Memo-Cell project

Introduction

The Memo-cell project is a novel and original approach to allow for limitless counting of the signals a cell receives.

Counting is an essential process in our daily life, and so humans have invented many ways to count, from very simple manual counters to more complex logic gates implemented within electrical circuits. Implementing a counter in bacteria is essential to a foundation on which more complex devices that produce different outputs according to a pre-set time sequence can be built.

Counting with single-cells is a concept that has already been tackled but which is still in its infancy. Some counters have already been designed and implemented, but could only count up to three. Extension of these existing systems to achieve counting to higher numbers has limited feasibility, as these systems are limited by the number of different transcription factors / recombination enzymes that are characterized and that do not interfere with each other or "cross-talk." Moreover, it is important to notice that for these designs, once an element has been used for counting, it can not be reused.

In contrast, the approach we took here is free from these constraints, because we're using the same regulatory circuits cyclically. The framework for this approach is simple. We implement memory in the bacteria using a sequential integration of the same DNA pieces into the bacteria chromosome, controlled spatially and temporally. Memory will then be hardcoded in the genome by the number of DNA pieces integrated one after other in the genome.

Our design hijacks the phage integration mechanism to allow the bacteria to integrate in its chromosome a specific piece of DNA at a specific location called attachment site, every time it detects a specific input signal. Then we regenerate this attachment site through a second recombination event, by again hijacking the transposon systems. The successive detection of signals will results in the successive integration of DNA pieces one after the other into the chromosome. The total number of DNA pieces integrated on the chromosome will then correspond to the number of times the signal has been detected. For instance, we could integrate the previously developed light sensing module and plug it to our memo-cell module. Hence, the memo-cell module would be triggered when there is light, and our bacteria will count the number of days.

To achieve this goal, we had to extensively tinker with the following natural systems to engineer our three sub-modules, which we have done successfully in nearly 4 months of hard work:

• 1. The recombination system of Phages Lambda and HK022;
• 2. The recombination system of Transposon Tn916;
• 3. The microcin C51 from a specific E.coli strain.

With all the key components working, we have the full confidence of realizing our full system to count a fairly large number.