Part:BBa_K4601251

CmR expression cassette under the control of the J23110 promoter and of the Na+ RiboSwitch v1

This part is a reporter expression cassette controlled by the Na⁺ riboswitch v1.

Usage and Biology

A recently discovered class of riboswitches exhibits a remarkable ability to selectively sense and respond to sodium ions (Na⁺), and regulate the expression of genes relevant to sodium biology [1]. This riboswitch class, previously referred to as the 'DUF1646 motif', represents a novel addition to the diverse array of riboswitches found in bacteria [2,3]. The Na⁺ riboswitch was identified through bioinformatic analyses, revealing its presence in various bacterial phyla, including Firmicutes, Proteobacteria, Acidobacteria, and Verrucomicrobia [4].

It was predicted to form a distinctive secondary structure with two extended base-paired substructures, which together constitute the ligand-binding aptamer domain of the riboswitch (Figure 1). Experimental evidence demonstrated that the Na⁺ riboswitch selectively binds sodium ions while strongly rejecting other alkali and alkaline earth cations. It exhibits a dissociation constant (KD) in the low millimolar range, emphasizing its specificity for sodium ions. This selectivity for Na⁺ was confirmed through various biochemical assays [1]. The Na⁺ riboswitch was found to function as a genetic ON switch in response to Na⁺ binding. It is typically associated with an intrinsic transcription terminator stem, and the binding of Na⁺ prevents the formation of this terminator stem, allowing transcription to proceed, leading to increased gene expression (Figure 1).

Furthermore, it was observed that the riboswitch's response to Na⁺ concentrations was more pronounced at higher pH levels. In alkaline environments, where the cellular concentration of Na⁺ is expected to be reduced due to Na⁺/H⁺ antiporters' action, the riboswitch plays a critical role in adjusting gene expression to maintain sodium homeostasis.

Figure 1. Na⁺ riboswitch secondary structure in the presence and absence of Na⁺ ions. The prediction was described in the literature [1] and graphically represented using the forna RNA secondary structure visualization tool [5]. Nucleotides were coloured based on their position. The exact nucleotides forming the Na⁺ binding pocket were not yet identified.

Design

Two distinct versions of the Na⁺ riboswitch, based on the 'DUF1646 motif' present upstream of the kefB gene of Clostridium acetobutylicum [1], were designed and employed to investigate the activity of the channelrhodopsins (Figure 2).

The Na⁺ riboswitch v1 (BBa_K4601021) includes the key elements of the Na⁺ riboswitch: the ligand-binding aptamer domain and the sequence forming the intrinsic transcription terminator stem (Figure 1). To assess how this riboswitch influences transcription termination and gene expression in response to Na⁺ ions, we placed this sequence upstream of a reporter gene and its corresponding RBS and downstream of the medium strength constitutive promoter (BBa_J23110) for transcription initiation.

The Na⁺ riboswitch v2 (BBa_K4601022) includes all the v1’s sequence and an additional 49 nucleotides present upstream of it in the genome of C. acetobutylicum. It was this sequence that was tested in vivo in Bacillus subtilis to drive the expression of the lacZ gene encoding the full length sequence of the β-galactosidase [1] and thus assess how the Na⁺ riboswitch influences gene expression in a cellular context in response to environmental factors and cellular conditions. It is to be noted that no promoter B. subtilis sequence was placed upstream of lacZ gene. Thus, as a control of the Na⁺ riboswitch v1, we placed no E. coli promoter either.

Figure 2. Na⁺ riboswitches reporter constructs.

Build

The expression cassettes of the two versions of the Na⁺ riboswitch were assembled in the pSB3T5 backbone with one of the reporter genes of sfGFP (BBa_K4601221 and BBa_K4601222), LacZ𝛼 (BBa_K4601231 and BBa_K4601232), the ampicillin resistance gene AmpR (BBa_K4601241 and BBa_K4601242) or chloramphenicol resistance gene CmR (BBa_K4601251 and BBa_K4601252). Those constructs when bound to the Na⁺ will allow for the transcription of those reporter genes, while, when there is no Na⁺ binding, no transcription will occur.

Test

The first functional tests were performed in E. coli NEB^® 5-alpha cells carrying the plasmids, in the pSB3T5 backbone, containing the Na⁺ riboswitches v1 and v2 followed by two different reporter genes: sfGFP or chloramphenicol resistance gene CmR.

