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<h2> I. Introduction </h2> | <h2> I. Introduction </h2> | ||
<p>Iron is an essential chemical element for cell survival. It plays important roles in the catalysis of many enzyme reactions in addition to stabilising the folding of many proteins. The total amount of iron found within an adult human body is relatively low, varying from 3 to 5 g. Furthermore, as the majority of iron in the human body is found in complexes with proteins, including haemoglobin and ferritin, the amount of free iron is as little as 10<sup>-24 </sup> M. Such limiting iron conditions are highly unfavourable for bacterial growth, therefore bacteria have developed numerous mechanisms to overcome this issue. <sup> 1 </sup> </p> | <p>Iron is an essential chemical element for cell survival. It plays important roles in the catalysis of many enzyme reactions in addition to stabilising the folding of many proteins. The total amount of iron found within an adult human body is relatively low, varying from 3 to 5 g. Furthermore, as the majority of iron in the human body is found in complexes with proteins, including haemoglobin and ferritin, the amount of free iron is as little as 10<sup>-24 </sup> M. Such limiting iron conditions are highly unfavourable for bacterial growth, therefore bacteria have developed numerous mechanisms to overcome this issue. <sup> 1 </sup> </p> | ||
<p>Bacteria actively acquire the iron they need for their survival through the production of siderophores. Siderophores are small, organic molecules that bind iron with high affinity, allowing bacteria to sequester iron from their environment. Following iron binding, siderophore-iron complexes are transported back to the bacterial cytoplasm, where iron is released in order to facilitate essential biochemical processes.<sup> 2 </sup> </p> | <p>Bacteria actively acquire the iron they need for their survival through the production of siderophores. Siderophores are small, organic molecules that bind iron with high affinity, allowing bacteria to sequester iron from their environment. Following iron binding, siderophore-iron complexes are transported back to the bacterial cytoplasm, where iron is released in order to facilitate essential biochemical processes.<sup> 2 </sup> </p> | ||
− | <p> As seen in <b> | + | <p> As seen in <b> Animation I (Bacteria compete with host for acquiring their iron) </b>, iron acquisition by pathogenic bacteria is not as straightforward, as bacteria have to compete with the host for the available free iron. Upon detection of certain pathogen-associated motifs, neutrophils produce lipocalin 2 (LCN2), a protein that binds to siderophores, thereby preventing iron transport into bacteria. <sup> 3 </sup> </p> |
+ | <center> | ||
+ | <img style="width:70%;float:none" src="https://static.igem.org/mediawiki/2016/6/65/T--Sheffield--Iron-Wars.gif"> | ||
+ | </center> | ||
+ | <center> <p style="text-align:center"><span> Animation I -Bacteria compete with host for acquiring their iron </span></p> | ||
+ | </center> | ||
+ | <p> </p> | ||
+ | |||
<p>The level of lipocalin 2 has been shown to be highly elevated in the case of bacterial infections and this protein has been suggested as a potential biomarker for distinguishing between a bacterial and a viral infection. Studies have shown that patients with a bacterial infection have serum lipocalin levels exceeding 155 μg l <sup> -1 </sup>, while in the cases of viral infections- the lipocalin 2 level is below this threshold. Using this cut-off in the lipocalin 2 level, bacterial infections have been accurately diagnosed in more than 92% of cases. <sup> 4, 5 </sup> Having the results of these studies as a starting point, we attempted to design a device that detects bacterial infections by indirectly measuring the level of lipocalin 2 in patient blood samples. </p> | <p>The level of lipocalin 2 has been shown to be highly elevated in the case of bacterial infections and this protein has been suggested as a potential biomarker for distinguishing between a bacterial and a viral infection. Studies have shown that patients with a bacterial infection have serum lipocalin levels exceeding 155 μg l <sup> -1 </sup>, while in the cases of viral infections- the lipocalin 2 level is below this threshold. Using this cut-off in the lipocalin 2 level, bacterial infections have been accurately diagnosed in more than 92% of cases. <sup> 4, 5 </sup> Having the results of these studies as a starting point, we attempted to design a device that detects bacterial infections by indirectly measuring the level of lipocalin 2 in patient blood samples. </p> | ||
