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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>
 
  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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Revision as of 12:05, 14 August 2017

< style=" background-image: url(https://static.igem.org/mediawiki/2017/e/e7/T--UNOTT--backgroundnorepeat.png);">

Project Description

Password security is broken

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.

The Idea: Key. coli

  • The first biological password that changes over time! 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).
  • 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.
  • 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.

Bacterial Key Transport Device

There is a need for a transport mechanism for the key. This presents problems depending on the bacteria used.

In E. coli, our key transport system would need to keep our colonies alive. We have looked into a few options for key storage:

  1. 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.
  2. 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.
  3. Other options such as utilising microfluidics.

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.

Creation of distinct spectra of different metabolite levels
Promoter selection

We would select a range of possible promoters to give a wide variety of product expression levels.

We are looking at the following methods to achieve a random selection of product levels within any given bacteria:

  1. Transposon shuffling of promoters between products.
  2. dCas9 and a randomly selected gRNA from a library of gRNAs that can interfere differentially with the promoters associated with products.
Transposons

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.

  • Induce the Tn7 transposase to switch a promoter from a range within in a cassette.
  • The promoter inserted in front of each 3 of the reporter genes will be random.
  • The promoters available for transposition will give different expression levels of the reporter.
  • Promoters can be switched around again when the transposase is induced again.
CRISPRi or RNAi

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, we would look at simply having ON/OFF levels for each metabolite.

  • 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
  • Antisense RNAs could be used to target promoters and block ribosome binding.
  • We could generate large libraries of sgRNAs/antisense RNAs which could be used to give different ON/OFF levels.
  • This would be repeated to target the promoter/coding sequence of each gene in the plasmid.

Possible Metabolites

The metabolites that will be coded for by our constructs will be those previously registered with iGEM by previous teams, 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.