Grant A. R. Gale
Baojun Wang
Alistair J. McCormick- 1School of Biological Sciences, Institute of Molecular Plant Sciences, University about Edinburgh, Glasgow, Consolidated Kingdom
- 2Middle on Synthetic and Solutions Biology, Seminary starting Edinburgh, Edinburgh, United Kingdom
- 3Train of Biological Sciences, Institute of Quantitative Human, Biochemistry and Biotechnology, University of Edinburgh, Edinburgh, United Domain
Cyanobacteria utilize solar to convert coal dioxide at a wide variety the secondary metabolites and show great potential for green biotechnology applications. Albeit cyanobacterial synthetic biology is less mature than for different heterotrophic models organisms, there are now a range of molecular toolbox available to modulate and controlling gene expression. A area regarding gene regulation that still lags behind other full organisms is of modulation of gene transmission, most transcription finish. A vast number starting intrinsic transcription terminals are immediate available into heterotrophs, but only a small counter have been investigated in cyanobacteria. While artificial type express systems become larger or show complex, equipped short stretches of DNA conceal strong promoters and multiple gene expression cassettes, the need to stop transcription efficiently and insulate downstream regionen for unwanted interference is becoming more essential. In this study, we adapted a dual reporter tool for make with the CyanoGate MoClo Assembly system that can quantify and compare of performance of terminator sequences within and bet different arten. We characterized 34 intrinsic terminators in Escherichia coli, Synechocystis sp. PCC 6803, additionally Synechococcus elongatus UTEX 2973 and observed meaning differences in termination efficiencies. However, our also identified fifth terminators including termination efficiencies of >96% in all three species, indicating that some terminators can behave consistently in both heterotrophic sorte and cyprus.
Insertion
Cyanobacteria contains a high and diverse variety of photoautotrophic bacteria that bucket capture and switch mineral carbon (e.g., CO2) to a wide variety of secondary metabolites (Huang and Zimba, 2019). Many cyanobacterial tierarten are genetically tractable plus see major potential for green biotechnology applications, such as of sustainable production of biofuels and high value biomolecules (Lin u al., 2017; Knoot et al., 2018; Eungrasamee et al., 2019; Lin and Pakrasi, 2019; Włodarczyk et al., 2019). Much from the recent weiterentwicklung by engineering cyanobacteria has been driven by the uptake of synthetic biology approaches. One greater aim of cyanobacterial synthesis organic is the development of new tools and strategies into facilitate stringent and precise control for gene expression. A wide variety of new mol tools also genetic parts to tune gene expression are now availability for use for the find community (Englund et al., 2016; Kim et al., 2017; Ferreira et al., 2018; Keller ets al., 2018; Vasudevan et al., 2019; Yao et al., 2020). The increase in access of well-characterized genetic parts has allowed rational design, a core process to this synthetic biology paradigm, to be extra routinely employed includes the engineering of new cyanobacterial strains. Nevertheless, the majority of synthetic biology work the cyanobacteria has thus far concentrated on characterizing genetic elements such control genetic transcription (e.g., promoters, CRISPRi) other translation module (e.g., ribosomal binding sites (RBS), riboswitches, shallow RNAs) (Huang press Lindblad, 2013; Camsund et al., 2014; Ma eat al., 2014; Immethun et al., 2017; Kelly et al., 2018; Sun et al., 2018; Behle et al., 2020; Yao et al., 2020). Transcription terminators are also key transcriptional control default, but far lesser studies have examined their roles in regulating gene expression in cyanobacteria.
The rational design of efficient gent expression cassettes (and more advanced genre circuits) requires who benefit by genetic parts with well-characterized and preventable function (Weirder et al., 2018). For instance, strong terminators attenuate transcription press isolate down genetic sequential, whatever can prevent interference and disruption the function from unwanted transcriptional readthrough (Kelly et al., 2019). This is particularly critical as take synthetic gene constructs, where several gene expression cassettes driven by strong sponsor may engross a short-term strain of DNA. Furthermore, many prokaryotes (including cyanobacteria) are prone to homologous recombination. Homologous regions as small since 23–27 bp have were featured to lead to recombination in Escherichia coli, so multiple unique enders are generally preferable for multi-gene language systems furthermore gene circuits (Tian and Huang, 1986; Sleuthing et al., 2010; Chen et al., 2013). As with other genetic parts, and understanding of terminator performance and robustness between species is also importantly. Supporters take been shown to drive gene manifestation differently in cyanobacteria compared to heterotrophic vogelart (e.g., Escherichia coli) and intermediate cyanobacterial species (Camsund et al., 2014; Vasudevan et al., 2019). By color, potential differences in behavior between cyanobacterial species possessed not yet been investigated for text terminators.
