Synthetic biology is among the most hyped research topics this century, and in 2010 it entered its teenage years. But rather than these being a problematic time, we've seen synthetic biology blossom and deliver many new technologies and landmark achievements. In 2020 synthetic biology turned 20 years old. It's first decade saw some impressive research papers, lots of visionary thinking and unprecedented excitement, but its second decade-from 2010 to 2020-was when the hype really needed to be replaced by some real achievements. So how has it done? The decade got off to a great start. Looking back at 2010, the biggest synthetic biology story of the year was the complete synthesis of a working bacterial genome by a team at the J. Craig Venter Institute (JCVI) 1. A landmark achievement that showed that DNA synthesis and DNA assembly could be scaled to megabase size, delivering on some of the biggest ambitions from the start of the century. However, just scaling DNA construction would not be enough to deliver the field's many other ambitions. 2010 also saw the publication of 'Five Hard Truths for Synthetic Biology' a critical article that examined how the lack of progress on engineering ambitions was slowing efforts to deliver on promises of reliability, standardisation and automated design 2. These were indeed problems for the field, and were highlighted in one of the first synthetic biology papers in Nature Communications which showed a robust genetic logic gate failing when moved into different E. coli strains 3. Could hard biological problems such as context, noise, burden and cross-reactivity really be solved to allow us to engineer cells like we wire-up electronic circuits? Well thanks to a lot of challenging technical biology and biological engineering work undertaken by many in the field, but especially MIT's Chris Voigt, the answer to this was yes. In 2016 Nielsen et al., published Cello, a remarkable end-to-end computer aided design system for logic circuit construction in E. coli 4. Of all the papers in the last decade, this is probably the most satisfying for hardcore synthetic biologists as it realises so much of the promised engineering of biology and does so through standardisation, characterisation and automated design. It's no coincidence that in the years preceding this paper Voigt's team worked tirelessly on delivering so many other foundational papers on E. coli synthetic biology, giving us algorithmic design of genetic parts, and professional characterisation of part libraries. While it is easy to focus on the many big landmark achievements of synthetic biology (Fig. 1), what has really helped the field deliver on the hype more than anything else has been a lot of hard technical work to improve our design and understanding of genetic parts alongside innovation and the discovery of new technologies that let us write, build, edit and share DNA code better than ever (Fig. 2). Indeed, looking back 10 years what is most striking is how the methods and tools have changed for those engineering life. Many...

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Natural organisms have evolved intricate regulatory mechanisms that sense and respond to fluctuating environmental temperatures in a heat- or cold-inducible fashion. Unlike dominant heat-inducible switches, very few cold-inducible genetic switches are available in either natural or engineered systems. Moreover, the available cold-inducible switches still have many shortcomings, including high leaky gene expression, small dynamic range (<10-fold) or broad transition temperature (>10°C). To address these problems, a high-performance cold-inducible switch that can tightly control target gene expression is highly desired. Here, we introduce a tight and fast cold-inducible switch that couples two evolved thermosensitive variants, TFts and TEVts, as well as an additional Mycoplasma florum Lon protease (mf-Lon) to effectively turn-off target gene expression via transcriptional and proteolytic mechanisms. We validated the function of the switch in different culture media and various Escherichia coli strains and demonstrated its tightness by regulating two morphogenetic bacterial genes and expressing three heat-unstable recombinant proteins, respectively. Moreover, the additional protease module enabled the cold-inducible switch to actively remove the pre-existing proteins in slow-growing cells. This work establishes a high-performance cold-inducible system for tight and fast control of gene expression which has great potential for basic research, as well as industrial and biomedical applications.