://c9.io/ab_igem/igem2014/workspace/Plasmid.png">
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            <h style="border:1px solid black;padding:5px;background-color:yellow"> Genes</h>
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we need an open reading frame (orf) a ribosome binding site (rbs), promoter, and terminator
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        <h style="border:3px solid black;padding:5px;background-color:green;border-radius:5px">Parts</h>
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at least 3-5 enzymes: pepsin, salivary amylase, trypsin, chymotripsin, pancreatic amylase
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        <h style="border:1px solid black;padding:5px;background-color:yellow;border-radius:5px">The Promoter</h>
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         <p>Message</p>
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        <p> A promoter is a DNA sequence that tends to recruit transcriptional machinery and lead to transcription of the downstream DNA sequence. The part we anticipated to use as a promoter was <a href="http://parts.igem.org/Part:BBa_J61002">BBa_J23100</a>; however, our Amylase ORF already comes with its own LacZ promoter, and ribosome binding site.</p>
 +
         <br></br>
 +
 
 +
        <h style="border:1px solid black;padding:5px;background-color:yellow;border-radius:5px">The Ribosome Binding Site</h>
 +
        <p>A ribosome binding site (RBS) is an RNA sequence found in mRNA to which ribosomes can bind and initiate translation in bacteria. The part we anticipated was <a href="http://parts.igem.org/Part:BBa_B0030">BBa_B0030</a>. This part is considered a strong RBS in terms of initiating transcription. It has many 'relatives', such as  BBa_B0031, BBa_B0032, BBa_B0033 and BBa_B0034.</p>
 +
        <br></br>
 +
 
 +
        <h style="border:1px solid black;padding:5px;background-color:yellow;border-radius:5px">The Open Reading Frames</h>
 +
        <p>Protein domains are portions of proteins cloned in frame with other proteins domains to make up a protein coding sequence. Some protein domains might change the protein's location, alter its degradation rate, target the protein for cleavage, or enable it to be readily purified. The open reading frame we actually ended up using in the <a href="https://2014hs.igem.org/Team:Acton-BoxboroughRHS/Labs">main lab and the high efficiency lab</a> was almost solely amylase, which as described, has its own transcription and secretion mechanisms.</p>
 +
        <br></br>
 +
 
 +
        <h style="border:1px solid black;padding:5px;background-color:yellow;border-radius:5px">The Terminator</h>
 +
        <p>The Terminator is the 'last' region of the plasmid, where RNA polymerase stops transcription. There are two main types of terminators in prokaryotes, rho-dependent terminators, and rho-independent terminators. Rho-dependent termination bases its function on the Rho protein, which causes the RNA polymerase to fall disassociate form the plasmid.
 +
<br>
 +
<br>
 +
        Rho-independent termination does not use Rho to disassociate the RNA polymerase, but instead stops by the DNA sequence, here's how it works. The DNA sequence contains a two regions that are rich in cytosine and guanine. When this is transcribed, those regions on the RNA form hydrogen bonds to itself: effectively forming a 'loop' which disassociates the DNA polymerase from the DNA.
 +
<br>
 +
<br>
 +
        Within the category of rho-independent terminators, are terminators that stop transcription on the forward strand in forward direction (forward terminators), on both strands and directions (bi-directional terminators), and on the reverse strand and direction only (reverse terminators).
 +
<br>
 +
<br>
 +
        One specific part that seems promising is the rho-independent, forward terminator <a href="http://parts.igem.org/Part:BBa_B1006">BBa_B1006</a>, which has a 99 per cent success rate of terminating the transcription, the highest efficiency of all the terminators.</p>
 +
        <br></br>
 +
 
 +
        <h style="border:1px solid black;padding:5px;background-color:yellow;border-radius:5px">The Backbone</h>
 +
        <p> A plasmid is a circular, double-stranded DNA molecules typically containing a few thousand base pairs that replicate within the cell independently of the chromosomal DNA. A plasmid backbone is defined as the plasmid sequence beginning with the BioBrick suffix, including the replication origin and antibiotic resistance marker, and ending with the BioBrick prefix.</p>
 +
        <br></br>
 +
        <br></br>
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 +
 