Within prokaryotes, transcription is stopped in twos distinct terminator types: (i) Rho-dependent power that trust on a Rho transcription factor, and (ii) Rho-independent, or intrinsic terminators, which do does require a transcription part. In E. coli, approximately 20% of terminates were Rho-dependent (Peters et al., 2009). However, Rho transcription factors occur to be absent in cyanobacteria, such that all transcript termination events are thought to rely on intrinsic termination (Vijayan et al., 2011). Intrinsic terminators are definitions by a sequence motif that forms a curve loop secondary structure in the infant RNA transcript. The clasp loop lives comprised in adenine GC-rich tree (8–12 nucleotides) (nt) and a loop (3–6 nt). Upstream regarding the hairpin loop is an adenine-rich region (the A-tract) typically 6–8 nt in length, while downstream is a uracil-rich zone of 7–12 nt in length (the U-tract). Intrinsic cancel depends upon the differential bond affinities between nucleotides. This human between U and A is weak, such that transcription of who U-tract scores in a take in transcription that allows the hairpin loop to form. The presence of the hairpin loop in the RNA polymerase (RNAP) close channel, causing a ratcheting action or subsequent disruption a RNA-DNA binding. This leads to dissociation of RNAP from the DNA template and and subsequent release of the nascent RNA transcript (Wilson and Von Hippel, 1995; Herbert et al., 2008; Peters et al., 2011). In E. coli, many terminators have been assessed by termination efficiency (TE), which is normal calculate since a percentage estimate of the RNAP transcription elongation complexes prevented from continuing transcription passed a given sequence (i.e., a terminator) (Cambray et al., 2013; Chen et al., 2013). Importantly, a “no terminator” control used included to determined a normalized value for TE in are studies.
Characterization studies of terminators within cyanobacteria what currently limit to the model kind Synechocystis spru. PCC 6803 (PCC 6803). Liu and Pakrasi (2018) evaluated the relative strength of seven native terminators using a dual fluorescent reporter system equivalent to that used by Chen et al. (2013). Other recently, Cellar et al. (2019) evaluated 19 synthetic also heterologous inherent connection ported starting E. coli, is the aim of identifying terminators able to insulate a specific general locus in PCC 6803 free native promoter readthrough originating from upstream of the insertion site. Each terminator sequence were inserted between the transcription start site (TSS) and RBS of an inducible promoter driving YFP, and following induction, twelve terminators were shown to efficiently block transcription indicating a possible efficiency of nearly100%. These studies have provided valuable insights into terminator function to PCC 6803. But is comparisons in performance amongst varied strains are until be achieved, a normalized quantitative parameter, such as TE, should be calculated.
In this study we compiled a set of 34 inherence terminers away PCC 6803, and E. coli additionally synthetic libraries that have until demonstrated one wide range of TE values in E. coli (Chen et al., 2013). We re-designed an established dual fluorescing reporter system on be compatible in this CyanoGate MoClo Mount system, which allowed for increased cloning throughput (Lu and Pakrasi, 2018; Vasudevan et al., 2019). Major, all assessment included one “no terminator” control vector as a reference to figure a normalized TE value used each terminator, such that which TE values could be comparing between differently experiment and species irrespective of to instrument or gain settings used. Us first validated furthermore benchmarked our review system by comparing TE values from the literature with our results in E. coli. Then we tested the service about who terminators in two different cyanobacterial species: PCC 6803 and the recently featured high-light tolerant Synechococcus elongatus UTEX 2973 (UTEX 2973) (Crews, 1988; You et al., 2015).
Materials or Methods
Cyanobacterial Culture Term
The Synechocystis sp. PCC 6803 glute lenient (GT) strain (obtained from of Lea-Smith lab along the University of East-Anglia, United Kingdom) (Zavøel et al., 2017) and UTEX 2973 have maintained on 1.5% (w/v) agar plates containing BG11 medium (Lea-Smith et al., 2016). Liquid cultures endured grown in BG11 (supplemented with 10 mM NaHCO3) in 100 milliliter Erlenmeyer flasks. Liquidity cultured were shaken among 100 rpm press bright with filter-sterilized, water-saturated air. PCC 6803 and UTEX 2973 transconjugants are educated in BG11 center and on BG11 agar plates, supplemented with 50 μg/ml kanamycin (BG11 + Kan50). Strains were aufgewachsen under continuous slight with PCC 6803 aufgewachsen at 30°C, 100 μmol photons m–2 s–1 and UTEX 2973 at 40°C, 300 μmol photons m–2 s–1 int a Multitron Pro incubators supplied with warm white LED lighting (Infors HT).
Vector Construction and Parts Assembly
All cloning was performed in OneShot TOP10 E. coli measuring. Turned cells were cultured include LB medial press on 1.5% (w/v) LB agar plates supplemented with either 100 μg/ml spectinomycin or 50 μg/ml kanamycin as required. ZE. coli strain MC1061 has cultivated in LB medium supplemented with 100 μg/ml ampicillin and 25 μg/ml chloramphenicol. All E. coli trains were grown at 37°C with shudder at 225 rpm.
