Difference between revisions of "Team:Leicester/Research"

Introduction

Due to the allotted lab time for the iGEM project, and various other constraints this project would not go to completion. Knowing this in advance required important decisions to be made in the early phases of the project's design, meaning sections that would be theoretical and those that were feasible for a lab based approach had to be defined. At this point decisions were made that determined the overall direction of the project, with the most important factor being what was possible within the timescale all the while ensuring the parts that were created were those which were most associated with the fundamental aim of the project. This page describes the theoretical sections of the project, taking into consideration why they would be required, ethical implications for their use as well as the steps and process that would have taken place to create them were the time available for a complete product. The reason as to why these particular components could not advance onto lab work stage will also be described.

Colonisation

Due to the allotted lab time for the iGEM project, and various other constraints, this project would not go to completion. Knowing this in advance required important decisions to be made in the early phases of the project's design, meaning sections that would be theoretical and those that were feasible for a lab based approach had to be defined. At this point decisions were made that determined the overall direction of the project, with the most important factor being what was possible within the timescale, all the while ensuring the parts that were created were those which were most associated with the fundamental aim of the project. This page describes the theoretical sections of the project, taking into consideration why they would be required, ethical implications for their use as well as the steps and process that would have taken place to create them were the time available for a complete product. The reasons as to why these particular components could not advance onto the lab work stage will also be described.

Escherichia coli Colonisation of The Intestinal Epithelial Layer

Maximising NAD uptake by mammalian cells is an essential aim of the project and also a requirement for the efficacy of the final product. It was therefore essential that the genetically modified (GM) E. coli were capable of colonising the intestine. The reason being, so they could be maintained in the environment for a longer period of time, ideally permanently and therefore have a longer lasting effect. However colonisation should also minimise NAD waste by reducing the distance between the GM E. coli and intestinal cells, this in turn should reduce the diffusion distance for NAD before uptake occurs. Another point to consider is that by doing this it may also prevent the human gut microbiome from utilising this additional NAD pool.

There are various pathogenic E. coli strains that have had their colonisation mechanism thoroughly researched, all of which colonise or invade the epithelial layer of the intestine. These would therefore be ideal for optimal NAD uptake, however the fact that they are pathogenic would prevent them from being a suitable option. Although with consultation from Dr Richard Haigh who had previously carried out research on various E. coli strain colonising mechanisms, it was decided that it might be possible to utilise an operon or edit one that is involved with colonisation. The strain suggested was enteropathogenic E. coli (EPEC); it has two initial colonisation capabilities. One is a plasmid based type four pili known as bundle forming pili (BFP) and the second is a type three secretion system (TTSS) contained within the genome. Figure one below shows a simplified colonisation mechanism (Kaper et al., 2004).

Kill Switch

There are two kill switch systems in the genetically engineered bacteria: One, a kill switch which causes the bacteria to die upon addition of an inducer; the other, a maintenance kill switch whereupon the bacteria die if they leave the gut. This dual containment system allows for the reduction in bacterial escape and horizontal gene transfer as well as the death of the bacteria as a backup option if it goes wrong.

Potentially could use a toxin-antitoxin system but with a modified antitoxin protein which incorporates a nonstandard amino acid that is vital for the toxin function. Therefore upon induction of the nonstandard amino acid the toxin will have the right amino acids needed for its correct synthesis and thus will kill the cell. It is easier and simpler than the altering 22 essential enzymes like Rovner et al, 2015 but more effective than toxin-antitoxin systems. However, this approach will need to add a stop codon for the nonstandard amino acid in a key hydrophobic region (if the nonstandard amino acid is hydrophobic) (Rovner et al, 2015) so that without the non-natural amino acid the protein cannot fold correctly and thus be subject to proteolysis. This results in cell death upon the addition of the non-standard amino acid. However this would require the changing of the stop codon used to another stop codon in all other genes as well as engineering a tRNA synthase that charges the non-natural amino acid to an edited tRNA molecule that is cognate for the stop codon used.

The CCDA/CCDB kill switch is in an operon system with CCDA under the control of a temperature sensitive RBS (Part BBa_K115002) whereas the CCDB is under the control of a generic RBS (Part BBa_K581008). This means that at under 37^OC (i.e. not in the human body) the translation rate of the CCDA will dramatically decrease relative to other genes in the bacteria at the same temperature, whilst CCDB will remain the same in respect to the fundamental decrease in rate due to the lower temperature. Thus there will be a high enough ratio of CCDB (once translated) to kill the cell. Summed up: When the temperature is too cold, the antitoxin (CCDA) doesn’t work, so the toxin (CCDB) kills the cell.

However, CCDB is patented so another toxin/antitoxin system would be preferred such as MazF/MazE. If no antitoxin is available then the antitoxin in the system described above could be replaced with a polymerase gene (e.g. T7 Polymerase) and the desired toxin under the control of a promoter for that polymerase (i.e. a T7 consensus promoter). This would give higher levels of transcription and would be perhaps a more sure system. For our iGEM team however, due to time requirements, the simpler the system the better.

Another Kill Switch which could be used to selectively kill the bacteria would be to use the X and Y expansion of the genetic code by Malyshev, et al 2014. This would use a toxin (Such as MazF toxin) that is dependent on the bases X and Y. This can be done by using long-range PCR for the MazF inside pUC19 but leaving a few bases in between the forward and the reverse primer. Then synthesis and PCR amplify an oligonucleotide which contains the primers and the X (dNaM)/Y (d5SICS) codon (Malyshev, et al 2014) for the non-natural amino acid for that codon (assuming there is a tRNA synthase for this). This can then be inserted into the PCR amplified pUC19 plasmid (with the gaps) through Gibson Assembly to then be transformed (Malyshev, et al 2014) as can be seen in figure 1. Proof will be needed for the incorporation of the X/Y through testing whether the transformed cells only die through addition of the X and/or Y base in a medium lacking these. This would significantly reduce the likelihood of the bacterium kill switch being activated naturally in the human microbiome. However, like through the addition of a nonstandard amino acid via stop codons, a tRNA molecule will be needed that can recognise the synthetic bases as well as a tRNA synthase which can accurately charge the nonstandard amino acid to this tRNA. As such this method can only be used in theory for our iGEM team. This will be a very useful application of synthetic biology once these key tRNA’s and tRNA synthases are ready.

Further Consideration

siRNA Inhibition

Not practical as there is no viable/practical clinical application.

Regulation of Cassette

With further work we hope to fine-tune the levels of NAD+ output by inserting negative feedback promotor sequences. We would vary the consensus sequence until assays had confirmed an optimal level of NAD+ was being secreted, levels of which could be determined by extensive literature research and further experiments in vivo and in vitro.

Further objectives

• Once the BFP Biobrick had been constructed and its functionality confirmed; it would then be necessary to see if it can work in tandem with the plasmid containing the genes responsible for increasing NAD output, as well as the killswitch.
• However as each plasmid would require a different origin or replication, it would be more likely that the contents of each plasmid would be combined. This meaning the killswitch, NAD genes, and BFP genes would be combined as one cassette in a specific order. The length of the overall construct would be nearing 20 kb by this point although, for iGEM parts the three components to the project would be submitted independently. The significant size of the construct may require a bacterial artificial chromosome to enable it to function effectively.
• However for this project to have real potential the three components required for effective treatment would need to be incorporated into the genome as this would prevent horizontal gene transfer, increasing efficacy

References:

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