Establishing a standardized genetic toolkit for the radiation-resistant extremophile Deinococcus radiodurans

Establishing a standardized genetic toolkit for the radiation-resistant extremophile Deinococcus radiodurans | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Establishing a standardized genetic toolkit for the radiation-resistant extremophile Deinococcus radiodurans View ORCID Profile Trevor R. Simmons , View ORCID Profile Antonio Cordova , Kobe B. Grismore , Laci C. Moline , Anna C. Stankes , Jennah Johnson , Christian Bök , View ORCID Profile Lydia M. Contreras doi: https://doi.org/10.1101/2025.06.09.658681 Trevor R. Simmons 1 McKetta Department of Chemical Engineering, The University of Texas at Austin , 200 E. Dean Keeton Street Stop C0400, Austin, TX 78712, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Trevor R. Simmons Antonio Cordova 2 Department of Molecular Biosciences, The University of Texas at Austin , Austin, TX 78712, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Antonio Cordova Kobe B. Grismore 1 McKetta Department of Chemical Engineering, The University of Texas at Austin , 200 E. Dean Keeton Street Stop C0400, Austin, TX 78712, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Laci C. Moline 3 Department of Biomedical Engineering, The University of Texas at Austin , 107 W Dean Keeton Street Stop C0800 Austin, TX 78712, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Anna C. Stankes 1 McKetta Department of Chemical Engineering, The University of Texas at Austin , 200 E. Dean Keeton Street Stop C0400, Austin, TX 78712, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jennah Johnson 2 Department of Molecular Biosciences, The University of Texas at Austin , Austin, TX 78712, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Christian Bök 4 Leeds School of Arts, Leeds Beckett University , Portland Way, Leeds LS1, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site Lydia M. Contreras 1 McKetta Department of Chemical Engineering, The University of Texas at Austin , 200 E. Dean Keeton Street Stop C0400, Austin, TX 78712, USA 2 Department of Molecular Biosciences, The University of Texas at Austin , Austin, TX 78712, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Lydia M. Contreras For correspondence: lcontrer{at}che.utexas.edu Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Deinococcus radiodurans is a highly radiation-resistant extremophile with potential for biomanufacturing and bioremediation in harsh environments, including extraterrestrial settings. However, genetic engineering in this organism has been constrained by limited genetic tools. Here, we establish a comprehensive genetic toolkit for D. radiodurans that enables tunable gene regulation with genome-engineering tools. We have standardized a library of 36 constitutive promoter sequences sourced from the native D. radiodurans genome and from synthetic sources, spanning 45-fold range gene expression in the context of plasmids. We have also identified 136 variants of ribosome binding sites (RBS), variants using a high-throughput screen for precise translational control across a 963-fold range of expression when used in our plasmid-based system. Additionally, we have developed a codon-optimizer script that we leverage to improve the function of four fluorescent proteins in D. radiodurans. Next, we characterized 9 small molecule-inducible promoter systems, and identified four key inducible promoter systems that achieve between 3-fold and 12-fold signal amplification, as well as titratability across induction concentrations in D. radiodurans. To engineer the D. radiodurans genome, we present a novel method for gene integrations, compatible with de novo sequences, up to 3kB in length, doing so with 87% efficiency. Lastly, we repurpose the RNA-directed nuclease, TnpB, as a novel post-transcriptional tool for programmable gene repression analogous to CRISPRi-based systems, and this tool can achieve up to 70% repression of its target. Collectively, this toolkit provides modular, standardized components for both plasmid engineering and chromosomal engineering in D. radiodurans to improve its genetic tractability and to facilitate its deployment. Download figure Open in new tab Introduction As synthetic biology advances, more microbes become genetically tractable, which opens the door for novel bioprocessing applications. Current model organisms have supported many advances in the field and provide robust chassis for prototyping novel tools. However, these organisms can struggle to perform outside of standard lab conditions in the absence of significant genetic modifications. An underexplored alternative is to deploy genetic tools in microbes that have been natively evolved to survive in harsh or unfavorable environmental conditions unsuitable for standard lab conditions; these include high salinity, harsh pH, extreme desiccation, and elevated radiation 1 – 4 . Commonly referred to as Next-Generation Industrial Biotechnology (NGIB) 5 , these microbes and associated engineering tools enable new opportunities in both bioprocessing and biomanufacturing. Two examples are Corynebacteria 6 and Halamonas species, which as high producers of N-acetylglucosamine and hyaluronic acid and native to sea water, respectively 7 , 8 , could find high relevance to cosmetic, food pharmaceuticals and agricultural industries. Likewise, use of microorganisms for extraterrestrial applications 9 is of high interest given the desire to explore Biomanufacturing in space, where the durability of current model organisms (e.g. Escherichia coli and Saccharomyces cerevisiae adds to the challenges of biomanufacturing logistics, and microbial resource allocation 10 . An important factor in the development of effective extraterrestrial biomanufacturing tools is the ability for microbes to withstand higher doses of radiation exposure, since most space travel lacks radiation shielding from an atmosphere 11 , 12 . Most bacteria struggle to survive prolonged radiation exposure, particularly those identified as GRAS or commensal bacteria 13 . One species of interest for extraterrestrial biomanufacturing is Deinococcus radiodurans 14 as it can withstand high levels of ionizing radiation 15 . Additionally, D. radiodurans has recently demonstrated the ability to survive for up to three years in space 16 . The ability of D. radiodurans to maintain its genome under ionizing radiation and oxidative stress endows it ability to grow in harsh environments, as well as to utilize its metabolism to degrade toxic chemicals 3 , 17 . For these reasons, several potential applications for both bioprocessing and bioremediation have been demonstrated using D. radiodurans ; some include degradation of compounds found in nuclear waste, remediation of mercury pollution in soil, and conversion of metallic waste to nanoparticles 2 , 3 , 18 . Given the promising utility of this organism, it is crucial to establish a standardized, genetic toolkit in D. radiodurans that includes all the critical features required for efficient user-controlled gene regulation and genome engineering, as it has been done for other industrially-relevant microbes 19 – 26 . The standardization of genetic components not only allows for improved transferability of work, but also allows the field to refine engineering applications in this organism. Currently, engineering efforts in D. radiodurans are currently limited by the number of characterized genetic components and tools available in this organism. Since its discovery and initial characterization in 1956 27 , only one plasmid for heterologous gene expression has been widely established 28 , and only a handful of promoters native to D. radiodurans have been studied 29 , 30 , with only one fully characterized ribosome binding site 29 . Currently, the only small molecule-inducible promoter that has been ported from model organisms into D. radiodurans is a LacI-based system developed by Lecointe and colleagues over two decades ago 31 . Since then, few efforts have been made to standardize or identify other small-molecule inducible promoter systems that are functional and titratable in D. radiodurans . Beyond plasmid-based gene expression, there has been minimal effort to develop a protocol for efficient genome control, through either editing or regulating genomic expression. Moreover, previous work has demonstrated that Cas-based genome editing is harmful to cellular health in D. radiodurans, and this problem has limited the development of tools for use in the organism 32 . In this work, we establish a genetic toolkit for precise gene regulation by first identifying 2 new origins of replication for plasmid-based expression in addition to the single previously identified origin of replication. We then standardize 36 promoter sequences, sourced from the native D. radiodurans genome and from synthetic sequences, all which span a 45-fold range of gene expression. We also design a library of ribosome-binding site (RBS) sequences that provide tunability across almost three orders of magnitude (963x) for precise gene control, both transcriptional and translational. Next, we identify 3 small molecule inducible transcriptional regulator constructs with robust functionality in D. radiodurans . Concurrently, we introduce two tools for genome engineering and regulation: (i) an optimized method for genomic integration up to 3kB in length and (ii) a genomic post-transcriptional control system that repurposes the native transposon-associated protein TnpB to allow for targeted repression in the genome of D. radiodurans . Overall, with this toolkit, we provide users with both plasmid-based control and genomic-target control, applicable to biological engineering in D. radiodurans . Results Screening putative and synthetic promoter sequences for standardized transcriptional control Historically, the only plasmid broadly used in D. radiodurans is the pRAD1 vector derived from work by Meima et al., 28 — a vector that contains the minimal origin of replication (ori) sequence from the SARK cryptic plasmid in D. radiodurans, a