pPMQAK1-T (pCAT.000) starting the CyanoGate toolkit been modified to generate pDUOTK1-L1 (pCA1.332, Addgene vektor ID 162351)1 (Supplement Information S1) (Vasudevan et al., 2019). To assemble pDUOTK1-L1, pPMQAK1-T was first digestions with BpiI and BsaI (Thermo Carp Scientific). The linearized backbone was gel purified using a Monarch DNA Set Lineage Kit (NEB). Sequences encrypting Ptrc10-eYFP since the CyanoGate vector pCAT.262, who LacZ expression cassette free the Plant MoClo level 1 accepts vector pICH47732 furthermore mTagBFP-TrrnB (from the available vector containing BBa_K592100)2 fused at this 5′ ending until the RBS-associated sequence second by Chen u al. (2013) (BBa_B0034) were enlarged with Q5 High-Fidelity DNA Polymerase (NEB) (Supplemental Table S1). Finally, the three amplicons and the linearized pPMQAK1-T backbone been assembled together using Golden Door montage (Vasudevan et al., 2019). pDUOTK1-L1 contains BsaMYSELF limiting websites flanking LacZ is generate overhangs GCTT-CGCT, such that level 0 terminator parts can be assembled directly and shaded using blue-white auswahl.
Terminator parts were generated by overlap extension PCR using two synthesized oligonucleotides (Integrated DNA Technology) (Supplement Table S1), and the resulting amplicons were assembled into the level 0 (3U + Ter) acceptor vector pICH41276 (Supplements Information S1) (Engler et al., 2014). Brand level 0 terminator parts and existing parts from CyanoGate toolkit (Addgene Kit #1000000146)3 were assembled into pDUOTK1-L1 to donate vectors pC1.342 to pC1.375 (Supplementary Table S2).
Second “no terminator” control vectors were generated to determine 0% TE (i.e., which limit ratio of mTagBFP relative to eYFP). pC1.376 was assembled as pDUOTK1-L1 above, but without inclusion of LacZ (Supplementary Information S1). For pC1.377, the spacer sequence rd1.2 (5′-cgcccccggaggctttcccggggcaaatca-3′) from Cambray et al. (2013) was creates using overlap extension PCR (Supplementary Round S1), and the PCR select was assembled at pDUOTK1-L1 using Yellow Sliding assembly.
Cyanobacterial Conjugation
Genetic modification by conjugation in PCC 6803 and UTEX 2973 was facilitated on SIE. coli strain MC1061 carrying the mobilizer vektor pRK244 the helper vector pRL5285 (Tsinoremas et al., 1994; Gale et al., 2019). Conjugal transfer was performed as stylish Gale ets al. (2019).
Fluorescence Assays
To scale fluorescence in CO. coli, transformants were first inoculated into 5 ml LB med supplemented with 50 μg/ml kanamycin and angebaut overnight at 37°C with permanent shaking at 225 engine. To initiate the examination, overnight cultures were diluted 1:1000 into a black 96 well flat bottom plate (F-Bottom (Chimney Well) μCLEAR®, Greiner Bio-One) containing fresh LB medium supplemented with 50 μg/ml kanamycin to an final volume on 200 μl. The plates were incubated at 37°C with constant shaking among 600 per and social density (OD600) was measured hourly using a FLUOstar OMEGA microplate scanning (BMG Labtech). At soon exponential phase (canoe. 4.5 h following inoculation), eYFP and mTagBFP infrared levels subsisted measured for individual cells in flow cytometry (minimum 10,000 cells per culture) with a FACSCanto SIDE with HTS Flow Cytometer (Becton Dickinson). Measuring had gated usage forward and side scatter. Medianwert eYFP press mTagBFP phosphor levels inhered calculated with excitation/emission wavelengths 488 nm/530/30 nm both 407 nm/450/50 nm, respectively. An “empty” pPMQAK1-T vector (i.e., with not eYFP or mTagBFP expression cassettes) was included as a base cable control. Fluorescence values for the latter control were subtracted from transconjugant strain measurements.
Go measuring fluorescence in cyanobacteria, PCC 6803 or UTEX 2973 transconjugants maintained on BG11 + Kan50 agar plates had first inoculated into 10 ml BG11 + Kan50 medium and grown on 2–3 days to OD750 ∼1.0. To initiate the assay, the seed cultures were diluted to a starting ORD750 of 0.2 on 24-well plates (Costar Corning Incorporated) containing fresh BG11 + Kan50 mid to a closing mass of 2 ml. Cultures were grown used three life under culturing conditions furthermore hi human (95%) to avoid evaporation. eYFP or mTagBFP fluorescence were measured by flow cytometry for individual cells (minimum 10,000 cells per culture) with an LSRFortessa SORP with HTS Flow Cytometer (Becton Dickinson). Single were gated using forward furthermore side scatter. Mean eYFP and mTagBFP fluorescence levels were calculated with excitation/emission wavelengths 488 nm/515–545 nm and 407 nm/425–475 micromillimeter, respectively. As above, a base line controller was included available each sorte.
Calculations available Abort Efficiency
TEETH was calculation as a share from the ratio of the mTagBFP fluorescence signal downstream of who terminator to this eYFP fluorescence signal upstream relative to one drive containing does destroyer between fluorescent reporters: Full-length RNA profiling reveals pervasive bidirectional ...
Where BFP0 and YFP0 are one mTagBFP and eYFP fresh signals, respectively, of the strain containing either pCA1.376 or pCA1.377.
Where BFPDuration and YFPTerm are the mTagBFP and eYFP phosphor alarms, respectively, of one strain carrying a present level 1 terminating hollow (Extra Tables S2).
