Difference between revisions of "Team:UNOTT/Description"

 
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<h1>Project Description</h1>
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<h4>Password security</h4>
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<p><font size="3" color="white">Due to the recent explosion in hacking incidents, we were interested at looking into how biological systems can be used to overcome the limitations of current passwords. Although there was the possibility to use certain DNA sequences as passwords, the speed of current sequencing technologies and problems with mutating the bacteria to a point where it could not survive were limiting to the success and randomness of this option. For that reason we turned to using physical properties of bacteria such as its metabolome to create a biological password.</font></p>
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<h4 color="white">The Idea: <i>Key. coli</i></h4>
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<li> <font size="3" color="white">The first biological password that changes over time!
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We are looking into transforming bacteria with a unique array of existing iGEM systems to produce a unique signal of secondary metabolites, initially using fluorescence as a proof of concept. Eventually, we will use the system to produce a unique and random configuration of products, as our "key". In order to produce this randomness, alteration of the activity/presence of promoters associated with these metabolites will be applied using one of a few methods currently being considered by the team (detailed below).</li>
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<li>This key will be used to open safes, secure doors and various other locks. Measurement of certain engineered metabolites such as volatiles will give a distinct mass spectrum. A combination of a detection technique such as gas chromotography-mass spectrometry with a data comparison software will compare the secondary metabolites of the "key" bacteria to the "reference/lock" from which it was taken. If the spectra of both colonies exceeds a threshold of similarity then the system is unlocked.</li>
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<li>After an amount of time, our Key will have to be renewed from the Lock colony, and when this occurs the configuration of the key is shuffled once again to ensure the key and lock are changing. </li>
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<h4>Bacterial Key Transport Device</h4>
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<p><font size="3" color="white">There is a need for a transport mechanism for the key. This presents problems depending on the bacteria used.</p>
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<p> In <i>E. coli</i>, our key transport system would need to keep our colonies alive. We have looked into a few options for key storage:</p>
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<li>We could freeze the cells after assignment of promoters to genes. Freezing is one of the best ways to store bacteria and the lower the temperature, the longer the culture will retain viable cells. Ice can damage cells due to localised accumulation of salt, and it can also rupture membranes so we would need to use glycerol as a cryoprotectant. Freeze-dried cells could also be useful.</li>
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<li>We are currently looking into a system of a similar design to a chemostat where a continual supply of medium will allow maintenance of a culture.
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<li>Other options such as utilising microfluidics. </li>
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<p>For the key to be practical it would need to be portable, this is where the design of our key transport device comes in. We will be contacting experts for advice on the design of a product.</font></p>
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<h4> Creation of distinct spectra of different metabolite levels </h4>
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<h4> Promoter selection </h4>
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<p><font size="3" color="white">We would select a range of possible promoters to give a wide variety of product expression levels.</p>
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<p>We are looking at the following methods to achieve a random selection of product levels within any given bacteria:</p>
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<li>Transposon shuffling of promoters between products. </li>
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<li>dCas9 and a randomly selected gRNA from a library of gRNAs that can interfere differentially with the promoters associated with products.</li>
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<h4> Transposons </h4>
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<p><font size="3" color="white">We are looking at using Tn7 transposase due to its specific target site selection, which is impossible in other transposon species, without this modular increases in promoter activity could not be achieved as random insertions would create a gradient rather than step wise expression pattern of proteins.</p>
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<li>Induce the Tn7 transposase to switch a promoter from a range within in a cassette.</li>
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<li>The promoter inserted in front of each 3 of the reporter genes will be random.</li>
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<li>The promoters available for transposition will give different expression levels of the reporter.</li>
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<li>Promoters can be switched around again when the transposase is induced again.</li>
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<h4 color="white">CRISPRi or RNAi</h4>
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<p><font size="3" color="white">The second idea is using dCas9 and a library of sgRNAs, or a library of interference RNAs. The sgRNA/iRNA that is randomly selected would target one of our promoters in a way that represses transcription of that product, which would lower the levels of that metabolite. Eventually, by adding mutations into these RNA, we would also give more combinations for repressive ability. This would give a large amount of different combinations of metabolite levels for use as our key. Initially however,
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we would look at simply having ON/OFF levels for each metabolite.</p>
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<li>dCas9 can be used as a transcriptional repressor as it is nuclease deficient. A sgRNA is used as a guide to target a specific region</li>
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<li>Antisense RNAs could be used to target promoters and block ribosome binding.</li>
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<li>We could generate large libraries of sgRNAs/antisense RNAs which could be used to give different ON/OFF levels.</li>
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<li>This would be repeated to target the promoter/coding sequence of each gene in the plasmid.</li>
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<p><span style="color: #ffffff;">&nbsp;</span></p>
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<p><span style="color: #ffffff;">&nbsp;</span></p>
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<p><span style="color: #ffffff;">&nbsp;</span></p>
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<h1 style="text-align: center;"><span style="color: #ffffff;">PROJECT DESCRIPTION</span></h1>
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<p><span style="color: #ffffff;">&nbsp;</span></p>
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<h1>What?</h1>
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<p><b><i>Key. coli</i> provides a new, more secure, form of key for accessing content.</b> It uses random ligations and large repertoires of possible components to generate unique combinations of expression profiles; this next generation biological key could be the next BIG thing in security; watch this space!
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<h4 color="white">Possible Metabolites</h4>
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<p><font size="3" color="white">The metabolites that will be coded for by our constructs will be those previously registered with iGEM by previous teams,
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as the focus of this project falls on the assortment and variety of expression levels rather than the specific product. Nevertheless, we are looking into selecting various products that will be detectable by techniques such as volatiles being detected by GCMS. We are also looking at products that will not interfere with eachother. We will also choose products that are easily distinguishable from eachother on spectra. </font></p>
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<h1>Why?</h1>
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<p><b>Major hacking incidents are increasingly common,</b> with accounts being hacked and sensitive information stolen. Many companies are moving away from conventional passwords, which are proving to be unreliable in the hands of the public. Banks are now using physical biometric authentication procedures to correctly identify account owners. This new direction opens a market for biological “passwords”. An ideal system would be as separate from online software programs as possible while maintaining the complexity and uniqueness of a biometric system. Cells are effectively living computers, so we can programme cells to act as a changeable biometric password. </p>
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<h1>How?</h1>
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<p><b>A key must be unique, measurable and unpredictable.</b> In <i>Key. coli</i>, all these requirements are achieved by the random generation of modular vectors that are expressed in Escherichia coli to produce a unique and detectable fluorescent pattern. This pattern is obtained when different fluorescent proteins (GFP, RFP, CFP) and various promoters, subjected to transcription interference by dCas9, are randomly combined during ligation and transformed into the cells to generate the key. </p>
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<p><span style="color: #ffffff;">&nbsp;</span></p>
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<p><span style="color: #ffffff;">&nbsp;</span></p>
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<img src="https://static.igem.org/mediawiki/2017/8/84/UNOTT2017-How1.png" alt="" width="100%" height="100%">
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<h5><b> Figure 1:</b> Two-plasmid modular process used to generate random<i>Key. coli</i> construct(s)<p>
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<p><span style="color: #ffffff;">&nbsp;</span></p>
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<p><span style="color: #ffffff;">&nbsp;</span></p><p><span style="color: #ffffff;">&nbsp;</span></p>
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<p><span style="color: #ffffff;">&nbsp;</span></p>
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<img src="https://static.igem.org/mediawiki/2017/b/bc/UNOTT2017-How2.png" alt="" width="100%" height="100%"></h5>
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<h5><b>Figure 2:</b> Random ligation process and colony picking allows large numbers of plasmid variants to be created for use in keys. </p> <p><span style="color: #ffffff;">&nbsp;</span></p></h5>
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<p><span style="color: #ffffff;">&nbsp;</span></p>
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<br>
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<p>A key transport device, based on freeze-dried <i>Key. coli</i>, allows the bacteria to survive and be transported anywhere with ease. Once entry to a lock is desired, the <i>Key. coli</i> device can be activated, and the output read in a suitable detection device. </p>
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<h1><i>Key. coli</i> Summary</h1>
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Latest revision as of 03:45, 2 November 2017