sequence known as RepU. Meima and colleagues added the ColE1 ori as well as antibiotic resistance cassettes to the sequence to create a shuttle vector between E. coli and D. radiodurans. This plasmid has served as the base vector for nearly all engineering applications in D. radiodurans over the last two decades 15 , 33 – 36 . The pRAD1 plasmid also contains the P GroES promoter native to D. radiodurans and its ribosome binding site (RBS) sequence. Here, we design a new vector by reducing the size of the original pRAD1 plasmid, removing the ampicillin resistance cassette as well as the truncated P CAT promoter (located upstream of the chloramphenicol resistance cassette). We replace the latter with the full P CAT promoter from the pAcyc184 plasmid. This plasmid design conserves the well characterized promoter and RBS from the original pRAD1 plasmid. To ensure functionality, we inserted a sfgfp CDS downstream and we observed constitutive sfGFP fluorescence. We have named this plasmid pAT00.20.001-sfGFP, and this construct serves as our base plasmid for all future constructs in this work ( Fig. 1a ). Download figure Open in new tab Figure 1. Screening native and synthetic constitutive promoters to establish a standardized set of constitutive promoters in D. radiodurans. A) Plasmid map for pAT00.20.001-sfGFP base plasmid. B) Schematic for testing each of the constitutive promoters from the native set as well as the synthetic set. The original P GroES promoter was replaced with each promoter, then fluorescence of sfGFP was measured. C) sfGFP signal after 24 hours of culturing under the control of each native promoter from D. radiodurans. D) sfGFP signal after 24 hours of culturing under the control of each synthetic Anderson Promoter. E) Compiled promoter library from the native and synthetic promoter sets from 1C and 1D plotted on the same graph for ease of comparison. Given that the P GroES promoter is one of the few well-characterized promoters known in D. radiodurans , users have been limited in their ability to precisely tune heterologous gene expression. Previous work done by our lab used computational approaches to identify 5’ untranslated regions (5’ UTRs) from the genome of D. radiodurans , assessing these regions as sequences that might contain putative promoters and ribosome binding sites 30 . We screened 30 candidate 5’ UTR sequences to generate a standardized set of constitutive promoters for D. radiodurans . Since in previously published work, the 5’ UTR sequences contained both the promoter and RBS, it was important that we isolated the promoter sequence to directly compare expression between each promoter. To accomplish this task, we ran the set of 30 candidate sequences through two computational promoter-prediction tools, BPROM 37 and bTSSfinder 38 . We then selected candidates that contained the same transcription start site identified in both BPROM and bTSSfinder, thus reducing our total number of putative promoters to 12 ( Fig. 1b , Supplementary Table S1). We replaced the P GroES promoter in the pAT00.20.001-sfGFP plasmid with each putative promoter sequence and measured sfGFP fluorescence after 24 hours of culturing. Across the 12 new promoters and the P GroES promoter, we observed an approximate 45-fold range of fluorescence, with P GroES being the relatively strongest promoter within the set. Importantly, 8 of the 12 new sequences demonstrated sfGFP fluorescence significantly greater than background fluorescence of R1 D. radiodurans cells in liquid media ( Fig. 1c ). Concurrently, we screened the well-established Anderson Promoter Collection ( https://parts.igem.org/Promoters/Catalog/Anderson ), because sequences in this catalogue have been shown to work in a wide range of bacterial species 39 – 42 . We again replaced the P GroES promoter with each of the promoters from the Anderson Promoter Collection and measured sfGFP signal after 24 hours. All the Anderson Promoters demonstrated significant fluorescence above the background of R1 cells ( Fig. 1d ). Interestingly, the relative strengths of the promoters differed from what was previously observed in E. coli . While the J23113 promoter remained the weakest in both E. coli and D. radiodurans , the J23104 promoter demonstrated the strongest fluorescence in D. radiodurans , compared to J23119 and J23100 being the strongest in E. coli . Importantly, the J23104 promoter demonstrated even stronger fluorescence in R1 compared to the P GroES promoter when compiling the two sets of promoters into a single graph ( Fig 1e ) which was created for ease of comparison between the two promoter sets. In total, the suite of 28 promoters provided precise tunability across a 45-fold signal range ( Fig. 1e , Supplementary Table S1). With this newly defined set of promoters, targeted gene-expression can be more precisely tuned. We provided a compiled subset of plasmids containing these constitutive promoters that offer 45-fold range of plasmid-based gene expression in Table 1 ( Table 1 - pAT00_33_001_sfGFP, pAT00_28_001_sfGFP, pAT00_08_001_sfGFP, pAT00_11_001_sfGFP, pAT00_04_001_sfGFP). View this table: View inline View popup Table 1. Compiled Plasmids and Strains from the Toolkit of Particular interest to the field. Plasmids and strains were compiled to provide most essential plasmids and strains derived from this toolkit to provide future users with maximum range of control and tunability. Sections are color-coded as the following: Light Blue – Constitutive Promoters, Light Green – RBS Library, Orange – Codon Optimized Genes, Pink – Inducible Promoters, Grey – Genomic Regulatory Plasmids. Sequences of interest, the relative performance, and link to a Benchling plasmid map are also provided. Establishing a composable Ribosome Binding Site (RBS) Library for translation control After establishing a suite of promoter sequences, we developed a suite of well-defined ribosome binding sites (RBS). Earlier computational models have been developed to accurately predict RBS strengths in model organisms, such as E. coli 43 – 45 . However, these predictive models are not optimized for D. radiodurans . Therefore, we chose to establish a library of composable ribosome binding site sequences for tunable translational control. To do so, we adapted a protocol previously utilized for generating an RBS Library in the non-model organism Cupriavidus necator 46 . In short, we replaced the GGAGG sequence in the GroES ribosome binding site sequence with an NNNNN sequence pool (N representing any nucleotide) and transformed the library into E. coli . During the transformation, we observed that at least 1,024 single colonies formed, ensuring that each potential 5-nucleotide sequence could be represented in the library. Six individual colonies were sent for sequencing, and all 6 plasmids contained different ribosome binding site sequences. These results gave us confidence that our library generated sufficient sequence diversity. The purified library was transformed into D. radiodurans R1, and 136 colonies were selected for screening. We chose 136 colonies because this number was greater than 10% of the potential sequence space but still constituted a manageable size such that we could test every sample in a single experiment ( Fig. 2a ). From our 136 samples, 127 generated fluorescent signals, which became our final library. From those 127 constructs, our library achieved a 963-fold signal range between the weakest, RBS93, and the strongest, RBS102 ( Fig. 2b , Supplementary Table S4). Additionally, all constructs demonstrated fluorescence significantly greater than R1 cells alone. After screening the library, we sequenced each colony and estimated the predicted RBS strength for each sequence in the library using the DeNovoDNA RBS Calculator. We found poor correlation (r 2 = 0.02) between the predicted RBS strength and the fluorescent output of each construct (Supplementary Figure S1). Lastly, we compiled key plasmids containing a subset of the RBS Library thatprovide the largest range of control in Table 1 for ease of access ( Table 1 - pAT00_20_042_sfGFP, pAT00_20_014_sfGFP, pAT00_20_025_sfGFP, pAT00_20_037_sfGFP, pAT00_20_102_sfGFP). Download figure Open in new tab Figure 2. Synthesizing a composable Ribosome Binding Site (RBS) Library for translation tunability in D. radiodurans. A) Workflow for generating the RBS Library. Briefly, the GGAGG sequence in the GroES RBS was replaced with an NNNNN sequence, where N is any nucleotide. The library was transformed into E. coli then purified via MidiPrep. The purified library was transformed into D. radiodurans , and sfGFP signal was measured after 24 hours of growth for 127 colonies in addition to the original RBS sequence. B) Relative fluorescence of sfGFP for each of the 127 RBS sequences. The original RBS sequence is denoted as the black bar on the plot. Overall, the resulting library generated a 963-fold range of sfGFP expression. Analyzing codon usage and start codon sequence preferences for codon optimization of heterologous genes Because D. radiodurans has a high GC-content (~66%) relative to microbes such as E. coli (50%), codon preferences could significantly differ between these organisms, meaning the sequences of the heterologous genes developed in E. coli could lead to imperfect protein translation. Therefore, we wanted to build a codon optimization tool that could recode sequences to match codon usages in D. radiodurans. To build our codon optimizer, we leveraged the codon usage data for D. radiodurans collected by the Lowe Lab 47 . We then developed a program that would analyze the DNA sequence for each codon in a CDS and modify the sequence as needed to ensure each codon of the CDS corresponded to the sequence with the highest usage rate in D. radiodurans (code provided in Supplementary Material). To validate this approach, we ran 4 fluorescent reporter sequences ( sfgfp, mcherry, morange , and mturquoise ) through our program to optimize for expression in D. radiodurans (Supplemental Methodologies, Supplementary Table S3). We then compared fluorescence of the original (WT) proteins against