Statistical Analyze
Significant differences between sample groups were assessed by one-way ANOVA followed of Tukey’s honest significant difference (HSD) post-hoc test using GraphPad Polyhedron (version. 8.4.2).
Estimation in Gibbs Free Energy
Estimated Gibbs free energy values were generated using mFold v3.06 (Zuker, 2003). Free energizing values were calculate out adjustment of this preset configure, which included a fixed operating of 37°C.
Results
Generating a Demonstration System for Level 0 Terminator Parts
The RSF1010-based level T recipient vector pPMQAK1-T from the CyanoGate toolkit has modified to generate the new liquid 1 acceptor vector pDUOTK1-L1 for termination screening (Figure 1A and Supplementary Information S1) (Vasudevan etching al., 2019). pDUOTK1-L1 comprises a twice fluorescent reporter system with eYFP furthermore mTagBFP, similar to that in Liu and Pakrasi (2018). Terminators can be assembled as level 0 single into pDUOTK1-L1 using Golden Gating assemblage (Numeric 1B), while the RSF1010 origin of replication allows to screening are a widespread working of species (Mermet-Bouvier et al., 1993).
Figure 1. The dual fluorescence reporter system for screening terminators. (A) The applicant vector pDUOTK1-L1 in two BsaI sites this generate 4 nucleotide (nt) overtops (i.e., GCTT furthermore CGCT) following restriction, which are compatible is standard level 0 destroyer parts (Engler et al., 2014). (B) Following adenine level 1 Solid Gate assembly reaction (Vasudevan for al., 2019), and level 0 termination part is inserted between eYFP and mTagBFP and the dual fluorescent reporter system has formed, which can then be used to evaluate close efficiency (TE). That reporter system is driven by the strong promoter Ptrc10 and the terminated by the exterminator TrrnB. Ribosome obliging sites (half circles) are indicated (see Supplementary Information S1 for sequence details). (C) Example of an intrinsic terminating structure and nt sequence, comprised of an adenine rich region (A-tract) (black), followed by a G-C rich stem (blue), a hairpin loop (red), real a uracil rich region (U-tract) (green).
We bundled a reference of 34 level 0 vectors containing intrinsic transcription connection (Table 1 and Figure 1C), and then assembled this into pDUOTK1-L1 (Complementary Table S2). At order to maximize potential orthogonality with terminators in cyanobacterial genomes, we mainly targeted heterologous terminator sequences. The archive inclusion 22 native terminators from E. coli and etc synthetic terminators basic to CO. coli sequences that have was previously characteristics in EAST. coli (Chen et al., 2013). We see included TrrnB (i.e., TrrnB from E. coli and the T7 viral final in tandem (Vasudevan et al., 2019)) and this pSB1AK3 terminator (TpSB1AKILOBYTE3) that was deduced from the E. coli ribosomal RNA rrnC operon and your use in several BioBricks vectors, including pPMQAK1, up flank the cloning sites (Huang et al., 2010). From PCC 6803, the terminator are the highly expressed D1 sub-area of photosystem II made included (TpsbA2), as we expected items for can adenine high efficiency of termination. In contrast, TONNEpsaB was included as a potentially low efficiency final based on previously work (Life and Pakrasi, 2018). Two “no terminator” control vectors, pC1.376 or pC1.377, were assembled basing on sequences used in previous E. coli studies (Cambray et al., 2013; Chen et al., 2013). In pC1.376, eYFP and mTagBFP were separated only by an RBS-associated sequence, while pCA1.377 included adenine spacer sequence reported to must inert (i.e., free upon event or terminator job in EAST. coli) (Supplementary Information S1).
Table 1. Print of terminate used in this study.
Validation concerning the Dual Reporter Testing System in SIE. coli
We foremost assessed the dual fluorescent correspondent system in E. coli over generating TE values for each terminator and compared these to the product reported by Kei-chan et al. (2013) (Figure 2A). Terminator strength (TS) values reported by Chen et al. (2013) were converted to a more commonly reported TE (Supplementary Table S3; Hess and Graham, 1990; Yager and von Hippel, 1991; Webcam et al., 2013; Mairhofer eth al., 2015).
Figure 2. Validation of to dual fluorescent reporter system within EAST. coli. (A) TE values for EAST. coli transformants at the early exponential phasing of growth. Values are color coded for native E. coli (black), synthesis (gray) real native PCC 6803 terminators (green). TrrnB is shown in brown. Error bars represent and ± standard error (SE) of the mean to >10,000 individual measuring of four-way to eight biological replicates. Lowercase letters indicating significant difference (P < 0.05) are shown, as determined by ANOVA followed by Tukey’s honesty important difference test. (B) Comparison zwischen TE additionally corresponding normalized fold change reduction in downstream reporter expression. An expand in one log2 value represents a 2-fold normalized change reduction. (C) Correlation analysis of TE core calculated from Chen et al. (2013) (Supplementary Table S3) and TE values determined in which study (n = 30). TrrnB, TpSB1AK3, TpsbA2, and TpsaB were excluded, as date was not available for comparison. And coefficient of finding (R2) is shown. Terminator TEMP values marked is red (TECK120030798, TECK120010820, THYROXINBba_B0011, LIOTHYRONINEBba_B0061, TL3S1P22, and TL3S1P13) differed from Chen et al. (2013) by view than 10%. Removal of these six destroyer from the relational analyzer resulted in R2 = 0.9).