 +
 
 +
        <h style="border:3px solid black;padding:5px;background-color:green;border-radius:5px">Anticipated Parts</h>
 +
        <br></br>
 +
        <h style="border:1px solid black;padding:5px;background-color:yellow;border-radius:5px">Pepsin</h>
 +
        <p>This gene will help digest protein in the coffee bean. We needed to find a pepsin gene more similar to the Paradoxurus hermaphroditus (Asian Palm Civet), because there are many different variations. Any secretion most likely means lysis with acid: we do not have the acid proof backbone in our plate.
 +
Although we planned on getting this pepsin part synthesized using the template from the existing planned pepsinogen part BBa_M143, analysis of the sequence revealed multiple errors. The part claims to be optimized for production in E. coli; however, it includes the human signal peptide which would not work if synthesized in E. coli. By the time we realized this, it was too late to plan and synthesize a new part. So we decided to just plan the part, submit it, and get it synthesized in the future.
 +
</p>
 +
         <!--
 +
        <h style="border:1px solid black;padding:5px;background-color:yellow;border-radius:5px">Trypsin</h>
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        -->
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        <br>
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        <br>
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        <h style="border:3px solid black;padding:5px;background-color:green;border-radius:5px">Resources</h>
 +
         <p>
 +
        <ul>
 +
            <li>Jumhawan, Udi et al, Selection of Discriminant Markers for Authentication of Asian Palm Civet Coffee(Kopi Luwak): A Metabolomics Approach
 +
 
 +
            <li>NCBI Taxonomy Browser
 +
 
 +
            <li>Martinez, Federico Luis, Quality Enhancement of Coffee with Acid and Enzyme Treatment
 +
 
 +
            <li>Quality Enhancement of Coffee with Acid and Enzyme Treatment: Patent Application
 +
 
 +
            <li>Marcone, Massimo F, Composition and properties of Indonesian palm civet coffee(Kopi Luwak) and Ethiopian Civet Coffee
 +
 
 +
 
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            function to_project(){
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                document.getElementById("content").innerHTML="Kopi Luwak coffee is the rarest \
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                and the most expensive type of coffee in the world. This beverage is made \
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                from the feces of the Asian Palm Civet (Paradoxurus hermaphroditus) or Luwak,\
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                a cat-like omnivore that fills the niche of a racoon in Asia. The Asian Palm Civet \
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                eats raw coffee berries. As the berries are digested, enzymes in the animal's\
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                digestive tract break down components of the coffee bean that are responsible for its\
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                bitter taste. However, the beans themselves are not digested. The civet only digests the\
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                fleshy outer layer, so when it defecates, it leaves clumps of coffee beans that have been\
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                processed by its enzymes. The beans are then cleaned, roasted, and brewed to make the Kopi\
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                Luwak coffee. Due to the complexity of this process, Kopi Luwak is a very expensive item\
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                at $600 per pound. The outrageous price has made Kopi Luwak a novelty for the rich. Its \
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                allegedly excellent flavor is sadly something that most people are unable to afford. Another\
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                drawback of Kopi Luwak is that demand has driven businesses to animal cruelty in order \
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                to keep up production. A small civet farming industry has tens of thousands of civets\
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                living in battery cages being force fed coffee berries. Civets, being omnivores, are no\
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                more capable of surviving on coffee than humans. As a result their population is diminishing.\
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                We propose to make this process more humane, efficient, and sanitary by using bacteria instead of\
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                civets to process coffee berries into Kopi Luwak beans. We will accomplish this by inserting genes\
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                that code for proteins found in the civet's digestive tract into a hardy bacteria that can withstand\
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                the pH levels required for the proteins to operate. We intend to add genes for salivary amylase, pepsin,\
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                pancreatic amylase, trypsin, and chymotrypsin, sequenced from palm civets or closely-related species.\
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                Once the bacteria have been transformed, we will attempt to simulate the digestive process of the civet\
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                on coffee berries and analyze our results. Although this project seems unconventional, putting animal\
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                enzymes in bacteria has been done before to great effect. Take rennet cheese for an example. In order\
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                to obtain the enzymes required to produce this cheese, a calf must be slaughtered; the material (rennet)\
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                is taken from the dead animal's digestive tract. Now, due to growing demand for this cheese, bacteria containing\
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                the rennet enzymes are used instead. Today, the sale of these cheeses is a popular and profitable industry.\
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                By putting animal enzymes in bacteria, we create a digestive platform that is capable of processing more than\
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                just coffee berries.";
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Latest revision as of 02:58, 14 September 2014

Welcome to the ABRHS iGEM team

ABRHS


Research

Pre-Research

E. Coli vs. Yeast

E.coli can live in a higher range of pH and should be easier to engineer, but may be less productive. Yeast will most likely be more be more productive in protein production in the long run but is otherwise inferior to the use of E. coli, so our decision is to use the latter. Also, we used a k12 strain of E. coli from Carolina Labs and a highly-competent strain of E. coli from New England BioLabs.



