Joining in the #NAF2020 #RSCposter session fun! Here is my poster, How to make your E Coli Count, explaining the process of making binary counters using recombinases in the @UofG_DNApeople labs. Walkthrough thread! #sciencecommunication #dna #syntheticbiology #synbio 1/12

Binary counters, such as those seen in computing, can be acheived in synthetic biology by using recombinase proteins. 2/12

The counters are created by adapting pieces of DNA to combine the attachment sites of the recombinase, promoters and genes of interest. This creates what we call a 'switch'. 3/12

Once recombinase based switches have been created their complexity can be built to create a toggle switch, which in turn works as a binary counter. To help improve the complexity of counters requires 'orthogonal toggles', a set of switches without cross reactivity. 4/12

By creating orthogonal binary counters, numerous counters can be adapted into a single system. This can create a four-state binary counter as shown below (it expands). By creating this we are creating a mechanism which can be used to control biological functions. 5/12

The functions of a biological counter could be:

- In industry, to create delays in multiple step biological synthesis processes.
- In medical settings to regulate genetic therapies.

However the counter is a tool, and therefore can be applied to many biomolecular uses. 6/12

HOWEVER, making the switches is only one part of the process. Synthetic biology requires, and investigates, knowledge of the molecular mechanisms we use. Therefore we need to understand and improve upon the recombinases as well. 7/12

Previous research explains the mechanism of large serine integrases (Rutherford et al, 2014, https://pubmed.ncbi.nlm.nih.gov/24509164/ ) and shows that the recombinase co factor, the Recombination Directionality Factor (RDF), is key in recombination. 8/12

So an obvious step is to create investigative mutants of the RDF of a well known recombinase, ΦC31 integrase. Deletion mutants were a good first step. 9/12

The results of the deletions led to the creation of C terminal mutants of that RDF, which themselves highlighted residues of the RDF important for recombination. 10/12

So what can we conclude? Binary counters made from recombinases are possible, and we can make them. However, we need to integrate our findings about RDF proteins into our toggles. Most importantly we need to combine orthogonal toggles into a single system to... 11/12

make our e coli count!

Thanks for following the thread, I hope scientists and non-scientists alike have had a snapshot into this work. References on the original poster. Please tweet any questions you have. 12/12