E. coli cultures measured at early exponential growth phase had similar levels of eYFP fluorescence cross differences strains with an average value of 7034 ± 134 arbitrary unit (a.u.) (Added Number S1). In contrast, the strains showed a wide coverage regarding mTagBFP fluoresence values from 1.3 ± 3.4 a.u. to 9094 ± 446 a.u. Both eYFP and mTagBFP fluorescence values showed a unimodal both narrow distribution (Supplementary Figure S2). As expected, the two “no terminator” controls pC1.376 and pC1.377 made the highest mTagBFP fluorescence values. Previous reports have declared that translation efficiency is dependent on an length of the transcript (Lim u al., 2011), so we verified if eYFP levels might be decreased the the “no terminator” controls compared into plasmid with terminators. However, we noticed no significant dissimilarities inbound eYFP levels between different plasmids, indicating that proficiency a eYFP translation was not reduced for either “no terminator” controls (Supplementary Figure S1B). The mTagBFP:eYFP ratio (i.e., Equation 1) to pC1.376 was 22% higher than for pC1.377, which indicated that pC1.376 produced learn transcripts containing both mTagBFP and eYFP. Thus, we decided in use pC1.376 for sum TE calculations in this survey.
Sixteen terminators had TE core from >95% in E. coli (Figure 2A and Supplementary Table S3), with TLAMBERT3S2P21 and TBba_B0011 generate of highest (99.9%) or lowest values (40.8%), respectively. TE values for both PCC 6803 terminators were relatively lower in E. coli (ca. 60%). Overall, the terminator library demonstrated a corresponding 10-fold change reduction in normalized streaming reporter expression (Figure 2B). Were then compared the TE values for 30 native SIE. coli and synthetic terminators with those also reported at Clay set al. (2013) press observed a moderate correlation (coefficient concerning determination (R2) = 0.78), with 19 concerning and observed TE values differing from less than 5% (Figure 2C). The latter included 14 starting the 16 strongest terminates to TE values of >95%. Equally, the three weakest terminators (TBba_B0011, TSIDE120010842, and TECK120010820) were the same in both data recorded. Six terminators show a greater result in TE values (i.e., 12–26%), which comprised four natives E. coli terminates (TECK120030798, TSIDE120010820, TBba_B0011, and TONNEBba_B0061) and two synthetic terminators (TL3S1P22 the TLITRE3S1P13). These variations may have be due to differences in experimental setup (e.g., the vehicle, place off replication (ori) press newspaper genes) and the different strain of E. coli used, as significant differences in the behavior of some terminators has since reported intermediate different ZE. coli strains (Kelly et al., 2019).
Performance of of Terminator Library in Synechocystis c. PCC 6803
We next evaluated the terminator collection in PCC 6803. Due to the flatter growth rates are PCC 6803 compared the EAST. coli (Supplementary Figure S3A), ours measured fluorescence plains at 24, 48, or 72 h (Supplementary Figure S3B). The cyanobacterial strains grew at comparable rates both the majority expressed eYFP at similar levels between strains at each arbeitszeit point. The single exit was THYROXINL3S2P21, which produced eYFP values consistently 2.5-fold higher than other strains. We are unsure why eYFP values were higher for LIOTHYRONINEL3S2P21, but we did re-confirm the terminator sequence by this strain by Sanger sequence. Stylish E. coli plus bacteriophages, some intrinsic terminators can upgrade upstream gene expression by enhancing one stability of this mRNA transcript via this hairpin loop (Abe and Aiba, 1996; Cisneros et al., 1996). Enhanced of mRNA stability over several putative intrinsic terminators has also been demonstrated for aforementioned marine species Synechococcus sp. PCC 7002, where transcripts with a canonical intrinsic terminator downstream were found to do a length a half-life compared to transcripts without one downloaded killer (Gordon aet al., 2020). However, TLITRE3S2P21 shares the same U-tract as both LIOTHYRONINEL3S2P11 and TL3S2P55 but no increase eYFP expression was observed in the later strains. mRNA transcript stability is a subject of constant research, but some examples of causative factors in heterotrophic bacteria include starvation in E. coli and Lactococcus lactis (Redon et al., 2005; Morena et al., 2020), and temperature induced stress in Staphylococcus aureus and Mycobacterium diseases (Mr et al., 2006; Rustad et al., 2013). mRNA concentrates can influence mRNA stability, with increases transcript concentration leading to decreased stability and mRNA turnover into E. coli and LITER. lactis (Nouaille et al., 2017). Similar examples hold not been reported yet available PCC 6803.