When the reporter gene was sfGFP, E. coli cells were grown overnight at 37 °C at 200 rpm in 96-deep-well plates with 1 mL of LB (Lennox) supplemented with 10 µg/mL tetracycline. The cells were then diluted by 40 times in LB NaCl-free media with 10 µg/mL tetracycline and after 4 hours of incubation at 37°C at 200 rpm, they were further diluted by 20 times in media containing 10 µg/mL tetracycline and increasing concentrations of NaCl in an opaque wall 96-well polystyrene microplate (COSTAR 96, Corning). This final media was either LB NaCl-free as such or buffered at pH 6, 7, 8 or 9 with 100 mM MES, PIPES, TAPS or AMPSO, respectively (all buffers were chosen based on literature [6] and prepared with KOH, not NaOH). We also tested the minimal salts (MS) media composed of 50 mM K₂HPO₄, 20 mM NH₄Cl, 4 mM Citric acid, 1 mM MgSO₄, 0.2% glucose, nitrilotriacetic acid 0.01 µM, CaCl₂ 3 µM, FeCl₃ 3 µM, MnCl₂ 1 µM ZnCl₂ 0.3 µM, Hsub>3</sub>BOsub>3</sub> 0.3 µM, CrClsub>3</sub> 0.3 µM, CoCl₂ 0.3 µM, CuCl₂ 0.3 µM, NiCl₂ 0.3 µM, Na₂MoO₄ 0.3 µM, Na₂SeO3 0.3 µM pH 7.2. The plate was then incubated at 37°C at 200 rpm and the sfGFP fluorescence (λ_excitation 488 nm and λ_emission 530 nm) and optical density at 600 nm (OD600) were measured every 10 minutes for 24 hours, in a CLARIOstar (BMGLabtech) plate reader. Fluorescence values were normalized by OD600.

When the reporter gene was CmR, E. coli cells were grown overnight at 37 °C at 200 rpm in LB (Lennox) supplemented with 10 µg/mL tetracycline. The cells were then diluted to an OD600nm of 1, 0.1 and 0.01 in LB NaCl-free and 5 µL of each suspension was spotted onto by LB agar plates containing 5 µg/mL tetracycline and increasing concentrations of chloramphenicol, followed by an overnight incubation at 37°C. Three different types of LB were used: LB NaCl-free, LB Lennox which contains 5 g/L NaCl and LB Luria-Miller which contains 10 g/L NaCl. When indicated, the LB agar was buffered at pH 6, 7, 8 or 9 with 100 mM MES, PIPES, TAPS or AMPSO, respectively (all buffers were chosen based on literature [6] and prepared with KOH, not NaOH).

Based on the results of these experiments (described below), we decided not to conduct any tests with LacZ𝛼 and AmpR as a reporter genes.

Learn

In the assessment of the Na⁺ riboswitches v1 and v2, we conducted a series of experiments to understand their functionality in E. coli NEB^® 5-alpha cells.

First, we examined the expression of sfGFP, a fluorescent reporter gene, under the control of these riboswitches in different media and at varying pH levels, while gradually increasing concentrations of NaCl were introduced (Figure 3). Surprisingly, we observed no significant changes in sfGFP fluorescence upon the addition of NaCl. This result suggests that the Na⁺ riboswitches v1 and v2 may not have a substantial impact on sfGFP expression under these conditions.

Additionally, we investigated the growth of E. coli NEB^® 5-alpha cells carrying the chloramphenicol acetyltransferase gene under the control of the Na⁺ riboswitches v1 and v2 in the presence of increasing concentrations of chloramphenicol (Figure 4). Notably, all cells appeared to grow similarly, regardless of the riboswitches' influence, indicating that the presence of Na⁺ riboswitches v1 and v2 is not significantly affecting chloramphenicol resistance.

However, it's important to note that our assessment could not be fully explored as, at pH 9, we observed no bacterial growth in liquid cultures, and growth was limited on plates. A similar trend was observed at pH 8, where bacterial growth remained very low. This suggests that the alkaline pH conditions may not be conducive to robust bacterial growth, potentially impacting the assessment of riboswitch functionality. The evaluation of riboswitch functionality at pH levels above 8 was challenging because E. coli exhibited difficulties in growing under such conditions. This limitation prevented a comprehensive assessment of riboswitch behavior at highly alkaline pH values in E. coli.

Figure 3. In vivo characterization of sfGFP expression by E. coli NEB^® 5-alpha cells carrying the Na⁺ riboswitches v1 and v2 (BBa_K4601221 and BBa_K4601222) in different media, at different pHs in the presence of increasing concentrations of NaCl. The data and error bars are the mean and standard deviation of at least three measurements on independent biological replicates.