 

 

 

PROJECT DESCRIPTION

 




What?

Key. coli provides a new, more secure, form of key for accessing content. It uses random ligations and large repertoires of possible components to generate unique combinations of expression profiles; this next generation biological key could be the next BIG thing in security; watch this space!

Why?

Major hacking incidents are increasingly common, with accounts being hacked and sensitive information stolen. Many companies are moving away from conventional passwords, which are proving to be unreliable in the hands of the public. Banks are now using physical biometric authentication procedures to correctly identify account owners. This new direction opens a market for biological “passwords”. An ideal system would be as separate from online software programs as possible while maintaining the complexity and uniqueness of a biometric system. Cells are effectively living computers, so we can programme cells to act as a changeable biometric password.

How?

A key must be unique, measurable and unpredictable. In Key. coli, all these requirements are achieved by the random generation of modular vectors that are expressed in Escherichia coli to produce a unique and detectable fluorescent pattern. This pattern is obtained when different fluorescent proteins (GFP, RFP, CFP) and various promoters, subjected to transcription interference by dCas9, are randomly combined during ligation and transformed into the cells to generate the key.

 

 

Figure 1: Two-plasmid modular process used to generate randomKey. coli construct(s)

 

 

 

 

Figure 2: Random ligation process and colony picking allows large numbers of plasmid variants to be created for use in keys.

 

 


A key transport device, based on freeze-dried Key. coli, allows the bacteria to survive and be transported anywhere with ease. Once entry to a lock is desired, the Key. coli device can be activated, and the output read in a suitable detection device.

Key. coli Summary