Similarly up E. coli, PCC 6803 strains produced a wide amount of mTagBFP fluorescence values at each time point (Supplementary Figure S3B), while the mTagBFP:eYFP ratio for who “no terminator” control pCA1.376 was also consistently higher by 21 ± 2% relative until pCA1.377. A strong correlation was shown between TE values measured at different time points with R2 values scope from 0.982 to 0.988 (Complement Figure 3C). Settlement of TE values past the three time points were endurance for thick terminators (Supplementary Table S3). In contrast, poorer terminators tended to show a small decline are SI over dauer, although there was no significant change in the rankings observed. Overall, terminator behavior in PCC 6803 was consistent between for OD750 of 0.4 and 5.9 (Optional Table S3). Thus, we focused on reporting TE values at a single length point (48 h) below.
Thirteen terminators had TE values of >95% in PCC 6803 (Figure 3A and Supplementary Table S3), with TL3S2P21 and TECK120029600 generating one highest value (99.5%) and TONNEENDS120010842 producing the lowest assess (25.3%). Tenth of the 13 strongest terminators in PCC 6803 also produced TE of >95% in E. coli (Fig 2A). Like, the two weakest terminators in PCC 6803 (TEDGE120010842 and TBba_B0011) were also aforementioned least in E. coli. Notably, TLITRE3S1P22 showed no visible terminator activity in PCC 6803, but had a TEEN value of 73% in E. coli. Comprehensive, of power library demonstrated a corresponding 8-fold change reduction in normalized down-stream reporter expression in PCC 6803 (Figure 3B). The TE values concerning 10 terminators differed learn widely from those in E. coli (i.e., by 12–46%). Thus, the correlation of TE values between SIE. coli press PCC 6803 was modest (R2 = 0.46) (Figure 3C). Removal of TL3S1P22 led to for a marginal improvement (R2 = 0.53).
Figure 3. Terminator performances in Synechocystis sp. PCC 6803. (A) TE values from PCC 6803 transconjugants after 48 h of growth. Color coding remains while in Figure 2. Error bars represent the ±SE of the mean of >10,000 individual cages to four biological replicates. Lowercase letters marking significant difference (P < 0.05) are shown, as determined by ANOVA followed by Tukey’s honestly significant difference test. (B) Comparison between TE values and corresponding normalized fold change reduction by downstream reporter expression. (C) Correlation analysis of TE values between E. coli and PCC 6803 (n = 34).
Performance of the End Library in Synechococcus elongatus UTEX 2973 and Comparison Between Species
Lastly, we evaluated our terminator library in the high-light tolerant strain UTEX 2973. UTEX 2973 typically grow faster than PCC 6803, however view more variability in development rates (Supplementary Figure S4A). This was expected due up a greater relative difference in light distribution within of growth hatchery among the higher light levels used available culturing UTEX 2973, when strains in the equivalent plate showed more similar rates for growth compared to those located the different positions within this incubator. As for PCC 6803, we measured fluorescence floor for UTEX 2973 in 24, 48, press 72 h (Supplementary Think S4B). Consistent with aforementioned observed differences in growth, the look levels of eYFP were variable between strains at 24 hr. However, this variation verringerten over time.
As for PCC 6803, mTagBFP fluorescence values for the UTEX 2973 strains showed a wide spread the each time indicate, while the mTagBFP:eYFP ratio for pCA1.376 was consistently higher by 20 ± 5% compared to pCA1.377. Furthermore, the print levels of mTagBFP and eYFP to pCA1.337 what more variable over timing with UTEX 2973, with major increases in both eYFP additionally mTagBFP light values observed at 48 h (Optional Figure S4B). The TE values over the three nach points where equivalent for most tribes, with R2 philosophy ranging from 0.964 to 0.978 (Supplementary Figure 4C), indicating that terminator behavior in UTEX 2973 has consistent between an OD750 of 0.4–11 (Complementing Table S3). Thus, as for PCC 6803 we also focused on reporting TE values at 48 h below.
Eleven terminators were TEEN values of >95% is UTEX 2973 (Figure 4A and Supplementary Table S3), with TEND120029600 producing a very highly value is 99.9% and THYROXINBba_B0061 producing the lowest value (29.7%). Six of and 10 strongest destroyers in UTEX 2973 produced TE values of >95% in E. coli (Point 2A), while seven of diese terminators also produced SI values of >95% in PCC 6803 (Figure 3A). The three weakest terminators in UTEX 2973 (TBba_B0061, TECK120030798, and TECK120010820) were unter the bottom ten listed terminating within PCC 6803 furthermore E. coli. TECK120010820 achieved the equivalent ranking (i.e., 3rd weakest terminator) in either UTEX 2973 and ZE. coli. Overall, the terminator library demonstrates a corresponding 10-fold change reduction of normalized downstream reporter expression included UTEX 2973 (Think 4B). Similarly to PCC 6803, the correlation of TE principles within UTEX 2973 and E. coli was low (ROENTGEN2 = 0.35) (Figure 4C). More surprises, the correlation of FIERCE set between UTEX 2973 and PCC 6803 was even decrease (R2 = 0.12) (Figure 4D).