their codon-optimized counterpart ( Figure 3A-D ). mCherry, mOrange, and mTurquoise all showed a significant increase in fluorescence, while sfGFP demonstrated an increase that was not statistically significant. To complement our codon-optimizer tool, we evaluated preferred start codon sequences in D. radiodurans and found that the “GTG” start codon led to significantly increased fluorescence compared to the start codons “ATG” and “TTG” in the sfGFP CDS ( Figure 3E ). Our codon-optimizer tool and our start codon preference work help translate protein-based systems previously developed in other hosts, into D. radiodurans. We provide our sfGFP and mCherry codon optimized plasmids in Table 1 for ease of access ( Table 1 - pAT00_20_001_sfGFP_CO, pAT00_20_001_mCherry_CO). Download figure Open in new tab Figure 3. Codon optimization of heterologous genes and start codon preference in D. radiodurans . A-D) Relative fluorescence of wild type fluorescent proteins versus their D. radiodurans codon optimized version. E) Relative fluorescence of WT sfGFP with the specific start codon ATG, TTG, or GTG. Asterisks indicate a p-value < 0.05 or less using a heteroscedastic T-test between experimental conditions. Screening and optimizing transcriptional regulators for small molecule-inducible gene expression While tuning gene expression through constitutive promoters is an important building block for developing a genetic toolkit, another key step is to develop components for user-controlled gene expression. The gold standard approach is to utilize small molecule-responsive transcriptional regulators such that, upon addition of a small molecule, transcription of a target sequence is initiated. Most transcriptional regulators currently utilized in synthetic biology are optimized for applications in E. coli or other model bacteria. As observed in the Anderson Promoter Collection, trends observed in E. coli do not necessarily correlate to those in D. radiodurans. Currently, there is one established transcriptional regulator system that has been previously shown to function in D. radiodurans —the Spac system 31 . In this previous work, the LacI regulatory protein was shown to inhibit transcription from the P spac promoter in D. radiodurans and, upon addition of Isopropyl β-D-1-thiogalactopyranoside (IPTG), transcription was activated. While this system has been a valuable foundation to build sophisticated genetic systems in D. radiodurans , it is important to have multiple, orthogonal transcriptional regulators, to precisely tune gene expression. Therefore, we screened several well-established transcriptional regulators and then subsequently optimized the most promising candidates for effective performance in D. radiodurans. We started with the “Marionette” promoter regulator collection developed by Meyer and colleagues. The “Marionette” collection consists of 14 optimized transcriptional reporters, all engineered to operate orthogonally of each other 48 . We hypothesized that this collection would be an effective place to start, since entries in this catalogue have been shown to function in other non-model organisms 46 , 49 ; moreover, this catalogue enabled us to survey a large number of small molecule-inducible promoters. To screen the “Marionette” promoter collection, we replaced the P GroES promoter from our pAT00.20.001-sfGFP vector with each of the inducible promoter sequences from the “Marionette” collection, and we inserted each corresponding regulatory protein directly downstream of the rrnBT1T2 terminator. Of the original 14 promoter constructs created in the “Marionette” promoter collection, we were able to clone 9 of them for our plasmid-based system. With the 9 inducible promoter designs, we titrated each respective small molecule inducer at different concentrations in liquid culture, and then we measured the fluorescent signal of the sfGFP protein after 24 hours of culturing at each induction condition. The anhydrotetracycline-(aTc), Cuminic acid-(CA), IPTG- and Salicylic Acid (SA)-inducible regulators all achieved at least 5-fold increase in signal between the uninduced conditions and the saturated, induced conditions. Additionally, the IPTG-, aTc-, and SA-inducible constructs demonstrated precise tunability between induction concentrations ( Fig. 4a, c, f ). The CA-inducible regulator demonstrated rapid increase in signal between the 100 nM conditions and the 1 μM conditions, but then saturated at all greater induction concentrations ( Fig. 4h ). Ultimately, after screening 9 systems, we identify four inducible promoter systems (IPTG, SA, CA, and aTc) that offer up to a 12-fold range of titratable gene expression via plasmids ( Table 1 – pAT00_40_001_sfGFP, pAT00_43_001_sfGFP, pAT00_44_001_sfGFP, and pAT00_45_001_sfGFP). Download figure Open in new tab Figure 4. Screening and optimizing small molecule-inducible transcriptional regulators in D. radiodurans . A-I) Titration curves for each small molecule inducer measured by sfGFP signal 24 hours after induction. J) Fold change in sfGFP expression for each small molecule inducer between the uninduced and fully induced conditions. Integrating stable expression of synthetic genes and biological signals in D. radiodurans In addition to plasmid-based genetic components, developing tools for precise genome editing is critical for making any microbe a versatile biological chassis given that integrations eliminate the need for plasmid-based expression and therefore eliminates the need to culture with antibiotic selection. There has been recent work to develop protocols for genomic deletions in D. radiodurans , all of which rely on conjugation-based techniques 50 , leveraging the bacterium’s natural double-crossover and homologous recombination abilities 15 , 33 , 51 . Additionally, due to its resistance to both oxidative stress and ionizing radiation, D. radiodurans is a top candidate for biological storage of data within its genome. However, there has yet to be a well-established method for efficient genome integrations in D. radiodurans . To develop our integration protocol, we selected a large (> 2kb) non-native synthetic protein, nicknamed “The Xenotext” (XenO) as a proof of concept. XenO is a synthetic, his-tagged protein designed in collaboration with Dr. Christian Bök, an experimental poet. XenO was developed for the purpose of encoding messages into the genome of a stress-resistant bacterium (Supplementary Table S5) 52 . We also chose to fuse XenO to a fluorescent mCherry protein, since the integrated XenO synthetic protein could then be tracked over time and easily identified without genome sequencing. The application of encoding messages through bacterial genomes has been previously established in model organisms 53 , 54 , and the stress-resistance properties of D. radiodurans position this bacterium as an ideal candidate for long-term biological storage of information regardless of external conditions 55 . With this goal in mind, we established and optimized a protocol for the integration of information into the genome of D. radiodurans . It is worth noting that a challenge with genomic engineering of D. radiodurans lies on the fact that it has multiple copies of its genome ranging from 8-10 at exponential phase 56 . Our protocol leverages the double-crossover, homologous recombination mechanism that D. radiodurans employs for genome repair and horizontal gene-transfer 57 . We chose to insert the XenO-mCherry fusion between the DR_0963 and DR_0937 coding sequences in the R1 genome, because there are approximately 200 base pairs between the two genes, and because there are no annotated open reading frames (ORFs) on the reverse strand. We hypothesized that these two factors combined would minimize disruption to the bacterial genome. The 1000 nucleotides upstream and downstream of the insertion site were then amplified from the genome to flank the 5’- and 3’-regions of the synthetic sequence, which contained the XenO-mCherry fusion sequence and a kanamycin resistance marker. This marker contained flanking sequences of both lox66 and lox71 for subsequent removal of the resistance marker, if desired ( Fig. 5a ). We then cloned the entire design into the pUC19mPheS plasmid ( Fig. 5a ). To begin the process of integration, we transformed this plasmid into D. radiodurans R1 cells (where the homology arms are recognized and homologous recombination occurs), then incubated the sample overnight. Strains containing the XenO-mCherry integration were selected by using TGY agar plates supplemented with 16µg/mL kanamycin and 5 mM 4-chloro-phenylalanine. The 4-chloro-phenylalanine and kanamycin selected for single colonies that had undergone successful recombination and had cured out the pUC19mPheS plasmid during the overnight incubation ( Fig. 5a ). Using this protocol, we were able to successfully integrate our XenO-mCherry fusion sequence into the genome of D. radiodurans with 70% efficiency after one round of selection, 86% efficiency after two rounds of selection, and 100% efficiency after three rounds of selection, for an average efficiency of 60%. These integrations were confirmed with colony PCR ( Fig 5b ) as well as full genome sequencing (Supplementary Figure S2). We chose to repeat this process to evaluate the number of rounds required to have all screened colonies fully saturated with the synthetic integration. To ensure proper functionality of our mCherry fusion protein, we tested multiple variations of the XenO sequence and found that fusing mCherry to the C-terminus of the XenO version 3 generated the greatest fluorescence signal (Supplementary Figure S2). We then validated the production of the XenO-mCherry fusion by measuring fluorescence of our strain in liquid culture ( Fig. 5c ), as well as by microscopy ( Fig. 5d ). Our results demonstrated that genome integrations of at least 2kB could be achieved using a single plasmid in a two-step process that yielded 60% efficiency for target integration and present a plasmid ( Table 1 - pUC19mPheS_GINT_C3C) that can be easily modified for the desired target of the user. Download figure