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| | <div id="page_title_holder_main_page" style="background:#09F"><h1>Project</h1></div> | | <div id="page_title_holder_main_page" style="background:#09F"><h1>Project</h1></div> |
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| - | <div id="page_title_holder_sub_page"><h2>The Problem</h2></div> | + | <div id="page_title_holder_sub_page"><h2>Colony Forming Unit Assay</h2></div> |
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| - | <p class="ContentParagraph">The World Health Organization (WHO) recently published a report on the dramatic increase of antibiotic resistances in bacteria around the world, stating that “A post-antibiotic era – in which common infections and minor injuries can kill – far from being an apocalyptic fantasy, is instead a very real possibility for the 21st Century”(1). Antibiotic resistances have serious medicinal and financial consequences. According to the Centres for Disease Control, approximately 70% of all people with bacterial infections in hospitals in the Unites(2) or 1.4 million people get bacterial infections that are resistant to at least one prevalently used antibiotic in hospitals in the United States every year. Furthermore, the National Academy of Science estimates an increase of $4 billion in healthcare costs every year in the United States associated with antibiotic resistant bacteria(2).</p>
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| - | | + | <h1>CFU Assay</h1> |
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| + | <p> In order to determine the number of viable cells in a liquid culture, we used a Colony Forming Unit ( CFU) assay. This simple assay involves the collection and serial dilution of samples of a liquid culture. These dilutions are then plated and colonies are counted. To determine the number of CFUs per milliliter, you use this formula:</p> |
| - | | + | <p> CFU/ mL = ( # of colonies*dilution factor)/ volume plated in mL’s</p> |
| - | <p class="ContentParagraph"> How do these antibiotic resistances develop? When bacteria are constantly exposed to high levels of antibiotics, they try to develop a defence mechanism against these antibiotics by taking in extracellular parts and even mutating their own DNA to produce an “anti antibacterial protein,” that disrupts the function of the antibiotics by either binding to it or degrading it. Those bacteria that are successful in warding off the antibiotics replicate through natural selection and are said to have an antibiotic resistance.</p>
| + | <p> We used this assay to generate a standard curve representing the effect of ampicillin concentration on CFU/mL of liquid <i>E. coli</i> cultures with no antibiotic resistance. </ p> |
| - | | + | <p>Here is the curve we have generated:</p> |
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| + | <img src="https://static.igem.org/mediawiki/2014hs/ c/ c2/ Lerthhs2014_graph_30984320849. png" alt="" width=" 1230px" /> |
| - | | + | <p>We plan to use this curve to determine the concentration of ampicillin in media that we treat with our project.</p> |
| - | <p class="ContentParagraph"> Lately the concentration of antibiotics in the water system, that would cause bacteria to develop antibiotic resistances, has been rising. One of the main ways antibiotics enter the water system is from run-off from feedlots. Antibiotics are fed to 83% of cattle feedlots and 84% of sheep farms in the United States in order to promote growth(3). Additionally, an estimated 75% of all antibiotics fed to animals are not fully digested and will eventually enter the environment(2).</p> | + | <div style="height: 20px;"></div> |
| - | | + | <h1>Dialysis</h1> |
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| + | <p> In order to remediate antibiotics in liquids without releasing cells into the liquid, we used a 1000KD molecular weight cut off (MWCO) dialysis tube. By placing our beta- lactamase producing cells in this tube and placing this tube in an ampicillin containing liquid, we hope to release the beta-lactamase enzyme into the liquid and degrade the ampicillin.</p> |
| - | | + | <p>We ran this test with 10ml (OD600 = 4.628) of cells containing an ampicillin resistant plasmid encoding beta-lactamase in the dialysis tube. We took samples of the ampicillin-containing media every hour and used those samples for another CFU assay.</p> |
| - | <p class="ContentParagraph"> A class of antibiotics that are commonly found in the water is penicillin. Penicillin is distinguished by its beta-lactam ring, which is a cyclic, 4-part amide ring attached to a carbonyl group, that prevents cell wall formation and thus kills the bacteria(4). In response to penicillin, the bacteria will produce the enzyme beta-lactamase, which degrades the beta-lactam ring in penicillin using hydrolysis (5).</p>
| + | <img src="https://static.igem.org/mediawiki/2014hs/ e/ e4/ Lethha2014_graph_23897423908423.png" alt="" width=" 1230px" /> |
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| + | <p> It is clear that the concentration of ampicillin in the media is decreasing, but there is still work to be done to be sure that it is the dialysis treatment and not heat or pH having this effect. We will have those results come the Jamboree!</p> |
| - | <p class="ContentParagraph">1. <a href=http://www.who.int/drugresistance/documents/surveillancereport/en/> WHO report on antimicrobial resistance</a></p>
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| - | <p class="ContentParagraph">2. <a href=http://www.sustainabletable.org/257/antibiotics/> Antibiotics and the Food Animal Industry</a></p>
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| - | <p class="ContentParagraph">3. <a href=https://www.govtrack.us/congress/bills/113/s1256/text/> Preventing Antibiotic Resistance Act of 2013</a></p>
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| - | <p class="ContentParagraph">4. <a href=http://en.citizendium.org/wiki/Lactam/> Lactam</a></p> | + | |