Figure 4. In vivo characterization of the growth of E. coli NEB^® 5-alpha cells carrying the chloramphenicol acetyltransferase gene under the control of Na⁺ riboswitches v1 and v2 (BBa_K4601251 and BBa_K4601252) in the presence of increasing concentrations of chloramphenicol (Cm). As controls, we used the chloramphenicol acetyltransferase gene under the control of J23110 and pOmpR promoters (BBa_K4601254 and BBa_K4601253).

Moreover, in order to better understand the not significant changes in the behavior of both sfGFP and CmR reporter genes when the Na⁺ riboswitches v1 and v2 were present upstream or not, at pH values lower than 8 or in non buffered LB conditions, we took a closer look at the Na⁺ riboswitches v1 and v2 sequences and analyzed them with state of the art available tools (Figure 5). Thus, within the Na⁺ riboswitch v1 sequence, an internal promoter was predicted using the De Novo DNA's Promoter Calculator v1.0 (sigma70) [7], with a calculated transcription initiation rate of 11786. Moreover, another promoter, with a similar strength (TIR 12153) was predicted with the same tool in the upstream additional 49 nucleotides of the Na⁺ riboswitch v2 sequence. These two promoters are thus strong ones, stronger than all Anderson’s library promoters evaluated with this same tool [7]. It is thus perfectly explainable why our reporter genes are expressed alike in all conditions tested.

Furthermore, no terminator was predicted using the ANRold tool [8], indicating a potential mechanism by which the riboswitches may control gene expression. Notably, the initial tests establishing the terminator activity of the Na⁺ riboswitch in the absence of Na⁺ were carried out in vitro using the E. coli RNA polymerase [6].

Figure 5. Sequence of the Na⁺ riboswitches v1 (BBa_K4601021) and v2 (BBa_K4601022). The secondary structure in the presence and absence of Na⁺ ions are represented in dot-bracket notation based on the prediction described in the literature [1]. The approximate 3’ termini of the transcript in the absence of Na⁺ [1] is highlighted in red. Two promoters were identified using the De Novo DNA's Promoter Calculator v1.0 (sigma70) [7] and the corresponding -35 and -10 boxes as well at the +1 transcription start are highlighted in purple, blue and green respectively.

In summary, our experiments shed light on the behavior of Na⁺ riboswitches v1 and v2 in E. coli NEB^® 5-alpha cells. While sfGFP fluorescence and chloramphenicol resistance did not appear to be significantly affected by the riboswitches under the tested conditions, the presence of internal promoters and the absence of terminators within the riboswitch sequences may explain the loss of function. Furthermore, challenges associated with bacterial growth at high pH levels expose the experimental limits of testing riboswitches in high pH conditions.

References

[1] White N, Sadeeshkumar H, Sun A, Sudarsan N, Breaker RR. Na⁺ riboswitches regulate genes for diverse physiological processes in bacteria. Nature Chemical Biology (2022) 18: 878–885.

[2] McCown PJ, Corbino KA, Stav S, Sherlock ME, Breaker RR. Riboswitch diversity and distribution. RNA (New York, N.Y.) (2017) 23: 995–1011.

[3] Breaker RR. The biochemical landscape of riboswitch ligands. Biochemistry (2022) 61: 137–149.

[4] Weinberg Z, Lünse CE, Corbino KA, Ames TD, Nelson JW, Roth A, Perkins KR, Sherlock ME, Breaker RR. Detection of 224 candidate structured RNAs by comparative analysis of specific subsets of intergenic regions. Nucleic Acids Research (2017) 45: 10811–10823.

[5] Kerpedjiev P, Hammer S, Hofacker IL. Forna (force-directed RNA): Simple and effective online RNA secondary structure diagrams. Bioinformatics (Oxford, England) (2015) 31: 3377–3379.

[6] Stancik Lauren M., Stancik Dawn M., Schmidt Brian, Barnhart D. Michael, Yoncheva Yuliya N., Slonczewski Joan L. pH-Dependent expression of periplasmic proteins and amino acid catabolism in Escherichia coli. Journal of Bacteriology (2002) 184: 4246–4258.

[7] LaFleur TL, Hossain A, Salis HM. Automated model-predictive design of synthetic promoters to control transcriptional profiles in bacteria. Nature Communications (2022) 13: 5159.

[8] Naville M, Ghuillot-Gaudeffroy A, Marchais A, Gautheret D. ARNold: a web tool for the prediction of Rho-independent transcription terminators. RNA biology (2011) 8: 11–13.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]