Open in new tab Figure 5. Genomic Integration protocol in D. radiodurans . A) pUC19mPheS plasmid schematic to integrate XenO-mCherry fusion (left) and mechanistic diagram of how region of interest is inserted into the D. radiodurans R1 Genome (right). B) cPCRs of colonies after two rounds of screening. The successful desired insert length is approximately 3 kB (highlighted in green), colonies with unsuccessful insertion attempts do not have that band (lanes 12 and 16). C) Fluorescence of R1 cells, a plasmid containing the XenO-mCherry fusion, and the final XenO strain containing the integrated mCherry fusion after 24 hours of culturing. D) Microscopy of R1 and XenO strains under brightfield imaging, as well as voltage for 561 nm to excite mCherry fluorescence. Repurposing the transposon-associated protein TnpB for programmable genetic knockdowns There have been considerable efforts towards genomic editing technology in bacteria in the last decade, particularly through Cas-based tools that allow up and down regulation of target genes in bacteria. Most efforts have leveraged dCas9 (or engineered derivatives) to selectively silence or activate gene expression 58 – 60 . Interestingly, expressing dCas9 in D. radiodurans is toxic and leads to a significant loss in colony forming units, and growth defects in liquid culture 32 . Therefore, finding alternate approaches for targeted gene-repression in D. radiodurans is essential for establishing similar technology. Recently, the transposon-associated protein TnpB was identified in D. radiodurans as an RNA-directed nuclease capable of being programmed to cleave target DNA sites in cells 61 . In this work, we showed that, within D. radiodurans , the catalytically inactive mutant (D191A, which we termed dTnpB), could be repurposed and leveraged in the same way that dCas9 engineering applications have been used in other bacteria 58 , 59 . To generate construct, we replaced the sfgfp CDS in our aTc-inducible plasmids and IPTG-inducible plasmids with that of the dtnpB CDS. We selected the aTc-, and IPTG-inducible systems, because these systems were two of the most titratable inducible promoter systems that we screened. Additionally, we integrated a constitutively expressed guiding RNA sequence defined by Karvelis and colleagues 61 , incorporating this sequence as the right end element RNA (reRNA) downstream of our regulator sequence ( Fig. 6a ). The reRNA sequence was approximately 150 nucleotides, wherein the last 16 nucleotides of the 3’-end contained the guide sequence for TnpB. According to Karvelis and colleagues, this guide sequence directs TnpB to cut at a “TTGAT” sequence, functioning similarly to a PAM site for Cas9 systems 61 . For our work, we initially designed the reRNA to target a sequence of 44 nucleotides upstream of the start codon: i.e. within the constitutive promoter upstream of our integrated XenO-mCherry sequence ( Fig. 6a ). Our reRNA sequence also contained a self-cleaving hepatitis delta ribozyme (HDV) sequence to ensure that the final 16 nucleotides of the reRNA sequence were our desired targeting sequence, which was shown to be critical for effective targeting 61 . To evaluate the ability of the dTnpB system to repress genomic expression of our target sequence, we tracked mCherry signal in liquid culture up to 24 hours after induction ( Fig. 6b-d ). Within four hours of dTnpB induction, we observed an approximate 30% reduction in mCherry signal in the IPTG-inducible construct. Maximum repression occurred 24 hours post-induction, when we observed a range of signal reduction (from 66% to 87%) in mCherry fluorescence between our induced samples and uninduced samples ( Fig 6e ). Ultimately, we constructed a plasmid-based system for dTnpB-guided repression of genomic targets ( Table 1 - pAT00_40_001_TnpB + 20_reRNA_mCherry) and these results serve as preliminary evidence that the catalytically inactivated dTnpB could be leveraged for targeted gene-silencing in D. radiodurans . Download figure Open in new tab Figure 6. Programmable dTnpB-assisted interference system for repression of genomic targets in D. radiodurans . A) Plasmid map and schematic for the dTnpB-based system. dTnpB is under small molecule induction, and the reRNA is constitutively expressed via the P GroES promoter. Upon expression of both components, the dTnpB and reRNA complex targets the promoter sequence of constitutively expressed mCherry fusion protein of the XENOTEXT strain. The binding of the dTnpB occludes RNA polymerase from binding and reduces transcription, therefore reducing mCherry signal. B) mCherry signal of cultures with dTnpB expression induced (aTc) and uninduced after 24 hours of growth. C) Time course of mCherry signal between the cultures with and without dTnpB induction under aTc. D) mCherry signal of cultures with dTnpB expression induced (IPTG) and uninduced after 24 hours of growth. E) Time course of mCherry signal between the cultures with and without dTnpB induction under IPTG. F) Relative mCherry signal of cultures under tetR and LacI regulation. Discussion In this work, we established a genetic toolkit in D. radiodurans that expands the available repertoire for origin of replications, standardized constitutive promoters, RBS sequences, and small molecule-inducible promoters. Additionally, we established a process for effective gene insertions as well as a tool for programmable gene repression. These new genetic components improve tunability of heterologous gene expression as well as the ability to edit and tune native gene expression. We have compiled a final list of the most versatile plasmids and strains and indicate their relative performance context in Table 1 below for ease of access and request. As a note, all sequences used in this study are compiled in the supporting information. Our standardized constitutive promoters provide a 45-fold range for tuning gene expression ( Figure 1 ) — a range comparable to other genetic toolkits recently established for non-model organisms 24 , 46 , 49 , 62 . These constitutive promoters can be used to express a heterologous target on a plasmid in D. radiodurans , making them useful in developing more complex biological control schemes. The ability to vary constitutively expressed components, such as the LacI regulatory protein or a small RNA transcript, provides the opportunity to improve coordination between components and can reduce the rate of failure as system complexity increases 63 . Separately, of the nine inducible promoter systems that we tested for plasmid-based control, the IPTG-, aTc-, CA-, and SA-inducible promoter systems from the Marionette collections inducible promoter systems all demonstrated at least 3-fold activation ( Figure 4a, c, f, h, j ), which is similar to magnitudes of fold activation for inducible promoter systems previously developed in D. radiodurans 31 . It is important to note that 5 of the inducible promoter systems did not demonstrate any titratable signal control ( Figure 4b, d, e, g, i ). This could be due to many factors such as low promoter strength or overexpression of the repressor protein. These features could be further explored if additional inducible promoter systems are needed; however, four functional systems provide a solid foundation for inducible transcription-based control in D. radiodurans . Additionally, because these systems are derived from the “Marionette” collection, they were designed to be orthogonal; therefore, they should experience minimal crosstalk between the systems, allowing for improved circuit dynamics. Overall, these inducible promoters lay the foundation of more complex genetic control, such as two-input logic gates or other bacterial computation, including BANDPASS Filters or three-input circuits 40 , 64 . Next, we generated a library of 127 composable RBS sequences that offered a 963-fold range of translational control ( Figure 2b ) — a standard that meets or significantly exceeds previous RBS libraries generated in non-model bacteria 46 , 65 . This RBS library is also the first of its kind in D. radiodurans and can be used in concert with the promoter library for extremely precise genetic tuning. In many model microbes, there are predictive computational tools that can accurately generate sequences for effective RBS tuning. However, in our work we found poor correlation between the in vivo fluorescent values and the in silico predicted RBS strengths for our library (Supplementary Figure S1). This may be due to a variety of variables: the high GC content of D. radiodurans ; the mechanism translation initiation; the environmental conditions; or other confounding factors. Importantly, these results demonstrate that additional work is required to understand the relationship between RBS and translation initiation in D. radiodurans . For now, our library provides the most effective option for precise RBS tuning. Additionally, our RBS library could service as a basis to develop computational models that more accurately predict RBS strength in D. radiodurans in the future. In addition to our RBS Library, our codon-optimization tool will be able to work in concert to ensure both efficient translation initiation as well as preferred codon usage to increase the likelihood of a correctly synthesized protein. Overall, this RBS library and codon-optimization tool will provide opportunities for precise optimization of heterologous pathways and other genetic tools originally developed in other host systems. We also sought to identify novel origin of replication (ori) sequences that were functional in D. radiodurans . Currently, the only plasmid ori is RepU derived from pRAD1. Identifying other origins of replications would allow for multi-plasmids strains or allow for users to vary copy number based on the ori, similar to how pMB1, p15a, and pSC101 are leveraged in E. coli. To find other origins of replication, we initially searched for sequences similar to repU , however no other homologous sequences were identified. Therefore, we took a rational engineering approach (Supplementary Figure S3a) by selecting three different