<h2>II. Brief device considerations</h2> | <h2>II. Brief device considerations</h2> | ||
<p> Our device is composed of a shoebox sized fluorimeter, as well as of disposable compartmented recipients . </p> | <p> Our device is composed of a shoebox sized fluorimeter, as well as of disposable compartmented recipients . </p> | ||
<h2> III. Structure of disposable recipients </h2> | <h2> III. Structure of disposable recipients </h2> | ||
− | <p> As seen in the <b> | + | <p> As seen in the <b> Animation II (Biochemical processes taking place in the 3 chambers) </b>, disposable recipients contain 3 interlinked chambers. In each of the chambers there are certain biochemical processes that take place in order to facilitate the final detection step. </p> |
+ | <center> <img style="width:70%;float:none" src="https://static.igem.org/mediawiki/2016/2/24/T--Sheffield--Chambers.gif"></center> | ||
+ | <center><p style="text-align:center"><span> Animation II-Biochemical processes taking place in the 3 chambers </span></p></center> | ||
<h2> <font size="16"> III.a. First chamber </font> </h2> | <h2> <font size="16"> III.a. First chamber </font> </h2> | ||
− | <p> | + | <p>Blood samples are inserted into this chamber. In the case of a bacterial infection, the level of lipocalin 2 in the input blood is expected to be high, while in the case of a non-bacterial infection, the level of lipocalin 2 is low. Lipocalin 2 is a soluble blood protein and designing of biosensors for direct protein detection represents a challenge for synthetic biology. In order to avoid these limitations, we came up with a converter system that allows indirect detection of lipocalin 2 level.</p> |
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<h2> <font size="16"> III.b. Second chamber </font> </h2> | <h2> <font size="16"> III.b. Second chamber </font> </h2> | ||
<p> This chamber acts as a signal converter. Signal conversion is achieved by adding the patient blood with unknown lipocalin 2 levels to a known concentration of iron-siderophore complexes. As lipocalin 2 binds to siderophores, a low concentration of free siderophores is expected to reflect a high lipocalin 2 concentration, while a high concentration of free siderophores is expected to reflect a low lipocalin 2 concentration. | <p> This chamber acts as a signal converter. Signal conversion is achieved by adding the patient blood with unknown lipocalin 2 levels to a known concentration of iron-siderophore complexes. As lipocalin 2 binds to siderophores, a low concentration of free siderophores is expected to reflect a high lipocalin 2 concentration, while a high concentration of free siderophores is expected to reflect a low lipocalin 2 concentration. | ||
− | Therefore, in the case of a bacterial infection, the level of lipocalin 2 will be high and the siderophore-iron complexes will be chelated; only | + | Therefore, in the case of a bacterial infection, the level of lipocalin 2 will be high and the siderophore-iron complexes will be chelated; only low amounts of siderophores will flow to the chamber 2. In contrast, in the case of a viral infection the level of lipocalin 2 is low, therefore only few siderophore-iron complexes will be chelated. The majority of siderophore-iron complexes will flow to the chamber 3. </p> |
<h2> <font size="16"> III.c. Third chamber </font> </h2> | <h2> <font size="16"> III.c. Third chamber </font> </h2> | ||
− | <p> This chamber contains enclosed <i> | + | <p> This chamber contains enclosed <i>E. coli </i> biosensors that are designed to sense the free siderophore-iron complexes incoming from chamber 1 and output detectable changes in fluorescence intensity. This change in fluorescence intensity involves srRNA-mediated degradation of the mRNA encoding GFP.</p> |
− | <p> < | + | <p> <u> In the case of a bacterial infection,</u> the concentration of siderophore-iron complexes that reach chamber 3 should be low, as the complexes have been previously bound by lipocalin 2. When little or no iron will enter the engineered <i>E. coli</i>, the Fur repressor will be inactive; therefore RyhB small interfering RNA will be transcribed. RyhB will determine the degradation of mRNA encoding for GFP, therefore the cells will lose their fluorescence.</p> |
