Harnessing the central dogma for stringent multi-level control of gene expression
Authors: Tobias J Erb
F Veronica Greco
Claire S Grierson
Thomas E Gorochowski
Amir Pandi
Journal: Nature Communications (2021)
Discipline: Multidisciplinary
Keywords: Gene expression; Inducible system; Central dogma; Transcription; Translation
Affiliations: School of Biological Sciences, University of Bristol, Bristol, UK., Department of Biochemistry and Synthetic Metabolism, Max Planck Institute for Terrestrial Microbiology, Marburg, Germany., SYNMIKRO Center of Synthetic Microbiology, Marburg, Germany., BrisSynBio, University of Bristol, Bristol, UK., School of Biological Sciences, University of Bristol, Bristol, UK. ., BrisSynBio, University of Bristol, Bristol, UK. .

Abstract:  Strictly controlled inducible gene expression is crucial when engineering biological systems where even tiny amounts of a protein have a large impact on function or host cell viability. In these cases, leaky protein production must be avoided, but without affecting the achievable range of expression. Here, we demonstrate how the central dogma offers a simple solution to this challenge. By simultaneously regulating transcription and translation, we show how basal expression of an inducible system can be reduced, with little impact on the maximum expression rate. Using this approach, we create several stringent expression systems displaying &gt;1000-fold change in their output after induction and show how multi-level regulation can suppress transcriptional noise and create digital-like switches between 'on' and 'off' states. These tools will aid those working with toxic genes or requiring precise regulation and propagation of cellular signals, plus illustrate the value of more diverse regulatory designs for synthetic biology. 

Retracted: No

PubPeer comments:
 In Abstract :  By simultaneously regulating transcription and translation, we show how **basal expression** of an inducible system **can be reduced**, with little impact on the maximum expression rate. Using this approach, we create several stringent expression systems displaying 1000-fold change in their output after induction From fig 4a: basal expression did not really reduce in 2 out of 3 stringent expression systems designed and seems to be already low in the native Ptac (black line). This is a misleading claim in the abstract which could have been worded better. I do see that table 1 reflects basal expression as a percentage of the maximum expression, so maybe this is what is being referred to in the abstract?  *Table 1* |Controller t|Type t |Basal (%) | |-------------|--------------------|----------------| |Ptac t|SLC t |0.5 | |THS t|MLC t |0.02 t| |STAR t|MLC t |1.45 t| |STAR2 t|MLC t |2.0 t| |DC t|MLC t |0.04t| , 
 Our statement in the abstract is indeed referring to the basal % expression of the construct. , 
 Ah, thanks for the clarification. I was searching for strategies to reduce the basal leaky expression in *absolute terms* and wasn t sure if I misunderstood the results of the paper. Also it would be really cool to see these constructs tested out for uninduced expression using a more sensitive reporter for really low expression- such as expressing a toxic protein and counting colonies, or expressing a protease and doing Western blot old school way to detect it s activity. Would love to know what you think about this. , 
 You are correct. It is exceptionally difficult to measure these things in absolute terms with the detection limits of flow cytometry. We tried to be forward about this problem, which was the reason for including the grey shaded regions in these plots (points where it is challenging to distinguish the value from zero). It would indeed be interesting to use a more sensitive methodology. We had thought about luciferase, but your suggestions are great too. ,