subsets of origin of replications: (i) common origins of replication used in E. coli (ColE1, and p15a), (ii) broad host range origins of replication (BBR1, R2K, RK6, RO1600), and (iii) origins of replications from plasmids used in Thermus thermophilus (UB110, MY1, TT8, TsYS45), a similar high GC-content extremophile. Of these origins of replication, we found that UB110 and RO1600 generated colony forming units in D. radiodurans , and those single colonies could be grown in liquid media to produce a fluorescent sfGFP signal (Supplementary Figure S3b). However, these liquid cultures could not be passaged a second time nor could copy numbers be quantified from these oris. Therefore, the RepU ori remains the only ori that stably maintains plasmids in D. radiodurans . Discovering or engineering novel oris compatible in D. radiodurans will be important for expanding the utility of plasmid-based gene expression in D. radiodurans . In parallel to building plasmid-based tools, we established methods for editing and tuning expression from the genome of D. radiodurans. Our protocol for gene integration leverages the natural competency and recombination of D. radiodurans to create novel gene-integration systems. Moreover, this approach achieves a 60% integration efficiency with only two rounds of screening ( Figure 5 ). These results are comparable to Cas-based genome engineering approaches in model bacteria 66 , 67 . Previous efforts have established a process for knocking out genes in D. radiodurans 50 ; however, our system is the first of its kind to achieve large (>2kB) genomic integrations. Additionally, our system only relies on a single plasmid and combines the genomic integration and plasmid curing into a single step ( Figure 5a ), reducing the time to develop new strains. This technology opens the door to engineering strains of D. radiodurans that can be used in large-scale metabolic engineering or bioprocessing applications, as synthetic systems integrated into the genome eliminate the need for continual plasmid maintenance. Concurrently, our programmable dTnpB-assisted gene repression system is equally useful, because it allows for targeted repression within the genome. This system achieves a range of repression between 66% and 87% of its target sequence relative to the uninduced system when using the LacI to control expression of the TnpB protein ( Figure 6d, e ) — a result comparable to Cas-based systems in both model organisms and non-model organisms 41 , 59 , 68 – 70 . This approach is also useful, because it can help simulate genomic deletions without requiring the labor to ensure that the gene is knocked out of all four chromosomes of D. radiodurans . One particular systems is the histone-like protector, HU, which plays a role in nucleoid compaction, but cannot be knocked out 71 . Therefore, this TnpB-based knockdown system could serve as a way to evaluate the phenotype of a genotype that cannot be otherwise synthesized in vivo . Previous work has demonstrated that dCas9 regulatory systems can function in D. radiodurans ; however, they greatly alter growth and prevent growth in liquid culture 32 . These effects are not observed with our application of the TnpB-based system. This outcome may occur because TnpB is native to D. radiodurans , conferring an additional benefit from using a protein in its native environment. Although TnpB has been recently used as a programmable gene-editing tool 61 , 72 , our work is the first demonstration of utilizing the catalytically inactive version of TnpB to repress genes from any genome in a targeted fashion. This system may be transferable to other organisms that also cannot utilize Cas-based system or other species of interest similar to D. radiodurans — species that, for example. do not have a robust gene-knockdown protocol, such as Deinococcus geothermalis . Additionally, this system provides a foundation for developing programmable gene-activation by fusing protein activators, such as transcription factors or subunits of RNA polymerase, to the dTnpB protein. Overall, we have built a toolkit that allows for precise user-control of gene-expression via the plasmid or the genome, as well as a novel protocol for editing the genome of D. radiodurans . We anticipate that this toolkit will be leveraged in future work to advance fundamental research and engineering applications of D. radiodurans. Importantly, all of the sequences, scripts, and methods utilized in this paper are provided in the Methods as well as in Supplementary Materials, and we encourage the research community to utilize these tools. This toolkit aims to advance the utility of D. radiodurans in bioprocessing, biomanufacturing, and biological data storage, as well as other applications in fundamental research and synthetic biology. Materials and Methods Bacterial strain and culture conditions Escherichia coli strain DH5α and its plasmid-containing derivatives were cultured aerobically at 37°C in Luria-Bertani (LB) liquid media (10 g/L tryptone, 10 g/L NaCl, and 5 g/L yeast extract) or LB solid (1.5% agar) medium. When necessary, chloramphenicol was used at a concentration of 34 μg/ml for E. coli strains. Deinococcus radiodurans R1 (ATCC 13939) and its plasmid-containing derivatives were cultured aerobically at 32°C in TGY liquid media (1% tryptone, 0.1% glucose, 0.5% yeast extract) or TGY solid (1.5% agar) medium. When necessary, antibiotics were used at a concentration of 34 μg/ml (Chloramphenicol in E. coli strains and 16 μg/ml (Kanamycin), 3.4 μg/ml (Chloramphenicol) for D. radiodurans strains All strains utilized and tested are listed in Supplementary Table S7. Plasmid Construction Plasmids were synthesized using Gibson Assembly of PCR fragments. Either Q5 Hot Start High-Fidelity 2x Master Mix (New England Biolabs) or Phusion Polymerase (New England Biolabs) were used. For Gibson Assemblies, NEBuilder® HiFi DNA Assembly Master Mix (New England Biolabs Inc.) was used to assemble full plasmids from their parts. Plasmids were purified from E. coli using the Qiagen QIAprep Spin MiniPrep Kit (Cat. No. 27104). Plasmids developed in this study are contained in Supplementary Table S7. Primers used in Plasmid development are contained in Supplementary Table S8. Measuring Fluorescent Signal from liquid culture of D. radiodurans To measure fluorescence of our synthetic toolkit constructs in D. radiodurans , sequence verified single colonies were grown in 5 mL of TGY supplemented with chloramphenicol for 24 hours in a shaking incubator at 32 °C. The next day a new 5 mL culture of TGY supplemented with chloramphenicol was inoculated with the saturated overnight, such that the final OD 600 was approximately 0.1- 0.2. These samples were grown for another 22 to 24 hours in a shaking incubator at 32 °C. Next, 1 mL of culture per sample was centrifuged for 5 minutes at 3000 rpms. The supernatant was discarded, and the remaining pellet was resuspended in 200 µL of 1x PBS, which was prepared according to the Cold Springs Harbor Protocol ( 63 ). The resuspension was then transferred into a Greiner 96-well µClear, F-Bottom, medium binding plate (Ref. No. 655096). Fluorescence was measured using a BioTek Cytation 3 Plate Reader. For sfGFP the excitation and emission wavelengths were 487 nm and 513 nm. For mCherry the excitation and emissions wavelengths were 579 nm and 616 nm. For mTurquoise the excitation and emission wavelengths were 434 nm and 479 nm. For mOrange the excitation and emission wavelengths were 544 nm and 570 nm. To measure OD 600 , absorbance was taken using a wavelength of 600 nm. All samples were taken in biological triplicate, unless otherwise specified. As a blank, 200 µL of PBS was used. Plasmid purification out of D. radiodurans Plasmids were purified from D. radiodurans using a three-phase protocol. First, single colonies were grown in 5 mL of TYG supplemented with appropriate antibiotics for 20-24 hours in a shaking 32 °C incubator. The entire volume of the cells was then pelleted via centrifugation at 3000 rpm for 5 minutes. After centrifugation, the supernatant was discarded and the pellet was resuspended in 1 mL of TGY and incubated at 95 °C for 10 minutes. After incubating, the cells were centrifuged again for 5 minutes at 3000 rpms, and the supernatant was discarded. Next, the pellet was resuspended in 250 µL of Qiagen P1 Buffer supplemented with 6 mg/mL Chicken Lysozyme (Muramidase - US Biological), which was freshly mixed. The suspension was incubated for 15 minutes at 37 °C, and could be extended up to 1 hour of incubation for hard to lyse samples. Next, these samples were purified using Qiagen QIAprep Spin MiniPrep Kit (Cat. No. 27104) starting from Step 3, which is the addition of Buffer P2 Step. As a last note, letting the P2 lysis step proceed for at least 3 minutes generated the highest plasmid yields. Plasmids were then sequenced using Plasmidsaurus or Eton Bioscience Inc. RBS Library Construction To create the RBS Library, a protocol was derived from previous work ( 41 ). To summarize, the pAT00.20.001 plasmid was amplified except for the region that includes the P GroES promoter, the RBS, and the first 153 nucleotides of the sfgfp sequence. This sequence was incubated overnight with DnpI (New England BioLabs) enzyme to digest any parent plasmid. Then, it was purified using the QIAquick PCR Purification Kit (Qiagen). A pool of gBlocks containing the P GroES promoter through the 153 rd nucleotide of the sfgfp CDS sequence was synthesized via Twist Bioscience, however the “GGAGG” sequence within the RBS was replaced with “NNNNN”, where N means any nucleotide. This sequence is listed in Supplementary Table S5. Our pool of gBlocks had 1024 combinations of possible RBS sequences. The two purified fragments were placed into NEBuilder® HiFi DNA Assembly Master Mix reactions totaling 20 µL. The fragments were added in a 5:1 molar ratio of RBS insert to pAT00 backbone. Two of these Gibson Assembly reactions were created. The reaction mixtures were then incubated at 50 °C for 1 hour. Next, 15 µL of product from each reaction were desalted using MF-Millipore 0.025 µm MCE Membranes (Millipore Sigma) for 15 minutes. Then, 10 µL of desalted Gibson Assembly Product was added to 100 µL of ElectroMAX DH10β cells (ThermoFisher) on for each reaction. The assemblies were transformed via electroporation using Gene Pulser/MicroPulser Cuvettes (Bio-Rad) and the GenePulse Xcell electroporator (Bio-Rad), set at a voltage of 1.8 kV. The corresponding time constants for each transformation were between 4.5-5. The transformations had 900 µL of S.O.C. media added, provided with the ElectroMAX DH10β Kit (ThermoFisher), before being recovered for 90 minutes in a 37 °C shaking incubator. The recoveries were then centrifuged for 5 minutes at 5000 rpm, the supernatant was discarded, and the cells were resuspended before each being plated on LB Agar plates supplemented with chloramphenicol. The petri dishes for the LB Agar plates used were 150 x 15 mm Falcon (Corning) plates. The plates were grown overnight at 37 °C. The next day, colony counts were made between both plates to ensure at least 1,024 single colonies were formed. 