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| - | <p class="ContentParagraph">5. <a href=http://www.pharmaresearchlibrary.com/wp-content/uploads/2013/07/PRL2013-IJPNM-1717.pdf/> Siddheshwar, S.S et al (2013). Need and Scope of Development of β-lactums</a></p>
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| - | <div id="page_title_holder" style="">
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| - | <div id="page_title_holder_main_page" style="background:#09F"><h1>Project</h1></div>
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| - | <div id="page_title_holder_sub_page"><h2>The Idea</h2></div>
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| - | <p class="ContentParagraph"> The Lethbridge HS iGEM team is attempting to find a solution to tackle the global issue of increased antibiotic resistances in bacteria by focusing on reducing the levels of antibiotics, primarily penicillin, in the water system. To do this, we are having <i>E.coli</i> excrete beta-lactamase into its periplasmic space. Once the penicillin and the <i>E.coli</ i> meet, and the pencillin enters into the < i> E.coli's</i> periplasmic space, the beta-lactamase will degrade the penicillin. This way the overall concentration of antibiotics in the water will start to decrease. Furthermore, we would like to characterize these signal sequences with other antibacterial resistance genes in order to have <i>E.coli</i> excrete other proteins, such as erythromycin esterase A, that would target a different class of antibiotics and therefore make our construct more effective. We hope to be able to apply our construct in a water treatment plant in the form of a bioreactor in order to decrease the levels of penicillin in the water system and limit the amount of time the bacteria will have to adapt to the stressed, antibiotic-rich environment.</p> | + | |
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| - | <img src="https://static.igem.org/mediawiki/2014hs/1/16/LethHS2014_IMG_4332.JPG" width="500px" height="400px"> | + | |
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| - | <a name="idea"></a>
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| - | <div id="page_title_holder" style=" "> | + | |
| - | <div id="page_title_holder_main_page" style="background:#09F"><h1>Project</h1></div>
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| - | <div id="page_title_holder_sub_page"><h2>Future Directions</h2></div>
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| - | <p class="ContentParagraph">There are many things we could continue with as an iGEM team. This includes additional correspondence with the water treatment plant, as it would be useful in solidifying how we could implement our project on a large scale, and how useful it would be in a real world. The characterization of our signal sequences with other enzymes would also be interesting because if our construct works with other “anti-antibiotics” we would be able to implement systems like ours in areas where antibiotics are prevalent in high concentrations. For our math model, we only showed one graph for our results of ampicillin concentration vs time spent in the system (where E0 = 3.7x1010); if we produced more graphs where E0 varied, we could define a rate constant for ampicillin degradation relative to the starting number of <i>E.coli</i>. | + | |
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| - | It would look something like this:
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| - | <p style="font-size: 22px;"> This would lead to more accurate values of how much ampicillin you have at a point in time. We also hope to continue to reach out to our community and make them aware of iGEM in general as well as what we are doing. One of our main goals for the future is to display our art pieces in an art exhibition for the city in November, even though that is outside of our season for this year. We feel that the support of our community is essential to our success as a team and the more people that we can involve, the more opinions we will gain, thus leading to a more informed and stronger project. </p > | + | |
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| - | <a href="#" class="back-to-top">Back to Top</a> | + | |
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Project
Colony Forming Unit Assay
CFU Assay
In order to determine the number of viable cells in a liquid culture, we used a Colony Forming Unit (CFU) assay. This simple assay involves the collection and serial dilution of samples of a liquid culture. These dilutions are then plated and colonies are counted. To determine the number of CFUs per milliliter, you use this formula:
CFU/mL = (# of colonies*dilution factor)/volume plated in mL’s
We used this assay to generate a standard curve representing the effect of ampicillin concentration on CFU/mL of liquid E. coli cultures with no antibiotic resistance.
Here is the curve we have generated:
We plan to use this curve to determine the concentration of ampicillin in media that we treat with our project.
Dialysis
In order to remediate antibiotics in liquids without releasing cells into the liquid, we used a 1000KD molecular weight cut off (MWCO) dialysis tube. By placing our beta-lactamase producing cells in this tube and placing this tube in an ampicillin containing liquid, we hope to release the beta-lactamase enzyme into the liquid and degrade the ampicillin.
We ran this test with 10ml (OD600 = 4.628) of cells containing an ampicillin resistant plasmid encoding beta-lactamase in the dialysis tube. We took samples of the ampicillin-containing media every hour and used those samples for another CFU assay.
It is clear that the concentration of ampicillin in the media is decreasing, but there is still work to be done to be sure that it is the dialysis treatment and not heat or pH having this effect. We will have those results come the Jamboree!
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