− | <p> < | + | <p> <u> In the case of a non-bacterial infection,</u> the concentration of siderophore-iron complexes that reach chamber 3 should be high. Siderophore-iron complexes will be taken up by the engineered <i>E.coli</i> cells and the iron will be released in the cytoplasm. In the cytoplasm, iron will bind to and activate the Fur transcriptional repressor, preventing the transcription of RyhB small interfering RNA. In the absence of RyhB, GFP will be constitutively expressed and cells will remain fluorescent. </p> |
− | <p> Such changes in the fluorescence will be measured by the | + | <p> Such changes in the fluorescence will be measured by the fluorometer and qualitative results would be inferred by quantitative fluorescence data. The system will be calibrated and the fluorescence emission corresponding to the 155 μg/l <sup> -1 </sup> lipocalin 2 level will be used as a cut off when interpreting results.</p> |
<h2> IV.References </h2> | <h2> IV.References </h2> | ||
<p> 1. Caza M, Kronstad JW. Shared and distinct mechanisms of iron acquisition by bacterial and fungal pathogens of humans. <i> Front Cell Infect Microbiol.</i> 2013;3(November):80. doi:10.3389/fcimb.2013.00080.</p> | <p> 1. Caza M, Kronstad JW. Shared and distinct mechanisms of iron acquisition by bacterial and fungal pathogens of humans. <i> Front Cell Infect Microbiol.</i> 2013;3(November):80. doi:10.3389/fcimb.2013.00080.</p> | ||
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+ | <div class="backToTop"><img src="https://static.igem.org/mediawiki/2016/5/55/T--Sheffield--TopArrow.png"></div> | ||
+ | <div class="glossary"><a href="https://2016.igem.org/Team:Sheffield/glossary"><img src="https://static.igem.org/mediawiki/2016/8/86/T--Sheffield--Glossary.png"></a></div> | ||
<!-- Bootstrap core JavaScript | <!-- Bootstrap core JavaScript | ||
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Latest revision as of 15:59, 18 October 2016
FINAL SYSTEM |
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I. Introduction
Iron is an essential chemical element for cell survival. It plays important roles in the catalysis of many enzyme reactions in addition to stabilising the folding of many proteins. The total amount of iron found within an adult human body is relatively low, varying from 3 to 5 g. Furthermore, as the majority of iron in the human body is found in complexes with proteins, including haemoglobin and ferritin, the amount of free iron is as little as 10-24 M. Such limiting iron conditions are highly unfavourable for bacterial growth, therefore bacteria have developed numerous mechanisms to overcome this issue. 1
Bacteria actively acquire the iron they need for their survival through the production of siderophores. Siderophores are small, organic molecules that bind iron with high affinity, allowing bacteria to sequester iron from their environment. Following iron binding, siderophore-iron complexes are transported back to the bacterial cytoplasm, where iron is released in order to facilitate essential biochemical processes. 2
As seen in Animation I (Bacteria compete with host for acquiring their iron) , iron acquisition by pathogenic bacteria is not as straightforward, as bacteria have to compete with the host for the available free iron. Upon detection of certain pathogen-associated motifs, neutrophils produce lipocalin 2 (LCN2), a protein that binds to siderophores, thereby preventing iron transport into bacteria. 3
Animation I -Bacteria compete with host for acquiring their iron
The level of lipocalin 2 has been shown to be highly elevated in the case of bacterial infections and this protein has been suggested as a potential biomarker for distinguishing between a bacterial and a viral infection. Studies have shown that patients with a bacterial infection have serum lipocalin levels exceeding 155 μg l -1 , while in the cases of viral infections- the lipocalin 2 level is below this threshold. Using this cut-off in the lipocalin 2 level, bacterial infections have been accurately diagnosed in more than 92% of cases. 4, 5 Having the results of these studies as a starting point, we attempted to design a device that detects bacterial infections by indirectly measuring the level of lipocalin 2 in patient blood samples.
II. Brief device considerations
Our device is composed of a shoebox sized fluorimeter, as well as of disposable compartmented recipients .