6 single colonies were purified via the Qiagen QIAprep Spin MiniPrep Kit (Cat. No. 27104), and sent for sequencing via Plasmidsaurus. All six single colonies had different RBS sequences in the “NNNNN” region, which provided confidence there was sequence diversity in the rest of the library. Next, both plates were scraped using 3 mL of ice-cold LB per plate and plastic plate scrapers. The solution of LB and scraped colonies were transferred into 50 mL of LB supplemented with chloramphenicol, and grown overnight at 37 °C in a shaking incubator. The next day, the culture was split into two 25 mL samples and the libraries were purified using the QIAGEN Plasmid Mini Kit (Qiagen) according to the user guide. 2 µg of Library DNA per sample was added to a fresh aliquot of D. radiodurans R1 competent cells and transformed according to the protocol below. The transformations were plated on TGY Agar plates supplemented with chloramphenicol. The petri dishes for the plates used were 150 x 15 mm Falcon (Corning) plates. After single colony formation (~2-3 days), 136 single colonies were grown for 24 hours in 5 mL of TGY supplemented with chloramphenicol. After 24 hours of growth, the fluorescence of each culture was measured in technical triplicate, according to the protocol for measuring sfGFP fluorescence in D. radiodurans stated above. Additionally, with the remaining liquid culture, the plasmids were purified and sequenced according to the plasmid purification out of D. radiodurans stated directly above. Once all sequences were returned, the RBS strengths were estimated using the Salis Lab RBS Calculator at ( https://salislab.net/software/predict_rbs_calculator ). The RBS sequences run through the calculator, their predicted strengths, as well as sfGFP signal measured in vivo are reported in Supplementary Table S4, and the correlation plot between predicted strength and fluorescent output is available in Supplementary Figure S2. Transformation of recombinant plasmids in D. radiodurans Recombinant plasmids were transformed into a chemically competent wild-type (R1 ATCC 13939) D. radiodurans strain as previously described ( 32 ). Briefly, D. radiodurans cells were grown overnight in 5 mL of TGY in a 32 °C shaking incubator. The next day, the saturated overnight was used to seed a fresh 5 mL of TGY to a final OD 600 of 0.2. The cultures were grown to late log phase (OD600 = 1.0), then 955 µL of cells were mixed with 45µL of 1M CaCl2 and 500 µL of ice cold 30% glycerol to create the chemically competent cell line. 300 µL of the mixture were aliquoted into sterile 1.7 mL microtubes (Olympus). Competent cells were either stored at −80 °C for future use immediately. For transformation into the competent cell stock, 1.5μg plasmid DNA was added, followed by an incubation of 1 hour on ice, and a subsequent incubation at 32°C shaking for 1 hour. Cells were then incubated at 32°C shaking overnight in 1 mL of fresh TGY media in test tubes. Post-incubation, cells were plated onto TGY plates with the appropriate antibiotic concentration (above), and incubated for 3 days at 32°C. Transformants were verified via polymerase chain-reaction (PCR) and sequencing (Plasmidsaurus, Eton Bioscience Inc.). Construction of genomic integration strains in D. radiodurans The genomic integration strains of D. radiodurans were constructed using a modified homologous recombination method reported previously for the deletion of genes 51 . In short, 1kb regions upstream and downstream from the selected genomic insertion site were amplified via PCR from the R1 genome, and cloned into the pUC19mPhes plasmid alongside the gene of interest to be inserted, a fragment containing a kanamycin cassette, and both lox66 and lox71 sequences. All fragments were then gel-purified using QIAquick Gel Extraction Kit (Qiagen) and assembled with a linearized, HindIII-digested, pUC19mPheS plasmid using NEBuilder® HiFi DNA Assembly Master Mix (New England Biolabs Inc.). The resulting plasmid was then transformed into E. coli DH5α strain and isolated via QIAprep Spin MiniPrep Kit (Qiagen). The isolate was subsequently transformed into D. radiodurans R1 chemically competent cells (above). Mutant strains were selected on TGY agar plates with the appropriate kanamycin concentration and 4-chlorophenylalanine (5 mM) for 3 rounds of selection. The total process selected for cells that underwent homologous recombination to replace the target region with the kanamycin cassette, and no longer contained the pUC19mPheS recombinant plasmid. The primers used for the gene integration processes are included in Supplementary Table S7. All plasmids utilized for the gene integration processes are listed in Supplementary Table S6. Measuring genomic repression of mCherry-XenO target using dTnpB To measure repression of the mCherry-XenO fusion protein, we transformed our dTnpB + reRNA-containing plasmid (pAT00.40.001-dTnpB + 20-reRNA1-mCherry) into competent cells of the XenO strain, according to the D. radiodurans competent cell preparation and transformation protocols. Single colonies were grown in a shaking incubator at 32 °C for 24 hours in 5 mL of TGY supplemented with chloramphenicol and kanamycin to ensure plasmid retention and select against any potential R1 contamination. After 24 hours of growth, 250 µL of saturated culture was added to 5 mL of fresh TGY supplemented with chloramphenicol, these cultures were grown for 10 minutes in a shaking incubator at 32 °C. Then to induce dTnpB expression, 5 µL of 1M IPTG, or 2.5 µL of 100 µM aTc were added and the samples were returned to the 32 °C shaking incubator. Periodically, fluorescence of the induced and uninduced samples were measured according to above protocols. Final fluorescent measurements were taken 24 hours post-induction. Imaging of bacterial strains Microscopy was performed via confocal microscopy using a Fluoview 1000 confocal microscope on an Olympus IX81 microscope base.The 543 nm laser line was used to observe mCherry fluorescence. The Fluoview settings for observation were as follows, Gain: 1500 (1.5x), Offset: 2000 (20%), PMT Voltage: 664-880. Funding This work was supported by the Air Force Office of Scientific Research (Grant FA9550-20-1-0131) and the Welch foundation (Grant F-1756). A.C., T.R.S., and K.B.G. were supported by the National Science Foundation Graduate Research Fellowships (Grant DGE-2137420). Acknowledgements We would like to thank Gina Partipilo, from the lab of Dr. Benjamin Keitz for her helpful discussion and insights on approaches for developing a protocol to synthesize the RBS Library, as well as providing support and guidance in generating the protocols to measure fluorescence of the D. radiodurans cultures. We would also like to thank Dr. Philip Sweet and other members of the Contreras Lab for helpful conversations and discussions during the ideation of the project, and drafting of the manuscript. We’d also like to thank Dr. Vernita Gordon, and Dr. Brandon Niese for their contributions in imaging the XenO strain. Lastly, we would like to thank Dr. Christian Bök for providing us with the inspiration to generate the XenO strain. Further reading regarding the Xenotext (XenO) and its inception are contained within THE XENOTEXT (BOOK 1) [ISBN: 9781552453216] and THE XENOTEXT (BOOK 2) [ISBN: 9781552454985]. Funder Information Declared United States Air Force Office of Scientific Research , FA9550-20-1-0131 Welch Foundation , F-1756 National Science Foundation , DGE-2137420 References (1). ↵ Ye , J.-W. ; Lin , Y.-N. ; Yi , X.-Q. ; Yu , Z.-X. ; Liu , X. ; Chen , G.-Q. Synthetic Biology of Extremophiles: A New Wave of Biomanufacturing . Trends in Biotechnology 2023 , 41 ( 3 ), 342 – 357 . doi: 10.1016/j.tibtech.2022.11.010 . OpenUrl CrossRef PubMed (2). ↵ Billi , D. ; Wright , D. J. ; Helm , R. F. ; Prickett , T. ; Potts , M. ; Crowe , J. H. Engineering Desiccation Tolerance inEscherichia Coli . Applied and Environmental Microbiology 2000 , 66 ( 4 ), 1680 – 1684 . doi: 10.1128/AEM.66.4.1680-1684.2000 . OpenUrl Abstract / FREE Full Text (3). ↵ Brim , H. ; McFarlan , S. C. ; Fredrickson , J. K. ; Minton , K. W. ; Zhai , M. ; Wackett , L. P. ; Daly , M. J. Engineering Deinococcus Radiodurans for Metal Remediation in Radioactive Mixed Waste Environments . Nat Biotechnol 2000 , 18 ( 1 ), 85 – 90 . doi: 10.1038/71986 . OpenUrl CrossRef PubMed (4). ↵ Daly , M. J. Engineering Radiation-Resistant Bacteria for Environmental Biotechnology . Current Opinion in Biotechnology 2000 , 11 ( 3 ), 280 – 285 . doi: 10.1016/S0958-1669(00)00096-3 . OpenUrl CrossRef PubMed Web of Science (5). ↵ Chen , G.-Q. ; Jiang , X.-R. Next Generation Industrial Biotechnology Based on Extremophilic Bacteria . Current Opinion in Biotechnology 2018 , 50 , 94 – 100 . doi: 10.1016/j.copbio.2017.11.016 . OpenUrl CrossRef PubMed (6). ↵ Chai , M. ; Deng , C. ; Chen , Q. ; Lu , W. ; Liu , Y. ; Li , J. ; Du , G. ; Lv , X. ; Liu , L. Synthetic Biology Toolkits and Metabolic Engineering Applied in Corynebacterium Glutamicum for Biomanufacturing . ACS Synth. Biol . 