DNA Sequences

We need an open reading frame (orf) a ribosome binding site (rbs), a promoter, at least 3-5 enzymes:(pepsin, salivary amylase, trypsin, chymotripsin, and pancreatic amylase), and a terminator, to create a fully functional plasmid to digest the coffee beans.

We planned on using the amylase orf that already existed in our ordered tray, in part to treat the coffee beans for breaking down complex carbohydrates to simple sugars. This will make the resulting coffee less bitter and more sweet. The part BBa_K523006 codes for amylase and also includes it's own secretion mechanism, which enables the displaying of amylase on the cell’s membrane.





This image shows the template for a typical plasmid requirement





Parts

The Promoter

A promoter is a DNA sequence that tends to recruit transcriptional machinery and lead to transcription of the downstream DNA sequence. The part we anticipated to use as a promoter was BBa_J23100; however, our Amylase ORF already comes with its own LacZ promoter, and ribosome binding site.



The Ribosome Binding Site

A ribosome binding site (RBS) is an RNA sequence found in mRNA to which ribosomes can bind and initiate translation in bacteria. The part we anticipated was BBa_B0030. This part is considered a strong RBS in terms of initiating transcription. It has many 'relatives', such as BBa_B0031, BBa_B0032, BBa_B0033 and BBa_B0034.



The Open Reading Frames

Protein domains are portions of proteins cloned in frame with other proteins domains to make up a protein coding sequence. Some protein domains might change the protein's location, alter its degradation rate, target the protein for cleavage, or enable it to be readily purified. The open reading frame we actually ended up using in the main lab and the high efficiency lab was almost solely amylase, which as described, has its own transcription and secretion mechanisms.



The Terminator

The Terminator is the 'last' region of the plasmid, where RNA polymerase stops transcription. There are two main types of terminators in prokaryotes, rho-dependent terminators, and rho-independent terminators. Rho-dependent termination bases its function on the Rho protein, which causes the RNA polymerase to fall disassociate form the plasmid.

Rho-independent termination does not use Rho to disassociate the RNA polymerase, but instead stops by the DNA sequence, here's how it works. The DNA sequence contains a two regions that are rich in cytosine and guanine. When this is transcribed, those regions on the RNA form hydrogen bonds to itself: effectively forming a 'loop' which disassociates the DNA polymerase from the DNA.

Within the category of rho-independent terminators, are terminators that stop transcription on the forward strand in forward direction (forward terminators), on both strands and directions (bi-directional terminators), and on the reverse strand and direction only (reverse terminators).

One specific part that seems promising is the rho-independent, forward terminator BBa_B1006, which has a 99 per cent success rate of terminating the transcription, the highest efficiency of all the terminators.



The Backbone

A plasmid is a circular, double-stranded DNA molecules typically containing a few thousand base pairs that replicate within the cell independently of the chromosomal DNA. A plasmid backbone is defined as the plasmid sequence beginning with the BioBrick suffix, including the replication origin and antibiotic resistance marker, and ending with the BioBrick prefix.





Anticipated Parts

Pepsin

This gene will help digest protein in the coffee bean. We needed to find a pepsin gene more similar to the Paradoxurus hermaphroditus (Asian Palm Civet), because there are many different variations. Any secretion most likely means lysis with acid: we do not have the acid proof backbone in our plate. Although we planned on getting this pepsin part synthesized using the template from the existing planned pepsinogen part BBa_M143, analysis of the sequence revealed multiple errors. The part claims to be optimized for production in E. coli; however, it includes the human signal peptide which would not work if synthesized in E. coli. By the time we realized this, it was too late to plan and synthesize a new part. So we decided to just plan the part, submit it, and get it synthesized in the future.



Resources

  • Jumhawan, Udi et al, Selection of Discriminant Markers for Authentication of Asian Palm Civet Coffee(Kopi Luwak): A Metabolomics Approach
  • NCBI Taxonomy Browser
  • Martinez, Federico Luis, Quality Enhancement of Coffee with Acid and Enzyme Treatment
  • Quality Enhancement of Coffee with Acid and Enzyme Treatment: Patent Application
  • Marcone, Massimo F, Composition and properties of Indonesian palm civet coffee(Kopi Luwak) and Ethiopian Civet Coffee

End of Page

Retrieved from "http://2014hs.igem.org/Team:Acton-BoxboroughRHS/Research"