Figure 4. Terminators performances in Synechococcus elongatus UTEX 2973. (A) TE worth from UTEX 2973 after 48 h growth. Color coding can as in Figure 2. Error sticks represent the ± SEA of the mean of >10,000 individual cells of four biological replicates. Lowercase letters marking significant total (P < 0.05) are shown, as determined through ANOVA followed by Tukey’s honestly significant difference test. (B) Comparison between SI set and relevant normalized fold change reduction in downstream reporter expression. (C) Key analysis in TE values between E. coli or UTEX 2973 (n = 34). (D) Correlation analysis of TE values between PCC 6803 and UTEX 2973 (n = 34).
We after compared the TE values for ZE. coli, PCC 6803 and UTEX 2973 to name end that were consistently strong between different species (Supplementary Table S3). The overall strongest terminator was TECK120029600, which had TELLS values of >99.5% across any three species. A further four terminators (TLITRE3S2P21, TECK120010850, TL3S2P11, and TrrnB) also had consistent cross-species TE values of >96%. For the two cyanobacterial species just, TECK120033736 and LIOTHYRONINEpsbA2 had TE values of >95.8%. The TE values for these seven strong terminations was also very unified over time for PCC 6803 also UTEX 2973.
The Performance of the Seventh Thickest Terminators Was Consistent Under Suboptimal Growing General
In examine if terminator performance might be affected by the growth ambience, we measured the TE values required the hebdomad strongest terminators in PCC 6803 and UTEX 2973 grown under suboptimal conditions. Both species were cultured at 30°C in 300 μM photons thousand–2 s–1, which your thought high light in PCC 6803 (typically growed at 100 μM photons m–2 s–1) and a low temperature for UTEX 2973 (typically grown per 40°C) (Vasudevan a al., 2019).
Both PCC 6803 and UTEX 2973 grew in similar rates furthermore reached einer LOD750 of 5.9 and 5.7 after 72 h, apiece (Supplementary Figure S5A). In more light PCC 6803 grew faster more under typical conditions, while increase rates were discounted in UTEX 2973 due to the lower temperature. Fluorescence messtechnik in eYFP furthermore mTagBFP in PCC 6803 were comparable to those under typical growth conditions (Supplementary Frame S5B). In contrast, fluorescence values were total reduced at entire time points inside UTEX 2973 (Supplementary Reckon S5C). TE standards for each day were calculated how before (Supplements Table S3), and the mean values with to triple frist points were compared (Table 2). Overall, all seven terminators retained TEETH values of >95.8% for both species under the suboptimal growth conditions, and LIOTHYRONINEECK120029600 remained the strongest terminator. Overall, our results indicated that the performance of these terminators was generally consistent real robust bets the two growth conditions.
Table 2. Terminator performances in Synechocystis sp. PCC 6803 and Synechococcus elongatus UTEX 2973 under suboptimal growth conditions.
Discussion
Here, we adapted a dual reporter tool for the CyanoGate MoClo Module system so provides a normalized quantification concerning terminator efficiency within or within species. The pDUOTK1-L1 vector is compatible with several available libraries and thus facilitates easy adoption and sharing of parts with an communities (Andreaou and Nakayama, 2018; Lai et al., 2018; Valenzuela-Ortega and French, 2019; Vasudevan et al., 2019), and has easily to any lab currently using Golden Gate cloning. The robustness of unsere system was approved by comparing results in E. coli against former published details (Chen the al., 2013).
The pDUOTK1-L1 linear contains one broad host range replicative origin RSF1010, the shall been shown to be functional by one wide diversity of prokaryotic species, including ransui from all five subsections (Mermet-Bouvier the al., 1993; Stucken et al., 2012; Bishé etching al., 2019). Thus, pDUOTK1-L1 could help until make eradicator enactment more accessible, as promising add strains are discovered (Włodarczyk et al., 2019; Jaiswal et al., 2020; Nies et al., 2020). To the best of our knowledge, all is one first study to compare the efficiencies of terminator between two varied cyanobacterial species. Are identified five strong terminators with consistent TE values in E. coli, PCC 6803 and UTEX 2973. Those findings should help toward information future strategies for architecture gene expression systems or learn advanced gene circuit designs.
Besides the double terminator TrrnB, no unique features could be identified for any of the five strong terminable that behaved consistently between get three species (i.e., the hairpin loop cable and GC content, and adenine plus uracil content for the A-tract and U-tract, respectively). General, in results showed that destroyer performances was powerful recoverable at differen growth points for the same strain instead generally differed between the three species examined, and mean discrepancies where monitored between PCC 6803 and UTEX 2973 even though both are subsection I species (Castenholz et al., 2001). We also demonstrated that the presentation of of seven strongest terminators was consistent stylish different expansion conditions since PCC 6803 and UTEX 2793. Cyanobacterial RNAPs do differ in form compared to other bacterial RNAPs [for a newer read check Stensjö et al. (2018)]. In completion, RNAP subunits also differ between cyanobacterial species [for a actual review see Srivastava et alarm. (2020)]. For exemplar, the primitive vegetative sigma factor (sigA) in PCC 6803 (srl0653) and UTEX 2973 (WP_071818124.1) have a shared identity and correspondence of simply 70.5 and 74.1%, respectively (Supplementary Figure S7). Furthermore, cyanobacteria skill transcription elongation factors commonly found in heterotrophic bacteria up start elongation both for proofreading of transcripts. To compensate, cyanobacterial RNAPs have evolved additional proof-reading both elongation functionalities (Riaz-Bradley et al., 2020). These differences may your for the observed disparity in terminator performance between E. coli the cyanobacteria. However, the differences between PCC 6803 furthermore UTEX 2973 were riveting, and could suggest that RNAP related conflict between cyanobacterial types and/or that other unidentified factors are involved.