2021 , 10 ( 12 ), 3237 – 3250 . doi: 10.1021/acssynbio.1c00355 . OpenUrl CrossRef PubMed (7). ↵ Liu , Y. ; Huo , K. ; Tan , B. ; He , X. ; Wu , Q. ; Li , Z.-J. Metabolic Engineering of Halomonas Bluephagenesis for the Production of Ethylene Glycol and Glycolate from Xylose . Journal of Biotechnology 2024 , 396 , 36 – 40 . doi: 10.1016/j.jbiotec.2024.10.006 . OpenUrl CrossRef PubMed (8). ↵ Tao , W. ; Lv , L. ; Chen , G.-Q. Engineering Halomonas Species TD01 for Enhanced Polyhydroxyalkanoates Synthesis via CRISPRi . Microbial Cell Factories 2017 , 16 ( 1 ), 48 . doi: 10.1186/s12934-017-0655-3 . OpenUrl CrossRef PubMed (9). ↵ Kruyer , N. S. ; Realff , M. J. ; Sun , W. ; Genzale , C. L. ; Peralta-Yahya , P. Designing the Bioproduction of Martian Rocket Propellant via a Biotechnology-Enabled in Situ Resource Utilization Strategy . Nat Commun 2021 , 12 ( 1 ), 6166 . doi: 10.1038/s41467-021-26393-7 . OpenUrl CrossRef PubMed (10). ↵ Averesch , N. J. H. ; Berliner , A. J. ; Nangle , S. N. ; Zezulka , S. ; Vengerova , G. L. ; Ho , D. ; Casale , C. A. ; Lehner , B. A. E. ; Snyder , J. E. ; Clark , K. B. ; Dartnell , L. R. ; Criddle , C. S. ; Arkin , A. P. Microbial Biomanufacturing for Space-Exploration—What to Take and When to Make . Nat Commun 2023 , 14 ( 1 ), 2311 . doi: 10.1038/s41467-023-37910-1 . OpenUrl CrossRef PubMed (11). ↵ Lu , Y. ; Fu , Y. ; Chen , L. ; Cui , J. ; Huang , M. ; Fu , Y. ; Liu , H. Combined Effect of Simulated Microgravity and Low-Dose Ionizing Radiation on Structure and Antibiotic Resistance of a Synthetic Community Model of Bacteria Isolated from Spacecraft Assembly Room . Life Sciences in Space Research 2023 , 38 , 29 – 38 . doi: 10.1016/j.lssr.2023.04.005 . OpenUrl CrossRef PubMed (12). ↵ Deshevaya , E. A. ; Fialkina , S. V. ; Shubralova , E. V. ; Tsygankov , O. S. ; Khamidullina , N. M. ; Vasilyak , L. M. ; Pecherkin , V. Y. ; Shcherbakova , V. A. ; Nosovsky , A. M. ; Orlov , O. I. Survival of Microorganisms during Two-Year Exposure in Outer Space near the ISS . Sci Rep 2024 , 14 ( 1 ), 334 . doi: 10.1038/s41598-023-49525-z . OpenUrl CrossRef PubMed (13). ↵ Daly , M. J. A New Perspective on Radiation Resistance Based on Deinococcus Radiodurans . Nat Rev Microbiol 2009 , 7 ( 3 ), 237 – 245 . doi: 10.1038/nrmicro2073 . OpenUrl CrossRef PubMed Web of Science (14). ↵ Cox , M. M. ; Battista , J. R. Deinococcus Radiodurans — the Consummate Survivor . Nat Rev Microbiol 2005 , 3 ( 11 ), 882 – 892 . doi: 10.1038/nrmicro1264 . OpenUrl CrossRef PubMed Web of Science (15). ↵ Cordova , A. ; Niese , B. ; Sweet , P. ; Kamat , P. ; Phillip , J. M. ; Gordon , V. ; Contreras , L. M. Quantitative Morphological Analysis of Deinococcus Radiodurans Elucidates Complex Dose-Dependent Nucleoid Condensation during Recovery from Ionizing Radiation . Applied and Environmental Microbiology 2024 , 90 ( 7 ), e00108 – 24 . doi: 10.1128/aem.00108-24 . OpenUrl CrossRef PubMed (16). ↵ Kawaguchi , Y. ; Shibuya , M. ; Kinoshita , I. ; Yatabe , J. ; Narumi , I. ; Shibata , H. ; Hayashi , R. ; Fujiwara , D. ; Murano , Y. ; Hashimoto , H. ; Imai , E. ; Kodaira , S. ; Uchihori , Y. ; Nakagawa , K. ; Mita , H. ; Yokobori , S. ; Yamagishi , A. DNA Damage and Survival Time Course of Deinococcal Cell Pellets During 3 Years of Exposure to Outer Space . Front. Microbiol . 2020 , 11 . doi: 10.3389/fmicb.2020.02050 . OpenUrl CrossRef (17). ↵ Lange , C. C. ; Wackett , L. P. ; Minton , K. W. ; Daly , M. J. Engineering a Recombinant Deinococcus Radiodurans for Organopollutant Degradation in Radioactive Mixed Waste Environments . Nat Biotechnol 1998 , 16 ( 10 ), 929 – 933 . doi: 10.1038/nbt1098-929 . OpenUrl CrossRef PubMed Web of Science (18). ↵ Chen , A. ; Hernandez-Vargas , J. ; Han , R. ; Cortazar-Martínez , O. ; Gonzalez , N. ; Patel , S. ; Keitz , B. K. ; Luna-Barcenas , G. ; Contreras , L. M. Small RNAs as a New Platform for Tuning the Biosynthesis of Silver Nanoparticles for Enhanced Material and Functional Properties . ACS Appl. Mater. Interfaces 2021 , 13 ( 31 ), 36769 – 36783 . doi: 10.1021/acsami.1c07400 . OpenUrl CrossRef PubMed (19). ↵ Shetty , R. P. ; Endy , D. ; Knight , T. F. Engineering BioBrick Vectors from BioBrick Parts . Journal of Biological Engineering 2008 , 2 ( 1 ), 5 . doi: 10.1186/1754-1611-2-5 . OpenUrl CrossRef PubMed (20). Lee , M. E. ; DeLoache , W. C. ; Cervantes , B. ; Dueber , J. E. A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly . ACS Synth. Biol . 2015 , 4 ( 9 ), 975 – 986 . doi: 10.1021/sb500366v . OpenUrl CrossRef PubMed (21). Cao , Y. ; Song , M. ; Li , F. ; Li , C. ; Lin , X. ; Chen , Y. ; Chen , Y. ; Xu , J. ; Ding , Q. ; Song , H. A Synthetic Plasmid Toolkit for Shewanella Oneidensis MR-1 . Front. Microbiol . 2019 , 10 . doi: 10.3389/fmicb.2019.00410 . OpenUrl CrossRef (22). Dundas , C. M. ; Walker , D. J. F. ; Keitz , B. K. Tuning Extracellular Electron Transfer by Shewanella Oneidensis Using Transcriptional Logic Gates . ACS Synth. Biol . 2020 , 9 ( 9 ), 2301 – 2315 . doi: 10.1021/acssynbio.9b00517 . OpenUrl CrossRef PubMed (23). Popp , P. F. ; Dotzler , M. ; Radeck , J. ; Bartels , J. ; Mascher , T. The Bacillus BioBrick Box 2.0: Expanding the Genetic Toolbox for the Standardized Work with Bacillus Subtilis . Sci Rep 2017 , 7 ( 1 ), 15058 . doi: 10.1038/s41598-017-15107-z . OpenUrl CrossRef PubMed (24). ↵ Rondthaler , S. N. ; Sarker , B. ; Howitz , N. ; Shah , I. ; Andrews , L. B. Toolbox of Characterized Genetic Parts for Staphylococcus Aureus . ACS Synth. Biol . 2024 , 13 ( 1 ), 103 – 118 . doi: 10.1021/acssynbio.3c00325 . OpenUrl CrossRef PubMed (25). Van Voorhis , A. F. ; Sherbo , R. S. Creating a Genetic Toolbox for the Carbon-Fixing, Nitrogen-Fixing and Dehalogenating Bacterium Xanthobacter Autotrophicus . ACS Synth. Biol . 2024 , 13 ( 11 ), 3658 – 3667 . doi: 10.1021/acssynbio.4c00455 . OpenUrl CrossRef PubMed (26). ↵ Caro-Astorga , J. ; Rogan , M. ; Malcı , K. ; Ming , H. ; Debenedictis , E. ; James , P. ; Ellis , T. SubtiToolKit: A Bioengineering Kit for Bacillus Subtilis and Gram-Positive Bacteria . Trends in Biotechnology 2025 , S0167779925000411 . doi: 10.1016/j.tibtech.2025.02.004 . OpenUrl CrossRef (27). ↵ Anderson , A. ; Nordon , H. ; Cain , R. F. ; Parrish , G. ; Duggan , D. ; Anderson , A. ; Nordan , H. ; Parish , G. ; Cullum-Dugan , D. Studies on a Radio-Resistant Micrococcus. I. Isolation, Morphology, Cultural Characteristics, and Resistance to Gamma Radiation . Food Technology 1956 . (28). ↵ Meima , R. ; Lidstrom , M. E. Characterization of the Minimal Replicon of a Cryptic Deinococcus Radiodurans SARK Plasmid and Development of Versatile Escherichia Coli-D. Radiodurans Shuttle Vectors . Applied and Environmental Microbiology 2000 , 66 ( 9 ), 3856 – 3867 . doi: 10.1128/AEM.66.9.3856-3867.2000 . OpenUrl Abstract / FREE Full Text (29). ↵ Meima , R. ; Rothfuss , H. M. ; Gewin , L. ; Lidstrom , M. E. Promoter Cloning in the Radioresistant BacteriumDeinococcus Radiodurans . Journal of Bacteriology 2001 , 183 ( 10 ), 3169 – 3175 . doi: 10.1128/jb.183.10.3169-3175.2001 . OpenUrl Abstract / FREE Full Text (30). ↵ Chen , A. ; Sherman , M. W. ; Chu , C. ; Gonzalez , N. ; Patel , T. ; Contreras , L. M. Discovery and Characterization of Native Deinococcus Radiodurans Promoters for Tunable Gene Expression . Applied and Environmental Microbiology 2019 , 85 ( 21 ), e01356 – 19 . doi: 10.1128/AEM.01356-19 . OpenUrl Abstract / FREE Full Text (31). ↵ Lecointe , F. ; Coste , G. ; Sommer , S. ; Bailone , A. Vectors for Regulated Gene Expression in the Radioresistant Bacterium Deinococcus Radiodurans . Gene 2004 , 336 ( 1 ), 25 – 35 . doi: 10.1016/j.gene.2004.04.006 . OpenUrl CrossRef PubMed (32). ↵ Misra , C. S. ; Pandey , N. ; Appukuttan , D. ; Rath , D. Effective Gene Silencing Using Type I–E CRISPR System in the Multiploid, Radiation-Resistant Bacterium Deinococcus Radiodurans . Microbiology Spectrum 2023 , 11 ( 5 ), e05204 – 22 . doi: 10.1128/spectrum.05204-22 . OpenUrl CrossRef (33). ↵ Han , R. ; Jiang , J. ; Fang , J. ; Contreras , L. M. PNPase and RhlB Interact and Reduce the Cellular Availability of Oxidized RNA in Deinococcus Radiodurans . Microbiology Spectrum 2022 , 10 ( 4 ), e02140 – 22 . doi: 10.1128/spectrum.02140-22 . OpenUrl CrossRef (34). Chen , A. ; Contreras , L. M. ; Keitz , B. K. Imposed Environmental Stresses Facilitate Cell-Free Nanoparticle Formation by Deinococcus Radiodurans . Applied and Environmental Microbiology 2017 , 83 ( 18 ), e00798 – 17 . doi: 10.1128/AEM.00798-17 . OpenUrl Abstract / FREE Full Text (35). Villa , J. K. ; Amador , P. ; Janovsky , J. ; Bhuyan , A. ; Saldanha , R. ; Lamkin , T. J. ; Contreras , L. M. A Genome-Wide Search for Ionizing-Radiation-Responsive Elements in Deinococcus Radiodurans Reveals a Regulatory Role for the DNA Gyrase Subunit A Gene’s 5′ Untranslated Region in the Radiation and Desiccation Response . Applied and Environmental Microbiology 2017 , 83 ( 12 ), e00039 – 17 . doi: 10.1128/AEM.00039-17 . OpenUrl Abstract / FREE Full Text (36). ↵ Villa , J. K. ; Han , R. ; Tsai , C.