Team:IIT Madras/Summary

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Our project PLASMID - Plasmid Locking Assembly for Sustaining Multiple Inserted DNA - introduces a new paradigm in gene regulation. The study is based on the concept of plasmid loss. Any episome (extrachromosomal genetic element) introduced into the cell shows a segregational asymmetry accompanied with differential growth rates in the absence and presence of episome leading to an overall loss of the episomal unit in the absence of any maintaining selective pressure. It is hypothesized that by appropriately controlling the external selective pressures, one can control the direction of plasmid loss in the cell, modifying the existing gene regulation system in a pre determined manner. It is also hypothesized that introducing negative selective pressures against certain other directions of plasmid loss, in the form of constitutively repressed endotoxins will help streamline the regulatory system even further.

If successful, this study allows for exquisitely delicate and precise multifactorial regulation of gene control in the future. This model can also be used to hide genes of commercial interest to protect it from unauthorized use (under some conditions).

Summary
Our project is based on the fundamental concept of plasmid instability in a novel way to conceal information or ‘lock’ a gene’s function in a cell until the correct combination of inputs is fed into the cell. We call this a ‘combinatorial lock’ or PLASMID. It involves the positive regulation of the gene of interest only on receiving the correct inputs from the user. We use plasmids which can confer resistance to certain antibiotics in the medium and link them up in a certain way (i.e, essentially designing a genetic circuit) so that they repress the expression of the gene of our interest. As the selection pressure is lifted from the media, the plasmids which have the repressors for the gene of interest are lost, hence revealing the gene on using the correct series of antibiotic washes. In essence, the process of unlocking would simply be the correct sequence of antibiotic media in which the cells should be washed.

We would be working with a 2 plasmid system and it is easy to see that this principle, theoretically, could be extended to N plasmids. In general the code length required to "unlock" is N-1 if the number of plasmids introduced are N. In our case, since the number of plasmids being introduced are 2, the code would essentially be just 1 unit long. Particularly in this case, the 1 unit of code corresponds to growing the cells in one correct antibiotic medium.



 Fig 1: ''The gene of interest is "locked" or repressed by the inhibitor in the plasmid 2. The plasmids are linked up in a certain way that the plasmids need to be lost only in a very specific order, else the cells die due to the release of a toxin. Thus the "unlocking" of the gene of interest requires a predetermined order of growth conditions which allows for a directional loss of the plasmids, and hence the repressor for the promoter that expresses the gene of interest.''

 However, in the experiments we have not incorporated any particular gene to be repressed. Instead we study how can we achieve a directed loss of plasmids which is the idea central to the working of the system. In place of a gene of interest, we have placed fluorescent reporters in each plasmid to monitor the presence or absence of any particular plasmid.