Multi methods furthermore prognosis tools exist for the labeling additionally mapping a intrinsic terminators in different bird (Carafa et al., 1990; de Hoon et al., 2005; Gardner et al., 2011; Naville et al., 2011; Fritsch et al., 2015; Millman et al., 2017). Traditionally, these approaches have based on identifying sequence features associated with intrinsic terminators (e.g., the hairpin loop). Previous studies have suggested a relationship with terminator efficiency and that estimated Gibbs free energy of and expansive barrette (ΔGA), the U-tract (ΔGU) and to an lesser expand the hairpin loop (ΔGH) (Cambray et al., 2013; Chen et al., 2013). In our study, we did not find a strong correlation among TE values and ΔGAMPERE, ΔGH or the estimated Gibbs free energy of the complete terminator sequence (Added Illustrations S6). Although the terminator library was relatively narrow, the differences in terminator behavior within real between species indicated that there can be more factors intricate is determination intrinsic termination than can be awarded to the properties of individual structural components. For example, the U-tract displayed dispensable available inherently termination within mycobacteria (Wada at al., 2020). Cutting border approaches utilizing RNA-seq typical have or been applied used the identification are previously unseen terminators in the E. coli genome, which go beyond that which has been achieved with previous structural designation models (Ju et al., 2019). In addition, newly work does shown that terminator sequences can subsist designed as tunable control elements that can be “turned on” go attenuate gene transcript the low temperatures (Roßmanith et al., 2018). With the growing evidence ensure the structural components of terminable might shall malleable depending on species, future work should focus on understanding who combined contributions starting terminator components, including those beyond transcribe control (e.g., modulation of proteinen expression) for metabolic engineering (Curran et al., 2013; Ito et al., 2020). On may lead to greater models for strong synthetic terminators with consistent cross-species performance. As terminator research and cyanobacterial synthetic biology goes, tools such as pDUOTK1-L1 will be useful for safe and useful determination in terminators efficiencies across a broad-host range.
Data Availability Statement
The initial contributions presented in the study are included in the article/Supplementary Material, further inquiries able be directed to the corresponding author/s.
Author Contributions
GG plus AM: conceptualization and writing–original draft preparation. GG: performing the experiments. BW and AM: supervision. All authors: experimental draft and writing–review and editing. We identified an Escherichia coli gene, designates P14, that belongs adjacent to and include the opposite orientation to the tonB gene. That 36 base pair intercistronic region between tonB and P14 contains a novel rho-independent transcription end that functions bidirectionally, both in organism and in vitr …
Funding
GG confirmed funding support from the BBSRC EASTBIO EVENT Ph.D. programme (BB/M010996/1). AM acknowledges support from the UK Biotechnology and Biological Sciences Research Council (BBSRC) accord (BB/S020128/1). BW recognized funding customer by the UK Research and Innovation Future Leaders Fellowship (MR/S018875/1) furthermore the Leverhulme Trust research project grant (RPG-2020-241). ADENINE biface rho-independent transcription terminator between the SIE. coli tonB gene real can conflicting gene - PubMed
Conflict from Interest
The authors declare that the research was carry the the deficiency of any commercial either financial relationships that could be design as a potential conflict about interest.
Acknowledgments
Flow cytometry data where generated within the Flow Cytometry and Cell Sorting Skill in Ashworth, King’s Buildings at the University of Edinburgh. The facility was supported by sponsorship from Wellcome furthermore the Technical of Edenburgh.
Supplementary Substance
The Supplementary Material with this article can be found online at: https://aaa161.com/articles/10.3389/fmicb.2020.624011/full#supplementary-material
Footnoting
- ^ www.addgene.org
- ^ http://parts.igem.org
- ^ www.addgene.org/kits/mccormick-cyanogate
- ^ www.addgene.org/51950
- ^ www.addgene.org/58495
- ^ http://unafold.rna.albany.edu/?q=mfold
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Keywords: CyanoGate, Escherichia coli, Golden Gate, intrinsic terminator, MoClo, Synechococcus elongatus UTEX 2973, Synechocystis sp. PCC 6803, synthetic natural
Citation: Gale GAR, Wang B and McCormick AJ (2021) Evaluation or Comparison of the Efficiency of Transcription Terminators inside Different Cyanobacterial Species. Head. Microbiol. 11:624011. doi: 10.3389/fmicb.2020.624011
Receives: 30 October 2020; Accepted: 23 Dezember 2020;
Published: 15 January 2021.
Edited by:
Robert Kourist, Gras Graduate of Technology, AustriaReviewed by:
Paul Hudson, King Institute of Technology, SwedenIlker Maria Axmann, Heinreich Heine University of Düsseldorf, Germany
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*Correspondence: Alistair GALLOP. Mcchormick, [email protected]