III. Structure of disposable recipients
As seen in the Animation II (Biochemical processes taking place in the 3 chambers) , disposable recipients contain 3 interlinked chambers. In each of the chambers there are certain biochemical processes that take place in order to facilitate the final detection step.
Animation II-Biochemical processes taking place in the 3 chambers
III.a. First chamber
Blood samples are inserted into this chamber. In the case of a bacterial infection, the level of lipocalin 2 in the input blood is expected to be high, while in the case of a non-bacterial infection, the level of lipocalin 2 is low. Lipocalin 2 is a soluble blood protein and designing of biosensors for direct protein detection represents a challenge for synthetic biology. In order to avoid these limitations, we came up with a converter system that allows indirect detection of lipocalin 2 level.
III.b. Second chamber
This chamber acts as a signal converter. Signal conversion is achieved by adding the patient blood with unknown lipocalin 2 levels to a known concentration of iron-siderophore complexes. As lipocalin 2 binds to siderophores, a low concentration of free siderophores is expected to reflect a high lipocalin 2 concentration, while a high concentration of free siderophores is expected to reflect a low lipocalin 2 concentration. Therefore, in the case of a bacterial infection, the level of lipocalin 2 will be high and the siderophore-iron complexes will be chelated; only low amounts of siderophores will flow to the chamber 2. In contrast, in the case of a viral infection the level of lipocalin 2 is low, therefore only few siderophore-iron complexes will be chelated. The majority of siderophore-iron complexes will flow to the chamber 3.
III.c. Third chamber
This chamber contains enclosed E. coli biosensors that are designed to sense the free siderophore-iron complexes incoming from chamber 1 and output detectable changes in fluorescence intensity. This change in fluorescence intensity involves srRNA-mediated degradation of the mRNA encoding GFP.
In the case of a bacterial infection, the concentration of siderophore-iron complexes that reach chamber 3 should be low, as the complexes have been previously bound by lipocalin 2. When little or no iron will enter the engineered E. coli, the Fur repressor will be inactive; therefore RyhB small interfering RNA will be transcribed. RyhB will determine the degradation of mRNA encoding for GFP, therefore the cells will lose their fluorescence.
In the case of a non-bacterial infection, the concentration of siderophore-iron complexes that reach chamber 3 should be high. Siderophore-iron complexes will be taken up by the engineered E.coli cells and the iron will be released in the cytoplasm. In the cytoplasm, iron will bind to and activate the Fur transcriptional repressor, preventing the transcription of RyhB small interfering RNA. In the absence of RyhB, GFP will be constitutively expressed and cells will remain fluorescent.
Such changes in the fluorescence will be measured by the fluorometer and qualitative results would be inferred by quantitative fluorescence data. The system will be calibrated and the fluorescence emission corresponding to the 155 μg/l -1 lipocalin 2 level will be used as a cut off when interpreting results.
IV.References
1. Caza M, Kronstad JW. Shared and distinct mechanisms of iron acquisition by bacterial and fungal pathogens of humans. Front Cell Infect Microbiol. 2013;3(November):80. doi:10.3389/fcimb.2013.00080.
2. Faraldo-Gómez JD, Sansom MSP. Acquisition of siderophores in gram-negative bacteria. Nat Rev Mol Cell Biol. 2003;4(2):105-116. doi:10.1038/nrm1015.
3. Flo TH, Smith KD, Sato S, et al. Lipocalin 2 mediates an innate immune response to bacterial infection by sequestrating iron. Nature. 2004;432(7019):917-921. doi:10.1038/nature03104.
4. Xu SY, Pauksen K, Venge P. Serum measurements of human neutrophil lipocalin (HNL) discriminate between acute bacterial and viral infections. Scand J Clin Lab Invest. 1995;55(2):125-131. doi:10.3109/00365519509089604.
5. Venge P, Håkansson LD, Garwicz D, Peterson C, Xu S, Pauksen K. Human neutrophil lipocalin in fMLP-activated whole blood as a diagnostic means to distinguish between acute bacterial and viral infections. J Immunol Methods. 2015;424:85-90. doi:10.1016/j.jim.2015.05.004.
How the synthetic biology works
Jamies stuff here