-H. ; Chen , A. ; Sweet , P. ; Franco , G. ; Vaezian , R. ; Tkavc , R. ; Daly , M. J. ; Contreras , L. M. A Small RNA Regulates pprM, a Modulator of Pleiotropic Proteins Promoting DNA Repair, in Deinococcus Radiodurans under Ionizing Radiation . Sci Rep 2021 , 11 ( 1 ), 12949 . doi: 10.1038/s41598-021-91335-8 . OpenUrl CrossRef PubMed (37). ↵ Solovyev , V. ; Salamov , A. ; Seledtsov , I. ; Vorobyev , D. ; Bachinsky , A. AUTOMATIC ANNOTATION OF BACTERIAL COMMUNITY SEQUENCES AND APPLICATION TO INFECTIONS DIAGNOSTIC ; 2025 ; pp 346 – 353 . (38). ↵ Shahmuradov , I. A. ; Mohamad Razali , R. ; Bougouffa , S. ; Radovanovic , A. ; Bajic , V. B. bTSSfinder: A Novel Tool for the Prediction of Promoters in Cyanobacteria and Escherichia Coli . Bioinformatics 2017 , 33 ( 3 ), 334 – 340 . doi: 10.1093/bioinformatics/btw629 . OpenUrl CrossRef PubMed (39). ↵ Rottinghaus , A. G. ; Ferreiro , A. ; Fishbein , S. R. S. ; Dantas , G. ; Moon , T. S. Genetically Stable CRISPR-Based Kill Switches for Engineered Microbes . Nat Commun 2022 , 13 ( 1 ), 672 . doi: 10.1038/s41467-022-28163-5 . OpenUrl CrossRef PubMed (40). ↵ Simmons , T. R. ; Partipilo , G. ; Buchser , R. ; Stankes , A. C. ; Srivastava , R. ; Chiu , D. ; Keitz , B. K. ; Contreras , L. M. Rewiring Native Post-Transcriptional Global Regulators to Achieve Designer, Multi-Layered Genetic Circuits . Nature Communications 2024 , 15 ( 1 ), 8752 . doi: 10.1038/s41467-024-52976-1 . OpenUrl CrossRef PubMed (41). ↵ Gordon , G. C. ; Korosh , T. C. ; Cameron , J. C. ; Markley , A. L. ; Begemann , M. B. ; Pfleger , B. F. CRISPR Interference as a Titratable, Trans -Acting Regulatory Tool for Metabolic Engineering in the Cyanobacterium Synechococcus Sp . Strain PCC 7002 . Metabolic Engineering 2016 , 38 , 170 – 179 . doi: 10.1016/j.ymben.2016.07.007 . OpenUrl CrossRef PubMed (42). ↵ Cook , T. B. ; Rand , J. M. ; Nurani , W. ; Courtney , D. K. ; Liu , S. A. ; Pfleger , B. F. Genetic Tools for Reliable Gene Expression and Recombineering in Pseudomonas Putida . Journal of Industrial Microbiology and Biotechnology 2018 , 45 ( 7 ), 517 – 527 . doi: 10.1007/s10295-017-2001-5 . OpenUrl CrossRef PubMed (43). ↵ Salis , H. M. ; Mirsky , E. A. ; Voigt , C. A. Automated Design of Synthetic Ribosome Binding Sites to Control Protein Expression . Nat Biotechnol 2009 , 27 ( 10 ), 946 – 950 . doi: 10.1038/nbt.1568 . OpenUrl CrossRef PubMed Web of Science (44). Seo , S. W. ; Yang , J.-S. ; Kim , I. ; Yang , J. ; Min , B. E. ; Kim , S. ; Jung , G. Y. Predictive Design of mRNA Translation Initiation Region to Control Prokaryotic Translation Efficiency . Metabolic Engineering 2013 , 15 , 67 – 74 . doi: 10.1016/j.ymben.2012.10.006 . OpenUrl CrossRef PubMed (45). ↵ Roots , C. T. ; Lukasiewicz , A. ; Barrick , J. E. ‘OSTIR’: Open Source Translation Initiation Rate Prediction . Journal of Open Source Software 2021 , 6 ( 64 ), 3362 . doi: 10.21105/joss.03362 . OpenUrl CrossRef PubMed (46). ↵ Mishra , S. ; Perkovich , P. M. ; Mitchell , W. P. ; Venkataraman , M. ; Pfleger , B. F. Expanding the Synthetic Biology Toolbox of Cupriavidus Necator for Establishing Fatty Acid Production . Journal of Industrial Microbiology and Biotechnology 2024 , 51 , kuae008 . doi: 10.1093/jimb/kuae008 . OpenUrl CrossRef (47). ↵ Kollmar , M. Chan , P. P. ; Lowe , T. M. tRNAscan-SE: Searching for tRNA Genes in Genomic Sequences . In Gene Prediction: Methods and Protocols ; Kollmar , M. , Ed.; Springer : New York, NY , 2019 ; pp 1 – 14 . doi: 10.1007/978-1-4939-9173-0_1 . OpenUrl CrossRef PubMed (48). ↵ Meyer , A. J. ; Segall-Shapiro , T. H. ; Glassey , E. ; Zhang , J. ; Voigt , C. A. Escherichia Coli “Marionette” Strains with 12 Highly Optimized Small-Molecule Sensors . Nat Chem Biol 2019 , 15 ( 2 ), 196 – 204 . doi: 10.1038/s41589-018-0168-3 . OpenUrl CrossRef (49). ↵ Venkataraman , M. ; Yñigez-Gutierrez , A. ; Infante , V. ; MacIntyre , A. ; Fernandes-Júnior , P. I. ; Ané , J.-M. ; Pfleger , B. Synthetic Biology Toolbox for Nitrogen-Fixing Soil Microbes . ACS Synth. Biol . 2023 , 12 ( 12 ), 3623 – 3634 . doi: 10.1021/acssynbio.3c00414 . OpenUrl CrossRef PubMed (50). ↵ Brumwell , S. L. ; Van Belois , K. D. ; Giguere , D. J. ; Edgell , D. R. ; Karas , B. J. Conjugation-Based Genome Engineering in Deinococcus Radiodurans . ACS Synth. Biol . 2022 , 11 ( 3 ), 1068 – 1076 . doi: 10.1021/acssynbio.1c00524 . OpenUrl CrossRef PubMed (51). ↵ Han , R. ; Fang , J. ; Jiang , J. ; Gaidamakova , E. K. ; Tkavc , R. ; Daly , M. J. ; Contreras , L. M. Signal Recognition Particle RNA Contributes to Oxidative Stress Response in Deinococcus Radiodurans by Modulating Catalase Localization . Front. Microbiol . 2020 , 11 . doi: 10.3389/fmicb.2020.613571 . OpenUrl CrossRef (52). ↵ Bök , C. The Xenotext: Book 1 , 1st ed. ; Coach House Books : Toronto , 2015 . (53). ↵ Shipman , S. L. ; Nivala , J. ; Macklis , J. D. ; Church , G. M. CRISPR–Cas Encoding of a Digital Movie into the Genomes of a Population of Living Bacteria . Nature 2017 , 547 ( 7663 ), 345 – 349 . doi: 10.1038/nature23017 . OpenUrl CrossRef PubMed Web of Science (54). ↵ Lear , S. K. ; Shipman , S. L. Molecular Recording: Transcriptional Data Collection into the Genome . Current Opinion in Biotechnology 2023 , 79 , 102855 . doi: 10.1016/j.copbio.2022.102855 . OpenUrl CrossRef (55). ↵ Yim , S. S. ; McBee , R. M. ; Song , A. M. ; Huang , Y. ; Sheth , R. U. ; Wang , H. H. Robust Direct Digital-to-Biological Data Storage in Living Cells . Nat Chem Biol 2021 , 17 ( 3 ), 246 – 253 . doi: 10.1038/s41589-020-00711-4 . OpenUrl CrossRef PubMed (56). ↵ Makarova , K. S. ; Aravind , L. ; Wolf , Y. I. ; Tatusov , R. L. ; Minton , K. W. ; Koonin , E. V. ; Daly , M. J. Genome of the Extremely Radiation-Resistant Bacterium Deinococcus Radiodurans Viewed from the Perspective of Comparative Genomics . Microbiology and Molecular Biology Reviews 2001 , 65 ( 1 ), 44 – 79 . doi: 10.1128/mmbr.65.1.44-79.2001 . OpenUrl Abstract / FREE Full Text (57). ↵ Ithurbide , S. ; Coste , G. ; Lisboa , J. ; Eugénie , N. ; Bentchikou , E. ; Bouthier de la Tour , C. ; Liger , D. ; Confalonieri , F. ; Sommer , S. ; Quevillon-Cheruel , S. ; Servant , P. Natural Transformation in Deinococcus Radiodurans: A Genetic Analysis Reveals the Major Roles of DprA, DdrB, RecA, RecF, and RecO Proteins . Front. Microbiol . 2020 , 11 . doi: 10.3389/fmicb.2020.01253 . OpenUrl CrossRef PubMed (58). ↵ Bikard , D. ; Jiang , W. ; Samai , P. ; Hochschild , A. ; Zhang , F. ; Marraffini , L. A. Programmable Repression and Activation of Bacterial Gene Expression Using an Engineered CRISPR-Cas System . Nucleic Acids Research 2013 , 41 ( 15 ), 7429 – 7437 . doi: 10.1093/nar/gkt520 . OpenUrl CrossRef PubMed Web of Science (59). ↵ Qi , L. S. ; Larson , M. H. ; Gilbert , L. A. ; Doudna , J. A. ; Weissman , J. S. ; Arkin , A. P. ; Lim , W. A. Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression . Cell 2013 , 152 ( 5 ), 1173 – 1183 . doi: 10.1016/j.cell.2013.02.022 . OpenUrl CrossRef PubMed Web of Science (60). ↵ Zalatan , J. G. ; Lee , M. E. ; Almeida , R. ; Gilbert , L. A. ; Whitehead , E. H. ; La Russa , M. ; Tsai , J. C. ; Weissman , J. S. ; Dueber , J. E. ; Qi , L. S. ; Lim , W. A. Engineering Complex Synthetic Transcriptional Programs with CRISPR RNA Scaffolds . Cell 2015 , 160 ( 1 ), 339 – 350 . doi: 10.1016/j.cell.2014.11.052 . OpenUrl CrossRef PubMed Web of Science (61). ↵ Karvelis , T. ; Druteika , G. ; Bigelyte , G. ; Budre , K. ; Zedaveinyte , R. ; Silanskas , A. ; Kazlauskas , D. ; Venclovas , Č. ; Siksnys , V. Transposon-Associated TnpB Is a Programmable RNA-Guided DNA Endonuclease . Nature 2021 , 599 ( 7886 ), 692 – 696 . doi: 10.1038/s41586-021-04058-1 . OpenUrl CrossRef PubMed (62). ↵ Lautenschläger , N. ; Schmidt , K. ; Schiffer , C. ; Wulff , T. F. ; Hahnke , K. ; Finstermeier , K. ; Mansour , M. ; Elsholz , A. K. W. ; Charpentier , E. Expanding the Genetic Toolbox for the Obligate Human Pathogen Streptococcus Pyogenes . Front. Bioeng. Biotechnol . 2024 , 12 . doi: 10.3389/fbioe.2024.1395659 . OpenUrl CrossRef PubMed (63). ↵ Brophy , J. A. N. ; Voigt , C. A. Principles of Genetic Circuit Design . Nat Methods 2014 , 11 ( 5 ), 508 – 520 . doi: 10.1038/nmeth.2926 . OpenUrl CrossRef (64). ↵ Rojano-Nisimura , A. M. ; Simmons , T. R. ; Lukasiewicz , A. J. ; Buchser , R. ; Ruzek , J. S. ; Avila , J. L. ; Contreras , L. M. Concentration-Dependent CsrA Regulation of the uxuB Transcript Leads to Development of a Post-Transcriptional Bandpass Filter . ACS Synth. Biol . 2025 , 14 ( 4 ), 1084 – 1098 . doi: 10.1021/acssynbio.4c00668 . OpenUrl CrossRef PubMed (65). ↵ Guiziou , S. ; Sauveplane , V. ; Chang , H.-J. ; Clerté , C. ; Declerck , N. ; Jules , M. ; Bonnet , J. A Part Toolbox to Tune Genetic Expression in Bacillus Subtilis . Nucleic Acids Research 2016 , 44 ( 15 ), 7495 – 7508 . doi: 10.1093/nar/gkw624 . OpenUrl CrossRef PubMed (66). ↵ Li , Y. ; Lin , Z. ; Huang , C. ; Zhang , Y. ; Wang , Z. ; Tang , Y. ; Chen , T. ; Zhao , X. Metabolic Engineering of Escherichia Coli Using CRISPR–Cas9 Meditated Genome Editing . Metabolic Engineering 2015 , 31 , 13 – 21 . doi: 10.1016/j.ymben.2015.06.006 . OpenUrl CrossRef PubMed (67). ↵ Pyne , M. E. ; Narcross , L. ; Melgar , M. ; Kevvai , K. ; Mookerjee , S. ; Leite , G. B. ; Martin , V. J. J. An Engineered Aro1 Protein Degradation Approach for Increased Cis,Cis-Muconic Acid Biosynthesis in Saccharomyces Cerevisiae . Applied and Environmental Microbiology 2018 , 84 ( 17 ), e01095 – 18 . doi: 10.1128/AEM.01095-18 . OpenUrl Abstract / FREE Full Text (68). ↵ Brödel , A. K. ; Charpenay , L. H. ; Galtier , M. ; Fuche , F. J. ; Terrasse , R. ; Poquet , C. ; Havránek , J. ; Pignotti , S. ; Krawczyk , A. ; Arraou , M. ; Prevot , G. ; Spadoni , D. ; Yarnall , M. T. N. ; Hessel , E. M. ; Fernandez-Rodriguez , J. ; Duportet , X. ; Bikard , D. In Situ Targeted Base Editing of Bacteria in the Mouse Gut . Nature 2024 , 632 ( 8026 ), 877 – 884 . doi: 10.1038/s41586-024-07681-w . OpenUrl CrossRef PubMed (69). Zhao , C. ; Shu , X. ; Sun , B. Construction of a Gene Knockdown System Based on Catalytically Inactive (“Dead”) Cas9 (dCas9) in Staphylococcus Aureus . Applied and Environmental Microbiology 2017 , 83 ( 12 ), e00291 – 17 . doi: 10.1128/AEM.00291-17 . OpenUrl Abstract / FREE Full Text (70). ↵ Call , S. N. ; Andrews , L. B. CRISPR-Based Approaches for Gene Regulation in Non-Model Bacteria . Front. Genome Ed . 2022 , 4 . doi: 10.3389/fgeed.2022.892304 . OpenUrl CrossRef PubMed (71). ↵ Nguyen , H. H. ; De La Tour , C. B. ; Toueille , M. ; Vannier , F. ; Sommer , S. ; Servant , P. The Essential Histone-like Protein HU Plays a Major Role in Deinococcus Radiodurans Nucleoid Compaction . Molecular Microbiology 2009 , 73 ( 2 ), 240 – 252 . doi: 10.1111/j.1365-2958.2009.06766.x . OpenUrl CrossRef PubMed (72). ↵ Wiegand , T. ; Hoffmann , F. T. ; Walker , M. W. G. ; Tang , S. ; Richard , E. ; Le , H. C. ; Meers , C. ; Sternberg , S. H. TnpB Homologues Exapted from Transposons Are RNA-Guided Transcription Factors . Nature 2024 , 631 ( 8020 ), 439 – 448 . doi: 10.1038/s41586-024-07598-4 . OpenUrl CrossRef PubMed View the discussion thread. Back to top Previous Next Posted June 11, 2025. Download PDF Supplementary Material Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. 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